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WO2006043271A1 - Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis - Google Patents

Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis Download PDF

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Publication number
WO2006043271A1
WO2006043271A1 PCT/IL2005/001096 IL2005001096W WO2006043271A1 WO 2006043271 A1 WO2006043271 A1 WO 2006043271A1 IL 2005001096 W IL2005001096 W IL 2005001096W WO 2006043271 A1 WO2006043271 A1 WO 2006043271A1
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WIPO (PCT)
Prior art keywords
seq
pea
polypeptide
sequence
humal
Prior art date
Application number
PCT/IL2005/001096
Other languages
French (fr)
Inventor
Amit Novik
Sarah Pollock
Zurit Levine
Dvir Dahary
Rotem Sorek
Osnat Sella-Tavor
Anat Cohen-Dayag
Shirley Sameach-Greenwald
Shira Walach
Original Assignee
Compugen Ltd.
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Publication date
Application filed by Compugen Ltd. filed Critical Compugen Ltd.
Publication of WO2006043271A1 publication Critical patent/WO2006043271A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention is related to novel nucleotide and protein sequences, and assays and methods of use thereof.
  • Diagnostic markers are important for early diagnosis of many diseases, as well as predicting response to treatment, monitoring treatment and determining prognosis of such diseases.
  • Serum markers are examples of such diagnostic markers and are used for diagnosis of many different diseases. Such serum markers typically encompass secreted proteins and/or peptides; however, some serum markers may be released to the blood upon tissue lysis, such as from myocardial infarction (for example Troponin-I). Serum markers can also be used as risk factors for disease (for example base-line levels of CRP, as a predictor of cardiovascular disease), to monitor disease activity and progression (for example, determination of CRP levels to monitor acute phase inflammatory response) and to predict and monitor drug response (for example, as shedded fragments of the protein Erb-B2).
  • risk factors for disease for example base-line levels of CRP, as a predictor of cardiovascular disease
  • CRP chronic myocardial infarction
  • Serum markers can also be used as risk factors for disease (for example base-line levels of CRP, as a predictor of cardiovascular disease), to monitor disease activity and progression (for example, determination of CRP levels to monitor acute phase inflammatory response) and to predict and monitor drug response (for example,
  • Immunohistochemistry is the study of distribution of an antigen of choice in a sample based on specific antibody-antigen binding, typically on tissue slices.
  • the antibody features a label which can be detected, for example as a stain which is detectable under a microscope.
  • the tissue slices are prepared by being fixed. IHC is therefore particularly suitable for antibody-antigen reactions that are not disturbed or destroyed by the process of fixing the tissue slices.
  • IHC permits determining the localization of binding, and hence mapping of the presence of the antigen within the tissue and even within different compartments in the cell. Such mapping can provide useful diagnostic information, including: 1) the histological type of the tissue sample
  • IHC information is valuable for more than diagnosis. It can also be used to determine prognosis and therapy treatment (as in the case of HER-2 in breast cancer) and monitor disease.
  • IHC protein markers could be from any cellular location. Most often these markers are membrane proteins but secreted proteins or intracellular proteins (including intranuclear) can be used as an IHC marker too.
  • IHC has at least two major disadvantages. It is performed on tissue samples and therefore a tissue sample has to be collected from the patient, which most often requires invasive procedures like biopsy associated with pain, discomfort, hospitalization and risk of infection. In addition, the interpretation of the result is observer dependant and therefore subjective. There is no measured value but rather only an estimation (on a scale of 1 -4) of how prevalent the antigen on target is.
  • the present invention provides, in different embodiments, many novel amino acid and nucleic acid sequences, which may optionally be used as diagnostic markers.
  • the present invention provides a number of different variants of known serum proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers.
  • the present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results.
  • serum markers require only a simple blood test and their result is typically a scientifically measured number.
  • IHC markers the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions.
  • the markers presented in the present invention can also potentially be used for in-vivo imaging applications.
  • the present invention also provides a number of different variants of known IHC proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers.
  • the present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results.
  • serum markers require only a simple blood test and their result is typically a scientifically measured number.
  • the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions.
  • a "marker-detectable disease” refers to a disease that may be detected by a particular marker, with regard to the description of such diseases below.
  • the markers of the present invention alone or in combination, show a high degree of differential detection between disease and non-disease states.
  • the present invention therefore also relates to diagnostic assays for disease detection optionally and preferably in a biological sample taken from a subject (patient), which is more preferably some type of body fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, secretions of the breast ductal system (and/or lavage thereof), broncho alveolar lavage, lavage of the reproductive system and/or lavage of any other part of the body or system in the body, and stool or a tissue sample.
  • body fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid,
  • the term may also optionally encompass samples of in vivo cell culture constituents.
  • the sample can optionally be diluted with a suitable eluant before contacting the sample to an antibody and/or performing any other diagnostic assay.
  • Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tmhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs.dtu.dyservices/TMHMM/TMHMM2.0b.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinformatics, www.ch.embnet.org/software/TMPRED_form.html) for transmembrane region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs
  • signalp_hmm and “signalp nn” refer to two modes of operation for the program SignalP: hmm refers to Hidden Markov Model, while nn refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor.
  • T - > C means that the SNP results in a change at the position given in the table from T to C.
  • M - > Q means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*).
  • a comment may be found in parentheses after the above description of the SNP itself.
  • This comment may include an FTId 5 which is an identifier to a SwissProt entry that was created with the indicated SNP.
  • An FTId is a unique and stable feature identifier, which allows construction of links directly from position-specific annotation in the feature table to specialized protein-related databases.
  • the FTId is always the last component of a feature in the description field, as follows: FTKKXXX number, in which XXX is the 3 -letter code for the specific feature key, separated by an underscore from a 6-digit number.
  • the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence.
  • SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
  • Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
  • Library-based statistics refer to statistics over an entire library, while EST clone statistics refer to expression only for ESTs from a particular tissue or cancer.
  • the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. Results are provided from microarrays using Affymetrix technology.
  • the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured.
  • the probe name begins with the name of the cluster (gene), followed by an identifying number.
  • Oligonucleotide microarray results taken from Affymetrix data were from chips available from Affymetrix Inc, Santa Clara, CA, USA (see for example data regarding the Human Genome U133 (HG-Ul 33) Set at www.affymetrix.com/products/arrays/specific/hgul33.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33av2.affx; and Human Genome Ul 33 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33plus.affx).
  • the probe names follow the Affymetrix naming convention.
  • nucleic acid sequences of the present invention refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below.
  • oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
  • disease includes any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.
  • marker in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients (subjects) having one of the herein-described diseases or conditions, as compared to a comparable sample taken from subjects who do not have one the above- described diseases or conditions.
  • a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT- based assays.
  • a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample.
  • the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
  • a relatively low amount of up-regulation may serve as the marker, as described herein.
  • One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
  • diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.”
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • detecting may also optionally encompass any of the above.
  • Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.
  • a biological sample obtained from the subject may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.
  • the term "level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
  • the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.
  • Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
  • Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
  • test amount of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or condition.
  • a test amount can be either in absolute amount (e.g., macOgram/ml) or a relative amount (e.g., relative intensity of signals).
  • control amount of a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition.
  • a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • Detect refers to identifying the presence, absence or amount of the object to be detected.
  • a “label” includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample.
  • the label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
  • the label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly.
  • the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
  • the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule.
  • the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
  • Exemplary detectable labels include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads.
  • the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically imrnunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein.
  • This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein.
  • amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion as in any of the previous claims.
  • the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
  • kits for detecting a Marker-detectable disease comprising a kit detecting specific expression of a splice variant as described herein.
  • the kit comprises a NAT-based technology.
  • the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein.
  • the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
  • the kit comprises an antibody as described herein.
  • the kit further comprises at least one reagent for performing an ELISA or a Western blot.
  • a method for detecting a Marker-detectable disease comprising detecting specific expression of a splice variant according to any of the above claims.
  • detecting specific expression is performed with a NAT-based technology.
  • detecting specific expression is performed with an immunoassay.
  • the immunoassay comprises an antibody as described herein.
  • a biomarker capable of detecting Marker-detectable disease comprising any nucleic acid sequence described herein or a fragment thereof, or any amino acid sequence described herein or a fragment thereof.
  • a method for screening for variant-detectable disease comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • a method for diagnosing a marker-detectable disease comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease comprising detecting cells affected by Marker- detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • a method of selecting a therapy for a marker-detectable disease comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
  • An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: HUMAl ACMJPE A 2 T21 (SEQ ID NO:1), HUMA1ACMJPEA_2_T27 (SEQ ID NO:2), or HUMA1ACM_PEA_2_T7 (SEQ ID NO:3).
  • An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: HUMAlACM_PEA_2_node_23 (SEQ ID NO:4), HUMAl ACM_PEA_2_node_41 (SEQ ID N0:5), HUMA IACM JPE A_2_node_51 (SEQ ID NO:6), HUMAlACM_PEA_2_node_0 (SEQ ID NO:7),
  • HUMAlACM JPEA_2_node_l (SEQ ID NO:8), HUMAl ACM_PEA_2_node_10 (SEQ ID NO:9), HUMAl ACM_PEA_2_node_l 1 (SEQ ID NO: 10),
  • HUMAlACM JPEA_2_node_12 (SEQ ID NC-.l l), HUMAlACM_PEA_2_node_13 (SEQ ID NO: 12), HUMAl ACM_PEA_2_node_14 (SEQ ID NO: 13), HUMAlACM_PEA_2_node_15 (SEQ ID NO:14), HUMAlACM_PEA_2_node_16 (SEQ ID NO:15), HUMAlACM_PEA_2_node_17 (SEQ ID NO:16), HUMAlACM_PEA_2_node_18 (SEQ ID NO:17), HUMAlACM_PEA_2_node_19 (SEQ ID NO: 18), HUMAlACM_PEA_2_node_2 (SEQ ID NO: 19), HUMAlACM_PEA_2_node_20 (SEQ ID NO:20),
  • HUMAlACM_PEA_2_node_7 SEQ ID NO:47
  • HUMAlACM_PEA_2_node_8 SEQ ID NO:48
  • HUMAl ACM_PEA_2_node_9 SEQ ID NO:49
  • HUMEGFRBB3_PEA_1_T2 SEQ ID NO:60
  • HUMEGFRBB3_PEA_1_T8 SEQ ID NO:61
  • HUMEGFRBB3_PEA_1_T9 SEQ ID NO:62
  • HUMEGFRBB3_PEA_l_T10 SEQ ID NO:63
  • HUMEGFRBB3JPEA_l_T20 SEQ ID NO:64
  • HUMEGFRBB3_PEA_1_T35 SEQ ID NO:65
  • HUMEGFRBB3_PEA_1_T38 SEQ ID NO:66
  • HUMEGFRBB3_PEA_l_T50 SEQ ID NO:67
  • HUMEGFRBB3_PEA_1_T54 SEQ ID NO:68
  • HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69).
  • HUMEGFRBB3_PEA_l_node_0 SEQ ID NO:70
  • HUMEGFRBB3_PEA_l_node_13 SEQ ID NO:71
  • HUMEGFRBB3_PEA_l_node_14 SEQ ID NO:72
  • HUMEGFRBB3_PEA_l_node_l 8 SEQ ID NO:73
  • HUMEGFRBB3_PEA_l_node_23 SEQ ID NO:74
  • HUMEGFRBB3_PEA_l_node_26 SEQ ID NO:75
  • HUMEGFRBB3_PEA_l_node_27 SEQ ID NO:76
  • HUMEGFRBB3_PEA_l_node_40 SEQ ID NO.77
  • HUMEGFRBB3_PEA_l_node_42 SEQ ID NO:78
  • HUMEGFRBB3_PEA_l_node_40 SEQ ID NO.77
  • HUMEGFRBB3_PEA_l_node_16 (SEQ ID NO:99), HUMEGFRBB3JPEAJ_node_17
  • HUMEGFRBB3_PEA_l_node_20 SEQ ID NO: 101
  • HUMEGFRBB3_PEA_l_node_21 SEQ ID NO:102
  • HUMEGFRBB3_PEA_l_node_22 (SEQ ID NO:103),
  • HUMEGFRBB3_PEA_l_node_24 (SEQ ID NO:104),
  • HUMEGFRBB3_PEA_l_node_28 (SEQ ID NO:105),
  • HUMEGFRBB3_PEA_l_node_30 (SEQ ID NO:106)
  • HUMEGFRBB3JPEA_l_node_31 (SEQ ID NO:107)
  • HUMEGFRBB3_PEA_l_node_34 (SEQ ID NO:108),
  • HUMEGFRBB3_PEA_l_node_35 (SEQ ID NO:109),
  • HUMEGFRBB3_PEA_l_node_37 (SEQ ID NO:110),
  • HUMEGFRBB3_PEA_l_node_39 (SEQ ID NO:111)
  • HUMEGFRBB3_PEA_l_node_44 (SEQ ID NO:112)
  • HUMEGFRBB3_PEA_l_node_45 (SEQ ID NO: 113),
  • HUMEGFRBB3_PEA_l_node_47 (SEQ ID NO:114),
  • HUMEGFRBB3_PEA_l_node_48 (SEQ ID NO:115),
  • HUMEGFRBB3_PEA_l_node_52 (SEQ ID NO:116)
  • HUMEGFRBB3_PEA_l_node_53 (SEQ ID NO:117)
  • HUMEGFRBB3_PEA_l_node_57 (SEQ ID NO:118),
  • HUMEGFRBB3_PEA_l_node_61 (SEQ ID NO:119),
  • HUMEGFRBB3_PEA_l_node_62 (SEQ ID NO:120),
  • HUMEGFRBB3_PEA_l_node_64 (SEQ ID NO:121
  • HUMEGFRBB3_PEA_l_node_71 (SEQ ID NO:122)
  • HUMEGFRBB3_PEA_l_node_73 (SEQ ID NO:123),
  • HUMEGFRBB3_PEA_l_node_74 (SEQ ID NO:124),
  • HUMEGFRBB3_PEA_l_node_78 (SEQ ID NO:125),
  • HUMEGFRBB3_PEA_l_node_80 (SEQ ID NO:126)
  • HUMEGFRBB3_PEA_l_node_81 (SEQ ID NO:127)
  • HUMEGFRBB3_PEA_l_node_82 (SEQ ID NO:128)
  • HUMEGFRBB3_PEA_l_node_84 (SEQ ID NO:129),
  • HUMEGFRBB3_PEA_l_node_85 (SEQ ID NO:130),
  • HUMEGFRBB3_PEA_l_node_90 (SEQ ID NO:131)
  • HUMEGFRBB3_PEA_l_node_91 (SEQ ID NO: 132)
  • HUMEGFRBB3_PEA_l_node_92 (SEQ ID NO: 133),
  • HUMEGFRBB3_PEA_l_node_96 (SEQ ID NO: 134), or
  • HUMEGFRBB3_PEA_l_node_99 SEQ ID NO: 135.
  • An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of: HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137), HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138), HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), HUMEGFRBB3JPEA_1_P46 (SEQ ID NO:142), HUMEGFRBB3_PEA_l_P50 (SEQ ID NO:143), HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), HUMEGFRBB3JPEAJ_P54 (SEQ ID NO: 145), or HUMEG
  • An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS AN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMA1ACM_PEA_2_P36
  • An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P36 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO:180) OF HUMAL ACM_PEA_2_P36 (SEQ ID NO:51).
  • An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%,homologous to a polypeptide having the sequence MERMLPLLALGLLAAG corresponding to amino acids 1 - 16 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90% homologous to
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%,homologous to the sequence MERMLPLLALGLLAAG of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
  • SEQ ID NO:51 An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
  • a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLV
  • An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2JP36 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
  • An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGN ASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDI LLQLGIEEAFTSKADLSGITGARNLAVSQV
  • An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
  • MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of
  • HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), second amino acid sequence being at least 90% homologous to
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2JP49 (SEQ ID NO:52).
  • SEQ ID NO: 182 sequence MERMLPLLALGLLAAG
  • SEQ ID NO: 182 sequence of HUMAl ACM_PEA_2JP49
  • a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDWKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequence
  • An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
  • FFKER (SEQ ID NO: 183) corresponding to amino acids 47 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
  • SEQ ID NO: 183 An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52).
  • An isolated chimeric polypeptide encoding for HUMAl ACM PEA 2 P59 comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLI
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
  • SEQ ID NO: 182 sequence MERMLPLLALGLLAAG
  • SEQ ID NO:53 An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
  • SEQ ID NO:185 sequence GECAWLGVQKRWISGPFLS
  • polypeptide being at least 70% homologous, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
  • SEQ ID NO:3 An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
  • a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLV
  • An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
  • An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P59 comprising a first amino acid sequence being at least 90 % homologous to
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACT HUM AN, which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to
  • An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P15 comprising a first amino acid sequence being at least 90% homologous to
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3JPEA 1 P15 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence SVMG (SEQ ID NO: 186) in HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P28 comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGD corresponding to amino acids 1 - 41 of ERB3 HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 41 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138), and a second amino acid sequence being at least 90% homologous to
  • QAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT corresponding to amino acids 918 - 1342 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 42 - 466 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • An isolated chimeric polypeptide encoding for an edge portion of HUMEGFRBB3_PEA_1_P28 comprising a polypeptide having a length "n", wherein n is at least about 10, 20, 30, 40 or 50 amino acids in length, wherein at least two amino acids comprise DA, having a structure as follows: a sequence starting from any of amino acid numbers 41-x to 41; and ending at any of amino acid numbers 42+ ((n-2) - x), in which x varies from 0 to n-2.
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1JP31 comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTK
  • YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) corresponding to amino acids 639 - 699 of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P31 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) in HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), comprising a first amino acid sequence being at least 90% homologous to
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P41 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) in HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P45 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) in HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140
  • WRDIVRDRDAEIVVKDNGRSC corresponding to amino acids 1 - 183 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 183 of HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), and a second amino acid sequence having the sequence KWP corresponding to amino acids 184 - 186 of HUMEGFRBB3 PEA 1JP45 (SEQ ID NO: 141), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), comprising a first amino acid sequence being at least 90% homologous to
  • ERB3_HUMAN ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 140 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
  • GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) corresponding to amino acids 141 - 183 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3JPEA 1JP46 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) in HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), comprising a first amino acid sequence being at least 90% homologous to
  • ERB3_HUMAN ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 204 of HUMEGFRBB3_PEAJ_P50 (SEQ ID NO: 143), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
  • CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) corresponding to amino acids 205 - 254 of HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_l_P50 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) in HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3JPEA_1_P53 comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTK
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3JPEA_1JP54 (SEQ ID NO: 145), comprising a first amino acid sequence being at least 90% homologous to
  • An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P54 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence RKCLRGRNNDQQ (SEQ ID NO: 191) in HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145).
  • An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P55 comprising a first amino acid sequence being at least 90% homologous to
  • polypeptide (SEQ ID NO: 146), comprising a polypeptide being at least 70%, optionally at least about
  • HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146).
  • a primer pair comprising a pair of isolated oligonucleotides capable of amplifying one of said amplicons.
  • the primer pair as described above comprising a pair of isolated oligonucleotides: SEQ NOs 54 and 55, or 57 and 58; or SEQ NOs 147 and 148, or 150 and 151.
  • kits for detecting a Marker-detectable disease comprising a kit detecting specific expression of a splice variant as described herein.
  • the kit comprises a NAT-based technology; optionally and preferably, this kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein; alternatively and optionally, the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
  • the kit comprises an antibody according to any of the above claims (optionally and preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
  • a method for detecting a Marker-detectable disease comprising detecting specific expression of a splice variant as described herein; optionally the marker-detectable disease is cluster HUMAlACM marker-detectable disease and is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer. Alternatively, the marker-detectable disease is cluster HUMEGFRBB3 marker-detectable disease and is selected from the group consisting of colon cancer, breast cancer and ovarian cancer.
  • Detecting specific expression is optionally performed with a NAT-based technology, and/or with an immunoassay (optionally comprising an antibody according to any of the above embodiments).
  • biomarker capable of detecting Marker- detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
  • a method for screening for variant-detectable disease comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments.
  • a method for diagnosing a marker-detectable disease comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments.
  • a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments.
  • a method of selecting a therapy for a marker- detectable disease comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments and selecting a therapy according to said detection.
  • a method may optionally be used when the marker-detectable disease is cluster HUMAlACM marker- detectable disease and is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer; and/or when the marker-detectable disease is cluster HUMEGFRBB3 marker- detectable disease and is selected from the group consisting of colon cancer, breast cancer and ovarian cancer.
  • any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
  • nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
  • the present invention provides isolated nucleic acid sequences comprising a sequence described herein.
  • the present invention provides amino acid sequences comprising a sequence described herein.
  • the present invention provides a head, tail, bridge or edge sequence described herein.
  • the present invention provides an antibody capable of specifically binding to an epitope of an amino acid sequence comprising sequences described herein.
  • the present invention further provides the antibody as above, wherein said amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion as described herein.
  • the present invention further provides the antibody as above, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
  • the present invention further provides a kit for detecting a Marker-detectable disease, comprising a kit detecting specific expression of a splice variant according to any of the above claims.
  • the present invention further provides the kit as above, wherein said kit comprises a NAT-based technology.
  • the present invention further provides the kit as above, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
  • the present invention further provides said kit, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
  • the present invention further provides said kit, wherein said kit comprises an antibody according to any of the above claims.
  • the present invention further provides said kit, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.
  • the present invention further provides a method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims.
  • the present invention further provides the method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims, wherein said detecting specific expression is performed with a NAT-based technology.
  • the present invention further provides the method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims, wherein said detecting specific expression is performed with an immunoassay.
  • the present invention further provides said method, wherein said immunoassay comprises an antibody according to any of the above claims.
  • the present invention further provides a biomarker capable of detecting Marker- detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
  • the present invention further provides a method for screening for variant- detectable disease, comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • the present invention further provides a method for diagnosing a marker- detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • the present invention further provides a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
  • the present invention further provides a method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
  • all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.
  • Figure 1 shows a schematic description of the cancer biomarker selection engine.
  • Figure 2 shows a schematic illustration, depicting grouping of transcripts of a given cluster based on presence or absence of unique sequence regions.
  • Figure 3 shows a schematic presentation of the oligonucleotide based microarray fabrication.
  • Figure 4 shows a schematic summary of the oligonucleotide based microarray experimental flow.
  • Figure 5 shows a schematic summary of quantitative real-time PCR analysis.
  • Figure 6 shows a graph of cancer and cell-line vs. normal tissue expression for HUMAlACM.
  • Figure 7 shows HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMA lACMseg26-3 IWT in normal and cancerous ovary tissues.
  • Figure 8 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous ovary samples relative to the normal samples.
  • Figure 9 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous lung samples relative to the normal samples.
  • Figure 10 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous lung samples relative to the normal samples.
  • Figure 10 is a histogram showing down regulation of the above-indicated serine
  • cysteine proteinase inhibitor or cysteine proteinase inhibitor
  • clade A alpha- 1 antiproteinase, antichymotrypsin
  • AACT_HUMAN member 3
  • Figure 11 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous colon samples relative to the normal samples.
  • Figure 12 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous colon samples relative to the normal samples.
  • Figure 13 shows the expression of HUMAlACM transcripts which are detectable by amplicons as depicted in sequence names HUMAlACMseg23 (SEQ ID NO:59) ( Figure 13A) and HUMAl ACMseg26-3 IWT ( Figure 13B) in different normal tissues.
  • Figure 14 shows a graph of cancer and cell-line vs. normal tissue expression for
  • Figure 15 is a histogram showing over expression of the amplicon for segl ⁇ (SEQ ID NO: 149) (node 18) Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts in cancerous Ovary samples relative to the normal samples.
  • Figure 16 is a histogram showing over expression of the amplicon for seg46 (node 46) Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts in cancerous Ovary samples relative to the normal samples.
  • the present invention provides variants, which may optionally be used as diagnostic markers.
  • variants are useful as diagnostic markers for marker-detectable (also referred to herein as “variant-detectable”) diseases as described herein.
  • variant markers are collectively described as "variant disease markers”.
  • the markers of the present invention can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker-detectable disease.
  • these markers may be used for staging the disease in patients (for example if the disease features cancer) and/or monitoring the progression of the disease.
  • the markers of the present invention alone or in combination, can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer.
  • one or more of the markers may optionally be used in combination with one or more other disease markers (other than those described herein).
  • Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
  • markers are specifically released to the bloodstream under conditions of a particular disease, and/or are otherwise expressed at a much higher level and/or specifically expressed in tissue or cells afflicted with or demonstrating the disease.
  • the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of a particular disease and/or a condition that is indicative of a higher risk for a particular disease.
  • the present invention therefore also relates to diagnostic assays for marker- detectable disease and/or an indicative condition, and methods of use of such markers for detection of marker-detectable disease and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
  • the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides.
  • bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
  • a "tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
  • a "head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
  • an edge portion refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein.
  • An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein.
  • a "bridge” may optionally be an edge portion as described above, but may also include a join between a head and a "known protein” portion of a variant, or a join between a tail and a "known protein” portion of a variant, or a join between an insertion and a "known protein” portion of a variant.
  • a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant.
  • the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).
  • bridges are described with regard to a sliding window in certain contexts below.
  • a bridge between two edges may optionally be described as follows: a bridge portion of CONTIG-N AME Pl (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-N AME Pl): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for
  • n is any number of amino acids between 10-50 amino acids in length.
  • the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1 , nor 50 + ((n-2) - x) (for example) greater than the total sequence length.
  • this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
  • antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
  • this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention.
  • this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
  • this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
  • this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
  • the splice variants described herein are non- limiting examples of markers for diagnosing marker-detectable disease and/or an indicative condition.
  • Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of marker-detectable disease and/or an indicative condition, including a transition from an indicative condition to marker-detectable disease.
  • any marker according to the present invention may optionally be used alone or combination.
  • Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker.
  • such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker.
  • the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
  • Panels of markers according to the present invention optionally with one or more known marker(s)
  • the present invention is of methods, uses, devices and assays for diagnosis of a disease or condition.
  • a plurality of biomarkers may be used with the present invention.
  • the plurality of markers may optionally include a plurality of markers described herein, and/or one or more known markers.
  • the plurality of markers is preferably then correlated with the disease or condition.
  • such correlating may optionally comprise determining the concentration of each of the plurality of markers, and individually comparing each marker concentration to a threshold level.
  • the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed)
  • the marker concentration correlates with the disease or condition.
  • a plurality of marker concentrations correlate with the disease or condition.
  • such correlating may optionally comprise determining the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and comparing the index value to a threshold level.
  • such correlating may optionally comprise determining a temporal change in at least one of the markers, and wherein the temporal change is used in the correlating step.
  • such correlating may optionally comprise determining whether at least "X" number of the plurality of markers has a concentration outside of a predetermined range and/or above or below a threshold (as described above).
  • the value of "X" may optionally be one marker, a plurality of markers or all of the markers; alternatively or additionally, rather than including any marker in the count for "X", one or more specific markers of the plurality of markers may optionally be required to correlate with the disease or condition (according to a range and/or threshold).
  • such correlating may optionally comprise determining whether a ratio of marker concentrations for two markers is outside a range and/or above or below a threshold.
  • the ratio correlates with the disease or condition.
  • a combination of two or more these correlations may be used with a single panel and/or for correlating between a plurality of panels.
  • the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects.
  • sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected out of the total number of negative samples present.
  • the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. More preferably, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects. Also more preferably, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms.
  • a marker panel may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value, or a value indicating a particular outcome. A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis.
  • diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or condition differentiating markers, etc. may be combined in a single assay or device.
  • Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different pu ⁇ ose(s).
  • Preferred panels comprise markers for the following purposes: diagnosis of a disease; diagnosis of disease and indication if the disease is in an acute phase and/or if an acute attack of the disease has occurred; diagnosis of disease and indication if the disease is in a non-acute phase and/or if a non-acute attack of the disease has occurred; indication whether a combination of acute and non-acute phases or attacks has occurred; diagnosis of a disease and prognosis of a subsequent adverse outcome; diagnosis of a disease and prognosis of a subsequent acute or non-acute phase or attack; disease progression (for example for cancer, such progression may include for example occurrence or recurrence of metastasis).
  • the above diagnoses may also optionally include differential diagnosis of the disease to distinguish it from other diseases, including those diseases that may feature one or more similar or identical symptoms.
  • one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s).
  • threshold level(s) of a diagnostic or prognostic indicator(s) can be established, and the level of the indicator(s) in a patient sample can simply be compared to the threshold level(s). The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test—they also depend on the definition of what constitutes an abnormal result.
  • Receiver Operating Characteristic curves are typically calculated by plotting the value of a variable versus its relative frequency in "normal” and “disease” populations, and/or by comparison of results from a subject before, during and/or after treatment.
  • a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease.
  • a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal.
  • the area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
  • the horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives.
  • the vertical axis of the curve represents sensitivity, which increases with the rate of true positives.
  • the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained.
  • the area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.
  • One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis/prognosis.
  • particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of the entire marker profile by plotting ROC curves for the sensitivity of a particular panel of markers versus 1 -(specificity) for the panel at various cutoffs.
  • a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) that an individual has had a disease, is at risk for developing such a disease, optionally the type of disease which the individual has had or is at risk for, and so forth etc.
  • a global probability expressed either as a numeric score or as a percentage risk
  • an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient.
  • Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.
  • markers and/or marker panels are selected to exhibit at least 70% sensitivity, more preferably at least 80% sensitivity, even more preferably at least 85% sensitivity, still more preferably at least 90% sensitivity, and most preferably at least 95% sensitivity, combined with at least 70% specificity, more preferably at least 80% specificity, even more preferably at least 85% specificity, still more preferably at least 90% specificity, and most preferably at least 95% specificity.
  • both the sensitivity and specificity are at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%.
  • Sensitivity and/or specificity may optionally be determined as described above, with regard to the construction of ROC graphs and so forth, for example.
  • individual markers and/or combinations (panels) of markers may optionally be used for diagnosis of time of onset of a disease or condition. Such diagnosis may optionally be useful for a wide variety of conditions, preferably including those conditions with an abrupt onset.
  • determining the prognosis refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient.
  • the term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome may be about 3%.
  • a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance.
  • the term "about” in this context refers to +/-1%.
  • associating a prognostic indicator with a predisposition to an adverse outcome is a statistical analysis.
  • a marker level of greater than 80 pg/mL may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to 80 pg/mL, as determined by a level of statistical significance.
  • a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events.
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value.
  • Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.
  • a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level.
  • a preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%.
  • the term "about” in this context refers to +/-10%.
  • a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome.
  • data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers.
  • the group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects.
  • the first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state.
  • this first set of patients may be those that have recently had a disease and/or a particular type of the disease.
  • the confirmation of this condition state may be made through more rigorous and/or expensive testing, preferably according to a previously defined diagnostic standard.
  • subjects in this first set will be referred to as "diseased".
  • the second set of subjects are simply those who do not fall within the first set.
  • Subjects in this second set may be "non-diseased;” that is, normal subjects.
  • subjects in this second set may be selected to exhibit one symptom or a constellation of symptoms that mimic those symptoms exhibited by the "diseased" subjects.
  • the data obtained from subjects in these sets includes levels of a plurality of markers.
  • data for the same set of markers is available for each patient.
  • This set of markers may include all candidate markers which may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required.
  • Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition.
  • the levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.
  • a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient.
  • An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve as described above.
  • ROC Receiveiver Operating Characteristic
  • data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response.
  • the data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file.
  • the database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased.
  • an artificial cutoff region may be initially selected for each marker.
  • the location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer.
  • the cutoff region is initially centered about the center of the overlap region of the two sets of patients.
  • the cutoff region may simply be a cutoff point.
  • the cutoff region may have a length of greater than zero.
  • the cutoff region may be defined by a center value and a magnitude of length.
  • the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects.
  • a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.
  • Each marker value for each patient may then be mapped to an indicator.
  • the indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis.
  • the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.
  • the relative importance of the various markers may be indicated by a weighting factor.
  • the weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer.
  • acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5.
  • the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.
  • a panel response may be calculated for each subject in each of the two sets.
  • the panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker.
  • an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker.
  • a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state.
  • the use of an indicator which is constant on one side of the cutoff region eliminates this concern.
  • the panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient. Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response. Having obtained panel responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the selection of an effective panel.
  • the objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.
  • the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function.
  • the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers.
  • other features of the ROC curve may be used to define the objective function.
  • the point at which the slope of the ROC curve is equal to one may be a useful feature.
  • the point at which the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee” may be used.
  • the sensitivity at the knee may be maximized.
  • the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of these ROC- curve features may be used.
  • one of the markers in the panel is specific to the disease or condition being diagnosed.
  • the panel response may be set to return a "positive" test result.
  • the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.
  • An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimums.
  • the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables.
  • the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.
  • the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.
  • the optimization algorithm may be provided with certain constraints as well.
  • the resulting ROC curve may be constrained to provide an area-under-curve of greater than a particular value.
  • ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets.
  • a minimum acceptable value such as 0.75
  • Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.
  • the iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function.
  • the number of iterations may be limited in the optimization process.
  • the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.
  • the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators.
  • certain markers may be eliminated from the panel.
  • the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers.
  • the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated.
  • Individual panel response values may also be used as markers in the methods described herein.
  • a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above).
  • Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker.
  • the sample values for each marker are added together to arrive at the panel response for that particular sample to be tested.
  • the resulting panel responses may be treated as if they were just levels of another disease marker.
  • Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003 (hereby incorporated by reference as if fully set forth herein), and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas.
  • suitable tests may exhibit one or more of the following results on these various measures: at least 75% sensitivity, combined with at least 75% specificity; ROC curve area of at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; and/or a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (l-sensitivity)/specif ⁇ city) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1.
  • a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting marker-detectable disease and/or an indicative condition, such that a biomarker may optionally comprise any of the above.
  • the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein.
  • Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges.
  • the present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
  • the present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
  • the present invention also relates to kits based upon such diagnostic methods or assays.
  • Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • the present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
  • the present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.
  • the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.
  • a "nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide” are used herein interchangeably to refer to a polymer of nucleic acids.
  • a polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • RNA sequence a complementary polynucleotide sequence
  • cDNA complementary polynucleotide sequence
  • genomic polynucleotide sequence e.g., a combination of the above.
  • complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
  • genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • composite polynucleotide sequence refers to a sequence, which is composed of genomic and cDNA sequences.
  • a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
  • Preferred embodiments of the present invention encompass oligonucleotide probes.
  • an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
  • Equipment and reagents for executing solid- phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.
  • Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.
  • the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.
  • the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
  • oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
  • oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non- natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms can also be used.
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts, as disclosed in U.S. Pat. Nos.
  • oligonucleotides which can be used according to the present invention, are those modified in both sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target.
  • An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference.
  • Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat.
  • Oligonucleotides of the present invention may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted
  • Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl
  • oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
  • a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element.
  • cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
  • any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
  • the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
  • cell type- specific and/or tissue-specific promoters include promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al, (1988) Adv. Immunol. 43:235- 275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729- 733] and immunoglobulins; [Banerji et al.
  • the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
  • the nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
  • the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
  • the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
  • retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter.
  • Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining elements), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct, hi addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
  • Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described).
  • RNA detection Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.
  • Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
  • the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
  • Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and
  • hybridization of short nucleic acids (below 200 bp in length, e.g.
  • hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 niM EDTA
  • TMACI 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the T m ;
  • H hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI 3
  • hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
  • labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
  • Probes can be labeled according to numerous well known methods.
  • Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
  • detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
  • detectable markers for use with probes which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
  • oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs)
  • streptavidin e.g., phycoerythrin-conjugated streptavidin
  • fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • RNAse A RNAse A prior to hybridization
  • the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods.
  • radioactive nucleotides can be incorporated into probes of the invention by several methods.
  • Non-limiting examples of radioactive labels include 3 H, 14 C, 32 P, and 35 S.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • NAT-based assays which involve nucleic acid amplification technology, such as PCR for example (or variations thereof such as real-time PCR for example).
  • a "primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab.
  • amplification techniques Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill.
  • Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid.
  • RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA.
  • the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.
  • the nucleic acid i.e. DNA or RNA
  • the nucleic acid for practicing the present invention may be obtained according to well known methods.
  • Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
  • the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al, 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N. Y.). It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
  • the polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail below).
  • the pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 0 C, most preferably less than 3 0 C, ideally between 3 0 C and 0 0 C.
  • Tm melting temperatures
  • PCR Polymerase Chain Reaction
  • PCR The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al, is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification.
  • This technology provides one approach to the problems of low target sequence concentration.
  • PCR can be used to directly increase the concentration of the target to an easily detectable level.
  • This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize.
  • the primers are extended with polymerase so as to form complementary strands.
  • the steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
  • the length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR- amplified.”
  • LCR Ligase Chain Reaction
  • LAR Ligase Amplification Reaction
  • LCR LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 Al (1990).
  • the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal.
  • the use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
  • the self-sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest.
  • the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest.
  • the use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
  • Q-Beta (Q ⁇ ) Replicase In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
  • thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • a successful diagnostic method must be very specific.
  • a straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction.
  • the basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle.
  • reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.85 ⁇ 0, or 220,513 copies of the starting material.
  • a PCR running at 85 % efficiency will yield only 21 % as much final product, compared to a reaction running at 100 % efficiency.
  • a reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product.
  • nucleic acid detection technologies such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences.
  • One method of the detection of allele-specif ⁇ c variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer.
  • An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
  • This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
  • the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
  • CPR cycling probe reaction
  • CPR Cycling probe reaction
  • the cycling probe reaction (CPR) uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
  • Branched DNA involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
  • labels e.g., alkaline phosphatase enzymes
  • the detection of at least one sequence change may be accomplished by, for example restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single- Strand Conformation Polymorphism (SSCP) analysis or Dideoxy fingerprinting (ddF).
  • RFLP analysis restriction fragment length polymorphism
  • ASO allele specific oligonucleotide
  • DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
  • SSCP Single- Strand Conformation Polymorphism
  • ddF Dideoxy fingerprinting
  • nucleic acid segments for mutations.
  • One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest.
  • amplified material e.g., PCR reaction products
  • a given segment of nucleic acid may be characterized on several other levels.
  • the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel.
  • a more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map.
  • the presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
  • Restriction fragment length polymorphism For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
  • RFLP restriction fragment length polymorphism
  • MCC Mismatch Chemical Cleavage
  • RFLP analysis When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
  • Allele specific oligonucleotide can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match.
  • Hybridization with radioactively labeled allelic specific oligonucleotides also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.
  • the ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
  • the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest.
  • DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
  • the fragments to be analyzed are "clamped” at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands.
  • the attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNA:RNA duplexes.
  • TGGE temperature gradient gel electrophoresis
  • Single-Strand Conformation Polymorphism (SSCP): Another common method, called “Single-Strand Conformation Polymorphism” (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
  • SSCP Single-Strand Conformation Polymorphism
  • the SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non- denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run.
  • This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
  • Dideoxy fingerprinting (ddF) The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP.
  • a dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
  • the ddF technique as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.
  • the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy fingerprinting.
  • Detection may also optionally be performed with a chip or other such device.
  • the nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group.
  • This reporter group can be a fluorescent group such as phycoerythrin.
  • the labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station, describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
  • the chip is inserted into a scanner and patterns of hybridization are detected.
  • the hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
  • polypeptide include glycoproteins, as well as non-glycoproteins.
  • Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N. Y.], after which their composition can be confirmed via amino acid sequencing. In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J.
  • the present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein.
  • the present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low-complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50.
  • NCBI National Center of Biotechnology Information
  • nucleic acid sequence homology/identity is determined by using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11.
  • NCBI National Center of Biotechnology Information
  • the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor- valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • the peptides of the present invention are preferably utilized in diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.
  • the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 3:17-311, Coruzzi et al. (1984) EMBO J.
  • Antibody refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)' 2 fragments.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • Monoclonal antibody development may optionally be performed according to any method that is known in the art. The method described below is provided for the purposes of description only and is not meant to be limiting in any way. Step 1 : Immunization of Mice and Selection of Mouse Donors for Generation of Hvbridoma Cells
  • Producing mAb requires immunizing an animal, usually a mouse, by injection of an antigen X to stimulate the production of antibodies targeted against X.
  • Antigen X can be the whole protein or any sequence thereof that gives rise to a determinant.
  • optionally and preferably such antigens may include but are not limited to any variant described herein or a portion thereof, including but not limited to any head, tail, bridge or unique insertion, or a bridge to such head, tail or unique insertion, or any other epitope described herein according to the present invention.
  • Injection of peptides requires peptide design (with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability) and synthesis.
  • the antigen is optionally and preferably prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes optionally used as immunogens. Other immunogens or adjuvants may also optionally be used.
  • mice are immunized every 2-3 weeks but the immunization protocols are heterogeneous. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.
  • serum antibodies are optionally and preferably obtained from mice for measurement of serum antibodies.
  • Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and/or immunoassays for example (for example a Western blot may optionally be used). If the antibody titer is high, cell fusion can optionally be performed. If the titer is too low, mice can optionally be boosted until an adequate response is achieved, as determined by repeated blood sampling.
  • ELISA enzyme-linked immunosorbent assay
  • flow cytometry for example a Western blot may optionally be used. If the antibody titer is high, cell fusion can optionally be performed. If the titer is too low, mice can optionally be boosted until an adequate response is achieved, as determined by repeated blood sampling.
  • mice When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.
  • Fusing antibody-producing spleen cells which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth.
  • Myeloma cells are immortalized cells that are optionally and preferably cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine- aminopterin-thymidine (HAT) selection medium used after cell fusion.
  • the selection growth medium contains the inhibitor aminopterin, which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cell line to which the normal antibody-producing cells are fused.
  • the HAT medium allows only the fused cells to survive in culture. A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth.
  • the antibody forming cells are isolated from the mouse's spleen and are then fused with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely.
  • a cancer cell such as cells from a myeloma
  • the resulting cell is called a hybridoma.
  • Step 4 Fusion of Myeloma Cells with Immune Spleen Cells and antibody screening
  • Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. Alternatively, the cells are centrifuged, the supernatant is discarded and PEG is then added. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and Kennedy 1994). Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells (Hoffman and others 1996).
  • hybridoma colonies reach a satisfactory cell count, the plates are assayed by an assay, eg ELISA or a regular immunoassay such as RIA for example, to determine which colonies are secreting antibodies to the immunogen.
  • an assay eg ELISA or a regular immunoassay such as RIA for example.
  • Cells from positive wells are isolated and expanded.
  • Conditioned medium from each colony is retested to verify the stability of the hybridomas (that is, they continue to produce antibody).
  • Step 5 Cloning of Hybridoma Cell Lines by "Limiting Dilution” or Expansion and Stabilization of Clones by Ascites Production
  • small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time.
  • Cloning consists of subcloonng the cells by either limiting dilution at an average of less than one cell in each culture well or by platingout the cells in a thin layer of semisolid agar of methyl cellulose or by single-cell manipulation. At each stage, cultures are assayed for production of the appropriate antibodies.
  • the secreted antibodies are optionally purified, preferably by one or more column chromatography steps and/or some other purification method, including but not limited to ion exchange, affinity, hydrophobic interaction, and gel permeation chromatography.
  • column chromatography steps and/or some other purification method including but not limited to ion exchange, affinity, hydrophobic interaction, and gel permeation chromatography.
  • the operation of the individual chromatography step, their number and their sequence is generally tailored to the specific antibody and the specific application.
  • Large-scale antibody production may also optionally and preferably be performed according to the present invention. Two non-limiting, illustrative exemplary methods are described below for the purposes of description only and are not meant to be limiting in any way.
  • In vivo production may optionally be performed with ascites fluid in mice. According to this method, hybridoma cell lines are injected into the peritoneal cavity of mice to produce ascitic fluid (ascites) in its abdomen; this fluid contains a high concentration of antibody.
  • An exemplary in vitro method involves the use of culture flasks.
  • monoclonal antibodies can optionally be produced from the hybridoma using gas permeable bags or cell culture flasks.
  • PCT Application No. WO 94/18219 and its many US equivalents, including US Patent No. 6096551 , all of which are hereby incorporated by reference as if fully set forth herein, describes methods for producing antibody libraries using universal or randomized immunoglobulin light chains, by using phage display libraries.
  • the method involves inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene for the purpose of producing light chain gene libraries for use in combination with heavy chain genes and gene libraries to produce antibody libraries of diverse and novel immunospecificities.
  • the method comprises amplifying a CDR portion of an immunoglobulin light chain gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide.
  • PCR polymerase chain reaction
  • the resultant gene portions are inserted into phagemids for production of a phage display library, wherein the engineered light chains are displayed by the phages, for example for testing their binding specificity.
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • a scFv antibody fragment is an engineered antibody derivative that includes heavy- and light chain variable regions joined by a peptide linker.
  • the minimal size of antibody molecules are those that still comprise the complete antigen binding site. ScFv antibody fragments are potentially more effective than unmodified IgG antibodies. The reduced size of 27-30 kDa permits them to penetrate tissues and solid tumors more readily.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • the chain could be the heavy or the light chain.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co- workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species, hi practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
  • An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination.
  • One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.
  • an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample.
  • This method comprises: providing an antibody that specifically binds to a marker; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample.
  • Antibodies that specifically bind to a protein marker can be prepared using any suitable methods known in the art.
  • a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays.
  • Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • EIA enzyme immune assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay e.g., Western blot assay
  • slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
  • solid supports include but are not limited to glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead.
  • Antibodies can also be attached to a solid support.
  • the mixture is washed and the antibody-marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent.
  • the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours.
  • the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like.
  • the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10 0 C to 40 0 C.
  • the immunoassay can be used to determine a test amount of a marker in a sample from a subject.
  • a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody-marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above.
  • the amount of an antibody-marker complex can optionally be determined by comparing to a standard.
  • the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and/or signal.
  • Preferably used are antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, for example.
  • Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies”.
  • Radioimmunoassay In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody
  • radiolabeled antibody binding protein e.g., protein A labeled with I
  • a precipitable carrier such as agarose beads.
  • the number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a labeled substrate and an unlabelled antibody binding protein are employed.
  • a sample containing an unknown amount of substrate is added in varying amounts.
  • the decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Enzyme linked immunosorbent assay This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
  • Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Immunohistochemical analysis This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies.
  • the substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
  • Fluorescence activated cell sorting This method involves detection of a substrate in situ in cells by substrate specific antibodies.
  • the substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • Both of these techniques are non-invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example.
  • PET positron emission tomography
  • SPECT can optionally be used with two labels simultaneously.
  • SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used.
  • US Patent No. 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
  • a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-
  • display vehicles such as phages, viruses or bacteria
  • GenBank sequences the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see www.ncbi.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST database in GenBank, may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
  • Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced. Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No. 10/426,002, published as US20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into "clusters" that represent genes or partial genes.
  • the GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
  • Dry analysis Library annotation - EST libraries are manually classified according to:
  • Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell-lines; normal tissues; cancer tissues; fetal tissues; and others such as normal cell lines and pools of normal cell-lines, cancer cell-lines and combinations thereof. A specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
  • Protocol of library construction various methods are known in the art for library construction including normalized library construction; non-normalized library construction; subtracted libraries; ORESTES and others. It will be appreciated that at times the protocol of library construction is not indicated. The following rules are followed:
  • Clusters having at least five sequences including at least two sequences from the tissue of interest are analyzed.
  • Clones no. score Generally, when the number of ESTs is much higher in the cancer libraries relative to the normal libraries it might indicate actual over-expression.
  • the algorithm —
  • Clones number score The total weighted number of EST clones from cancer libraries was compared to the EST clones from normal libraries. To avoid cases where one library contributes to the majority of the score, the contribution of the library that gives most clones for a given cluster was limited to 2 clones. The score was computed as
  • tissue libraries/sequences were compared to the total number of libraries/sequences in cluster. Similar statistical tools to those described in above were employed to identify tissue specific genes. Tissue abbreviations are the same as for cancerous tissues, but are indicated with the header "normal tissue”.
  • Each cluster includes at least 2 libraries from the tissue T. At least 3 clones (weighed - as described above) from tissue T in the cluster; and 2. Clones from the tissue T are at least 40 % from all the clones participating in the tested cluster
  • a Region is defined as a group of adjacent exons that always appear or do not appear together in each splice variant.
  • a “segment” (sometimes referred also as “seg” or “node”) is defined as the shortest contiguous transcribed region without known splicing inside. Only reliable ESTs were considered for region and segment analysis. An EST was defined as unreliable if:
  • Each unique sequence region divides the set of transcripts into 2 groups:
  • S 1 is significantly enriched by cancer EST clones compared to S2;
  • Region 1 common to all transcripts, thus it is not considered; Region 2: specific to Transcript 1 : T_l unique regions (2+6) against T_2+3 unique regions (3+4); Region 3: specific to Transcripts 2+3: T_2+3 unique regions (3+4) against Tl unique regions (2+6); Region 4: specific to Transcript 3: T_3 unique regions (4) against T 1+2 unique regions (2+5+6); Region 5: specific to Transcript 1+2: T_l+2 unique regions (2+5+6) against T3 unique regions (4); Region 6: specific to Transcript 1 : same as region 2.
  • Microarray fabrication Microarrays were printed by pin deposition using the MicroGrid II MGII 600 robot from BioRobotics Limited (Cambridge, UK). 50-mer oligonucleotides target sequences were designed by Compugen Ltd (Tel-Aviv, IL) as described by A. Shoshan et al, "Optical technologies and informatics", Proceedings of SPIE. VoI 4266, pp. 86-95 (2001).
  • the designed oligonucleotides were synthesized and purified by desalting with the Sigma-Genosys system (The Woodlands, TX, US) and all of the oligonucleotides were joined to a C6 amino-modified linker at the 5' end, or being attached directly to CodeLink slides (Cat #25-6700-01. Amersham Bioscience, Piscataway, NJ, US).
  • the 50- mer oligonucleotides, forming the target sequences were first suspended in Ultra-pure DDW (Cat # 01 -866- IA Kibbutz Beit-Haemek, Israel) to a concentration of 50 ⁇ M. Before printing the slides, the oligonucleotides were resuspended in 30OmM sodium phosphate (pH 8.5) to final concentration of 15OmM and printed at 35-40% relative humidity at 21 0 C.
  • Each slide contained a total of 9792 features in 32 subarrays. Of these features, 4224 features were sequences of interest according to the present invention and negative controls that were printed in duplicate. An additional 288 features (96 target sequences printed in triplicate) contained housekeeping genes from Human Evaluation Library2, Compugen Ltd, Israel. Another 384 features are E.coli spikes 1-6, which are oligos to E- CoIi genes which are commercially available in the Array Control product (Array control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
  • Slides were treated for blocking of the residual reactive groups by incubating them in blocking solution at 50°C for 15 minutes (lOml/slide of buffer containing 0.1M Tris, 5OmM ethanolamine, 0.1% SDS). The slides were then rinsed twice with Ultra-pure DDW (double distilled water). The slides were then washed with wash solution (10ml/slide. 4X SSC, 0.1% SDS)) at 50°C for 30 minutes on the shaker. The slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 rpm.
  • the slides were treated with Ventana Discovery hybridization station barcode adhesives.
  • the printed slides were loaded on a Bio-Optica (Milan, Italy) hematology staining device and were incubated for 10 minutes in 50ml of 3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess fluid was dried and slides were then incubated for three hours in 20 mm/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc. Redding CA).
  • the following protocol was then followed with the Genisphere 900-RP (random primer), with mini elute columns on the Ventana Discovery HybStationTM, to perform the microarray experiments. Briefly, the protocol was performed as described with regard to the instructions and information provided with the device itself. The protocol included cDNA synthesis and labeling. cDNA concentration was measured with the TBS-380 (Turner Biosystems. Sunnyvale, CA.) PicoFlour, which is used with the OHGreen ssDNA Quantitation reagent and kit.
  • Hybridization was performed with the Ventana Hybridization device, according to the provided protocols (Discovery Hybridization Station Tuscon AZ).
  • Figure 4 shows a schematic method for performing the microarray experiments. It should be noted that stages on the left-hand or right-hand side may optionally be performed in any order, including in parallel, until stage 4 (hybridization). Briefly, on the left-hand side, the target oligonucleotides are being spotted on a glass microscope slide (although optionally other materials could be used) to form a spotted slide (stage 1). On the right hand side, control sample RNA and cancer sample RNA are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled probes. It should be noted that the control and cancer samples come from corresponding tissues (for example, normal prostate tissue and cancerous prostate tissue).
  • the tissue from which the RNA was taken is indicated below in the specific examples of data for particular clusters, with regard to overexpression of an oligonucleotide from a "chip” (microarray), as for example "prostate” for chips in which prostate cancerous tissue and normal tissue were tested as described above.
  • the probes are mixed.
  • hybridization is performed to form a processed slide.
  • stage 5 the slide is washed and scanned to form an image file, followed by data analysis in stage 6.
  • Certain splice variants described herein are potential markers for ovarian cancer. Other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
  • a metastasis of unknown origin which originated from a primary ovarian cancer, for example gastric carcinoma (such as Krukenberg tumor), breast cancer, colorectal carcinoma and pancreatic carcinoma.
  • gastric carcinoma such as Krukenberg tumor
  • breast cancer colorectal carcinoma
  • pancreatic carcinoma for example gastric carcinoma (such as Krukenberg tumor)
  • ovary related markers include but are not limited to: cancers of the endometrium, cervix, fallopian tubes, pancreas, breast, lung and colon; nonmalignant conditions such as pregnancy, endometriosis, pelvic inflammatory disease and uterine fibroids.
  • Non-malignant causes of pelvic mass Including, but not limited to: benign (functional) ovarian cyst, uterine fibroids, endometriosis, benign ovarian neoplasms and inflammatory bowel lesions
  • b. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, skeletal or abdominal pain, paraneoplastic syndrome, c. Ascites.
  • Certain splice variants described herein are potential markers for lung cancer. Other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
  • the assessment of a malignant tissue residing in the lung and is from a non-lung origin including, but not limited to: osteogenic and soft tissue sarcomas; colorectal, uterine, cervix and corpus tumors; head and neck, breast, testis and salivary gland cancers; melanoma; and bladder and kidney tumors.
  • Conditions which have similar symptoms, signs and complications as lung cancer and where the differential diagnosis between them and lung cancer is of clinical importance including but not limited to: a Non-malignant causes of lung symptoms and signs. Symptoms and signs include, but are not limited to: lung lesions and infiltrates, wheeze, stridor.. b. Other symptoms, signs and complications suggestive of lung cancer, such as tracheal obstruction, esophageal compression, dysphagia, recurrent laryngeal nerve paralysis, hoarseness, phrenic nerve paralysis with elevation of the hemidiaphragm and Horner syndrome. c.
  • Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, hypophosphatemia, hyponatremia, syndrome of inappropriate secretion of antidiuretic hormone, elevated ANP, elevated ACTH, hypokalemia, clubbing, neurologic-myopathic syndromes and thrombophlebitis.
  • CA 125 may optionally be used for a number of diagnostic assays, such as detection of sepsis (and/or similar bacterial infections) and/or monitoring of the course of infection (as described with regard to PCT Application No. WO 03/048776, hereby incorporated by reference as if fully set forth herein) for example.
  • Ovarian cancer markers according to the present invention which may also optionally have this utility include but are not limited to: ErbB-2 variants, EGFR variants, BCMP variants, Stromelysin-3 variants, TGFBl variants, and/or Inhibin beta variants.
  • This section relates to examples of sequences according to the present invention, including illustrative methods of selection thereof.
  • the markers of the present invention were tested with regard to their expression in various cancerous and non-cancerous tissue samples.
  • a description of the samples used in the ovarian cancer testing panel is provided in Table 2 below.
  • a description of the samples used in the colon cancer testing panel is provided in Table 3 below.
  • a description of the samples used in the lung cancer testing panel is provided in Table 4 below.
  • a description of the samples used in the breast cancer testing panel is provided in Table 5 below.
  • a description of the samples used in the normal tissue panel is provided in Table 6 below. Tests were then performed as described in the "Materials and Experimental Procedures" section below.
  • Table 2 Tissue samples in ovarian cancer testing panel
  • RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ USA 07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA www.biochain.com), ABS (Wilmington, DE 19801, USA, www.absbioreagents.com), Ambion (Austin, TX 78744 USA, www.ambion.com), or GOG for ovary samples— Pediatic Cooperative Human Tissue Network, Gynecologic Oncology Group Tissue Bank, Children Hospital of Columbus (Columbus OH 43205 USA).
  • RNA was generated from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion).
  • RT PCR - Purified RNA (1 ⁇ g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 ⁇ M dNTP in a total volume of 15.6 ⁇ l. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 ⁇ l of 5X SuperscriptII first strand buffer (Invitrogen), 2.4 ⁇ l 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 °C, followed by further incubation at 42 0 C for 2 min.
  • Real-Time RT-PCR analysis- cDNA (5 ⁇ l), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche).
  • the amplification was effected as follows: 50 0 C for 2 min, 95 0 C for 10 min, and then 40 cycles of 95 0 C for 15sec, followed by 60 0 C for 1 min. Detection was performed by using the PE Applied Biosystem SDS 7000. The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and was used to calculate the relative transcript quantity in the RT reactions.
  • Ct threshold level
  • the efficiency of the PCR reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes.
  • RT reverse transcription
  • HSKP housekeeping
  • Threshold Cycle point which is the cycle that the amplification curve crosses the fluorescence threshold that was set in the experiment. This point is a calculated cycle number in which PCR product signal is above the background level (passive dye ROX) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geometrically increasing phase, during which measurements are most accurate, followed by a linear phase and a plateau phase; for quantitative measurements, the latter two phases do not provide accurate measurements).
  • the y-axis shows the normalized reporter fluorescence. It should be noted that this type of analysis provides relative quantification.
  • SDHA Forward primer (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG
  • SDHA Reverse primer (SEQ ID NO: 154) CCACCACTGCATCAAATTCATG
  • SDHA-amplicon (SEQ ID NO: 155): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC
  • PBGD GenBank Accession No. BC019323; SEQ ID NO: 194)
  • PBGD Forward primer (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG
  • PBGD Reverse primer (SEQ ID NO: 157)
  • CCAGGGTACGAGGCTTTCAAT PBGD-amplicon (SEQ ID NO: 158)
  • GAPDH GenBank Accession No. BC026907, SEQ ID NO: 196)
  • GAPDH Forward primer (SEQ ID NO: 162) TGCACC ACCAACTGCTTAGC
  • GAPDH Reverse primer (SEQ ID NO: 163) CCATC ACGCC ACAGTTTCC
  • GAPDH-amplicon (SEQ ID NO: 164) :
  • PBGD Forward primer (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG PBGD Reverse primer: (SEQ ID NO: 157) CCAGGGTACGAGGCTTTCAAT PBGD-amplicon: (SEQ ID NO: 158) TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGAC GGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
  • HPRTl (GenBank Accession No. NM_000194, SEQ ID NO:195), HPRTl Forward primer: (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO:160) GGTCCTTTTCACCAGCAAGCT HPRTl-amplicon: (SEQ ID NO:161)
  • G6PD GenBank Accession No. NM_000402, SEQ ID NO: 197) G6PD Forward primer: (SEQ ID NO: 165) gaggccgtcaccaagaacat G6PD Reverse primer: (SEQ ID NO: 166) ggacagccggtcagagctc G6PD-amplicon: (SEQ ID NO: 167) gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcggga gggacctgcagagctctgaccggctgtccc
  • RPS27A (GenBank Accession No. NM_002954, SEQ ID NO: 198)
  • Ubiquitin (GenBank Accession No. BC000449, SEQ ID NO: 199)
  • Ubiquitin Forward primer (SEQ ID NO:171) ATTTGGGTCGCGGTTCTTG
  • Ubiquitin Reverse primer (SEQ ID NO: 172) TGCCTTGACATTCTCGATGGT Ubiquitin-amplicon (SEQ ID NO: 173)
  • SDHA Forward primer (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG SDHA Reverse primer: (SEQ ID NO: 154) CCACCACTGCATC AAATTCATG SDHA-amplicon (SEQ ID NO: 155): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC CAGTAGTGGATCATGAATTTGATGCAGTGGTGG
  • PBGD Forward primer (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG
  • PBGD Reverse primer (SEQ ID NO: 157)
  • CCAGGGTACGAGGCTTTCAAT PBGD-amplicon (SEQ ID NO: 158)
  • HPRTl GenBank Accession No. NM_000194, SEQ ID NO:195
  • HPRTl Forward primer (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO: 160) GGTCCTTTTC ACCAGC AAGCT HPRTl-amplicon: (SEQ ID NO: 161)
  • G6PD GenBank Accession No. NM_000402, SEQ ID NO: 197)
  • G6PD Forward primer (SEQ ID NO: 165) gaggccgtcaccaagaacat
  • G6PD Reverse primer (SEQ ID NO: 166) ggacagccggtcagagctc
  • G6PD-amplicon (SEQ ID NO: 167) : gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcggga gggacctgcagagctctgaccggctgtcc
  • SDHA (GenBank Accession No. NM_004168; SEQ ID NO: 193) SDHA Forward primer: (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG SDHA Reverse primer: (SEQ ID NO: 154) CCACCACTGCATCAAATTCATG SDHA-amplicon (SEQ ID NO :155):
  • PBGD GenBank Accession No. BC019323; SEQ ID NO: 194
  • PBGD Forward primer (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG
  • PBGD Reverse primer (SEQ ID NO: 157)
  • CCAGGGTACGAGGCTTTCAAT PBGD-amplicon (SEQ ID NO: 158)
  • RPL 19 (GenBank Accession No. NM_000981, SEQ ID NO:200)
  • RPL19Reverse primer (SEQ ID NO:175)
  • TGATCAGCCCATCTTTGATGAG RPL19-amplicon (SEQ ID NO:176):
  • TATA box (GenBank Accession No. NM_003194, SEQ ID NO:201), TATA box Forward primer (SEQ ID NO: 177): CGGTTTGCTGCGGTAATCAT
  • TATA box Reverse primer (SEQ ID NO: 178) TTTCTTGCTGCCAGTCTGGAC
  • TATA box -amplicon (SEQ ID NO: 179)
  • Ubiquitin (GenBank Accession No. BC000449, SEQ ID NO: 199)
  • Ubiquitin Forward primer (SEQ ID NO:171) ATTTGGGTCGCGGTTCTTG
  • Ubiquitin Reverse primer (SEQ ID NO: 172) TGCCTTGACATTCTCGATGGT Ubiquitin-amplicon (SEQ ID NO: 173)
  • SDHA Forward primer (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG
  • SDHA Reverse primer (SEQ ID NO: 154) CCACC ACTGCATC AAATTC ATG
  • SDHA-amplicon (SEQ ID NO :155) :
  • Cluster HUMAlACM features 3 transcript(s) and 46 segment(s) of interest, the names for which are given in Tables 7 and 8, respectively, the sequences themselves are given at the end of the application.
  • the selected protein variants are given in table 9.
  • HUMAIACM-PEA _2_node_34 (SEQ ID NO:29)
  • variants of the known protein Alpha- 1-antichymotrypsin precursor SEQ ID NO:50
  • SwissProt accession identifier AACTJHUMAN known also according to the synonyms ACT
  • the variant proteins according to the present invention are variants of a known diagnostic marker, called Alpha 1 antichymotrypsin (Enzymes) for Chronic lung diseases and HISTOMARKER a-1 antichymotrypsin Histiocytic marker.
  • Protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO:50) is known or believed to have the following function(s): Although its physiological function is unclear, it can inhibit neutrophil cathepsin G and mast cell chymase, both of which can convert angiotensin I to the active angiotensin II.
  • the sequence for protein Alpha- 1- antichymotrypsin precursor is given at the end of the application, as "Alpha- 1- antichymotrypsin precursor amino acid sequence".
  • Known polymorphisms for this sequence are as shown in Table 10.
  • Protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO:50) localization is believed to be Extracellular.
  • annotation(s) apply to the previously known protein.
  • the following annotation(s) were found: acute-phase response; inflammatory response; regulation of lipid metabolism, which are annotation(s) related to Biological Process; DNA binding; serine protease inhibitor; protein binding, which are annotation(s) related to Molecular Function; and extracellular; intracellular, which are annotation(s) related to
  • the GO assignment relies on information from one or more of the
  • Alpha- 1 -antichymotrypsin is the major plasma inhibitor of neutrophil leukocyte elastase (NE) in the serum.
  • NE neutrophil leukocyte elastase
  • cluster HUMAlACM can be used as a diagnostic marker for diseases such as chronic lung diseases, pulmunary embolysm and stroke.
  • Alpha- 1 -antichymotrypsin is an extracellular protein synthesized in the liver. Its concentration increases in the acute phase of inflammation or infection. In the serum, Alpha- 1 -antichymotrypsin can be found bound to prostate specific antigen (PSA). Therefore, cluster HUMAlACM can optionally be used as a diagnostic marker for prostate cancer.
  • PSA prostate specific antigen
  • Cluster HUMAlACM can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods.
  • the term "number" in the left hand column of the table and the numbers on the y-axis of the figure below refer to weighted expression of ESTs in each category, as "parts per million” (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
  • cluster HUMAlACM features 3 transcript(s), which were listed in Table 7 above. These transcript(s) encode for protein(s) which are variant(s) of protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO: 50). A description of each variant protein according to the present invention is now provided.
  • Variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMAl ACMJPE A_2_T7 (SEQ ID NO:3).
  • An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows: Comparison report between HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) and Q96DW8 (SEQ ID NO:202):
  • LAn isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDS AAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMAl ACM_PEA_2_P36 (SEQ
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS AN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) OF HUMA1ACM_PEA_2JP36 (SEQ ID NO:51).
  • TGARNLAVSQV corresponding to amino acids 1 - 341 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
  • AAA51559 (SEQ ID NO.204):
  • chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYG
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence
  • chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO: 51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGN ASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDI
  • LLQLGIEEAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 357 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 357 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SL corresponding to amino acids 358 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans ⁇ membrane region..
  • Variant protein HUMA1ACM_PEA_2_P36 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Table 13 - Amino acid mutations Single Nucleotide Polymorphisms
  • Variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) is encoded by the following transcript(s): HUMAl ACMJ 5 E A_2_T7 (SEQ ID NO:3), for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HUMAl ACM_PEA_2_T7 (SEQ ID NO:3) is shown in bold; this coding portion starts at position 84 and ends at position 1160.
  • the transcript also has the following SNPs as listed in Table 14 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMA1ACM_PEA_2_T21 (SEQ ID NO:1).
  • An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPD
  • polypeptide encoding for a head of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDBGMVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACMJPE A_2
  • chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
  • LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKER (SEQ ID NO:183) corresponding to amino acids 47 - 216 of HUMA1ACM_PEA__2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACM PEA 2JP49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ER corresponding to amino acids 215 - 216 of HUMA1ACM_PEA_
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans ⁇ membrane region- Variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 15, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) sequence provides support for the deduced sequence of this variant protein according
  • Variant protein HUMA1ACM_PEA_2JP49 (SEQ ID NO:52) is encoded by the following transcript(s): HUMAl ACMJPE A_2_T21 (SEQ ID NO: I) 5 for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HUMAl ACMJPE A_2_T21 (SEQ ID NO:1) is shown in bold; this coding portion starts at position 84 and ends at position 731.
  • the transcript also has the following SNPs as listed in Table 16 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HUMA1ACMJPEA_2_P59 (SEQ ID NO:53) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMA1ACM_PEA_2_T27 (SEQ ID NO:2).
  • An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • chimeric polypeptide encoding for HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDF AFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND
  • YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 214 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
  • polypeptide encoding for a head of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACMJPE A_2_P59 (SEQ ID NO:53).
  • MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACMJPEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino
  • polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
  • LAn isolated chimeric polypeptide encoding for HUMA1ACMJPEA_2_P59 comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
  • FFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • AACT_HUMAN SEQ ID NO:205:
  • polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO.53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans ⁇ membrane region..
  • Variant protein HUMAl ACM JPEA_2_P59 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 17, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMAl ACM JPEA_2_P59 (SEQ ID NO:53) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • Variant protein HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) is encoded by the following transcript(s). HUMAl ACM_PEA_2_T27 (SEQ ID NO:2), for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) is shown in bold; this coding portion starts at position 84 and ends at position 782.
  • the transcript also has the following SNPs as listed in Table 18 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P59 (SEQ ID NO: 53) sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • cluster HUMAlACM features 46 segment(s), which were listed in Table 8 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
  • Segment cluster HUMAl ACM_PEA_2_node_23 (SEQ ID NO:4) according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T27 (SEQ ID NO:2). Table 19 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAl ACMJPE A_2_node_41 (SEQ ID NO:5) according to the present invention is supported by 13 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 20 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAl ACMJPE A_2_node_51 (SEQ ID NO:6) according to the present invention is supported by 203 libraries. The number of libraries was determined as previously described. This segment can be found in the following transc ⁇ pt(s): HUMAl ACMJPEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 21 below describes the starting and ending position of this segment on each transcript.
  • short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
  • Segment cluster HUMAlACM_PEA_2_node_0 (SEQ ID NO:7) according to the present invention is supported by 199 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM JPEA 2JT27 (SEQ ID NO:2) and HUMA IACM J 3 E A_2_T7 (SEQ ID NO.3). Table 22 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_l (SEQ ID NO:8) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACMJPE A_2_T27 (SEQ ID NO:2) and
  • HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 23 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAl ACM_PEA_2_node_10 (SEQ ID NO:9) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and
  • HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 24 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_ll (SEQ ID NO:10) according to the present invention is supported by 244 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 25 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_12 (SEQ ID NO:11) according to the present invention is supported by 230 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 26 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_13 (SEQ ID NO:12) according to the present invention is supported by 209 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 27 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_14 (SEQ ID NO: 13) according to the present invention is supported by 216 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 28 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_15 (SEQ ID NO:14) according to the present invention is supported by 211 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACMJPEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 29 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_16 (SEQ ID NO: 15) according to the present invention is supported by 222 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 30 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACMJPEA_2_node_17 (SEQ ID NO: 16) according to the present invention is supported by 224 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 31 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_18 (SEQ ID NO: 17) according to the present invention can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3).
  • Table 32 below describes the starting and ending position of this segment on each transcript. Table 32 - Segment location on transcripts
  • Segment cluster HUMAlACM_PEA_2_node_19 (SEQ ID NO: 18) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 33 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_2 (SEQ ID NO: 19) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACMJPEA_2_T27 (SEQ ID NO:2) and
  • HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 34 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_20 (SEQ ID NO:20) according to the present invention is supported by 216 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripts): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM JPEA_2_T27 (SEQ ID NO.2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 35 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_21 (SEQ ID NO:21) according to the present invention is supported by 218 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 36 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_22 (SEQ ID NO:22) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACMJPEA_2_T7 (SEQ ID NO:3).
  • Table 37 describes the starting and ending position of this segment on each transcript. Table 37 - Segment location on transcripts
  • Segment cluster HUMAlACM_PEA_2_node_26 (SEQ ID NO:23) according to the present invention is supported by 231 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 38 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_27 (SEQ ID NO:24) according to the present invention is supported by 238 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 39 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_28 (SEQ ID NO:25) according to the present invention is supported by 248 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 40 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_29 (SEQ ID NO:26) according to the present invention is supported by 257 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 41 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_30 (SEQ ID NO:27) according to the present invention can be found in the following transcript(s): HUMA1ACMJPEA_2_T7 (SEQ ID NO:3). Table 42 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_31 (SEQ ID NO:28) according to the present invention is supported by 256 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 43 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HUMAlACM_PEA_2_node_34 (SEQ ID NO:29) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 44 below describes the starting and ending position of this segment on each transcript.

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Abstract

Novel splice variants, amino acid sequences and nucleotide sequences thereof, and methods of using same.

Description

NOVEL NUCLEOTIDE AND AMINO ACID SEQUENCES, AND ASSAYS AND METHODS OF USE THEREOF FOR DIAGNOSIS
FIELD OF THE INVENTION The present invention is related to novel nucleotide and protein sequences, and assays and methods of use thereof.
BACKGROUND OF THE INVENTION
Diagnostic markers are important for early diagnosis of many diseases, as well as predicting response to treatment, monitoring treatment and determining prognosis of such diseases.
Serum markers are examples of such diagnostic markers and are used for diagnosis of many different diseases. Such serum markers typically encompass secreted proteins and/or peptides; however, some serum markers may be released to the blood upon tissue lysis, such as from myocardial infarction (for example Troponin-I). Serum markers can also be used as risk factors for disease (for example base-line levels of CRP, as a predictor of cardiovascular disease), to monitor disease activity and progression (for example, determination of CRP levels to monitor acute phase inflammatory response) and to predict and monitor drug response (for example, as shedded fragments of the protein Erb-B2).
Immunohistochemistry (IHC) is the study of distribution of an antigen of choice in a sample based on specific antibody-antigen binding, typically on tissue slices. The antibody features a label which can be detected, for example as a stain which is detectable under a microscope. The tissue slices are prepared by being fixed. IHC is therefore particularly suitable for antibody-antigen reactions that are not disturbed or destroyed by the process of fixing the tissue slices.
IHC permits determining the localization of binding, and hence mapping of the presence of the antigen within the tissue and even within different compartments in the cell. Such mapping can provide useful diagnostic information, including: 1) the histological type of the tissue sample
2) the presence of specific cell types within the sample
3) information on the physiological and/or pathological state of cells (e.g. which phase of the cell-cycle they are in)
4) the presence of disease related changes within the sample 5) differentiation between different specific disease subtypes where it is already known the tissue is of disease state (for example, the differentiation between different tumor types when it is already known the sample was taken from cancerous tissue).
IHC information is valuable for more than diagnosis. It can also be used to determine prognosis and therapy treatment (as in the case of HER-2 in breast cancer) and monitor disease.
IHC protein markers could be from any cellular location. Most often these markers are membrane proteins but secreted proteins or intracellular proteins (including intranuclear) can be used as an IHC marker too. IHC has at least two major disadvantages. It is performed on tissue samples and therefore a tissue sample has to be collected from the patient, which most often requires invasive procedures like biopsy associated with pain, discomfort, hospitalization and risk of infection. In addition, the interpretation of the result is observer dependant and therefore subjective. There is no measured value but rather only an estimation (on a scale of 1 -4) of how prevalent the antigen on target is.
SUMMARY OF THE INVENTION
The present invention provides, in different embodiments, many novel amino acid and nucleic acid sequences, which may optionally be used as diagnostic markers. For example, the present invention provides a number of different variants of known serum proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers. The present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results. For example, serum markers require only a simple blood test and their result is typically a scientifically measured number. As IHC markers, the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions. The markers presented in the present invention can also potentially be used for in-vivo imaging applications. The present invention also provides a number of different variants of known IHC proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers. The present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results. For example, serum markers require only a simple blood test and their result is typically a scientifically measured number. As IHC markers, the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions.
Other variants are also provided by the present invention as described in greater detail below.
The diseases for which such variants may be useful diagnostic markers are described in greater detail below for each of the variants. The variants themselves are described by "cluster" or by gene, as these variants are splice variants of known proteins. Therefore, a "marker-detectable disease" refers to a disease that may be detected by a particular marker, with regard to the description of such diseases below. The markers of the present invention, alone or in combination, show a high degree of differential detection between disease and non-disease states.
The present invention therefore also relates to diagnostic assays for disease detection optionally and preferably in a biological sample taken from a subject (patient), which is more preferably some type of body fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, secretions of the breast ductal system (and/or lavage thereof), broncho alveolar lavage, lavage of the reproductive system and/or lavage of any other part of the body or system in the body, and stool or a tissue sample. The term may also optionally encompass samples of in vivo cell culture constituents. The sample can optionally be diluted with a suitable eluant before contacting the sample to an antibody and/or performing any other diagnostic assay. Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tmhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs.dtu.dyservices/TMHMM/TMHMM2.0b.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinformatics, www.ch.embnet.org/software/TMPRED_form.html) for transmembrane region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, www.cbs.dtu.dk/services/SignalP/background/prediction.php) for signal peptide prediction. The terms "signalp_hmm" and "signalp nn" refer to two modes of operation for the program SignalP: hmm refers to Hidden Markov Model, while nn refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor. In some cases for the manual inspection of cellular localization prediction inventors used the ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman, Dan Graur and Amit Novik; (2004) "Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis." Cell Biology International 2004;28(3):171-8.], which predicts protein localization based on various parameters including, protein domains (e.g., prediction of trans-membranous regions and localization thereof within the protein), pi, protein length, amino acid composition, homology to pre-annotated proteins, recognition of sequence patterns which direct the protein to a certain organelle (such as, nuclear localization signal, NLS, mitochondria localization signal), signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.
Information is given in the text with regard to SNPs (single nucleotide polymorphisms). A description of the abbreviations is as follows. "T - > C", for example, means that the SNP results in a change at the position given in the table from T to C. Similarly, "M - > Q", for example, means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*). As part of the description of an SNP, a comment may be found in parentheses after the above description of the SNP itself. This comment may include an FTId5 which is an identifier to a SwissProt entry that was created with the indicated SNP. An FTId is a unique and stable feature identifier, which allows construction of links directly from position-specific annotation in the feature table to specialized protein-related databases. The FTId is always the last component of a feature in the description field, as follows: FTKKXXX number, in which XXX is the 3 -letter code for the specific feature key, separated by an underscore from a 6-digit number. In the table of the amino acid mutations of the wild type proteins of the selected splice variants of the invention, the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence. SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker. Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
Information given in the text with regard to the Homology to the known proteins was determined by Smith- Waterman version 5.1.2 using special (non default) parameters as follows: -moder=sw.model -GAPEXT=O -GAPOP= 100.0
-MATRIX=blosumlOO
Information is given with regard to overexpression of a cluster in cancer based on ESTs. A key to the p values with regard to the analysis of such overexpression is as follows:
- library-based statistics: P- value without including the level of expression in cell-lines (Pl)
- library based statistics: P-value including the level of expression in cell-lines (P2)
- EST clone statistics: P-value without including the level of expression in cell-lines (SPl)
- EST clone statistics: predicted overexpression ratio without including the level of expression in cell-lines (R3) - EST clone statistics: P-vaiue including the level of expression in cell-lines
(SP2)
- EST clone statistics: predicted overexpression ratio including the level of expression in cell-lines (R4)
Library-based statistics refer to statistics over an entire library, while EST clone statistics refer to expression only for ESTs from a particular tissue or cancer.
Information is given with regard to overexpression of a cluster in cancer based on microarrays. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. Results are provided from microarrays using Affymetrix technology. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. For microarrays prepared according to a design by the present inventors, the probe name begins with the name of the cluster (gene), followed by an identifying number. Oligonucleotide microarray results taken from Affymetrix data were from chips available from Affymetrix Inc, Santa Clara, CA, USA (see for example data regarding the Human Genome U133 (HG-Ul 33) Set at www.affymetrix.com/products/arrays/specific/hgul33.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33av2.affx; and Human Genome Ul 33 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33plus.affx). The probe names follow the Affymetrix naming convention. The data is available from NCBI Gene Expression Omnibus (see www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et al, Nucleic Acids Research, 2002, Vol. 30, No. 1 207-210). The dataset (including results) is available from www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1133 for the Series GSEl 133 database (published on March 2004); a reference to these results is as follows: Su et al (Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6062-7. Epub 2004 Apr 09).
It should be noted that the terms "segment", "seg" and "node" (abbreviated as "N" in the names of nodes) are used interchangeably in reference to nucleic acid sequences of the present invention, they refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below. Optionally and preferably, they are examples of oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
As used herein the phrase "disease" includes any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.
The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients (subjects) having one of the herein-described diseases or conditions, as compared to a comparable sample taken from subjects who do not have one the above- described diseases or conditions.
The phrase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having one of the herein-described diseases or conditions as compared to a comparable sample taken from patients who do not have one of the herein-described diseases or conditions. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT- based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present. Optionally, a relatively low amount of up-regulation may serve as the marker, as described herein. One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
As used herein the phrase "diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above.
Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below. As used herein, the term "level" refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.
Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or condition. A test amount can be either in absolute amount (e.g., miciOgram/ml) or a relative amount (e.g., relative intensity of signals). A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition. A control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals). "Detect" refers to identifying the presence, absence or amount of the object to be detected.
A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample. The label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly. For example, the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry. Exemplary detectable labels, optionally and preferably for use with immunoassays, include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
"Immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide (or other epitope), refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically imrnunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
According to preferred embodiments of the present invention, there is provided an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein.
Optionally the amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion as in any of the previous claims.
Optionally the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
According to preferred embodiments of the present invention, there is provided a kit for detecting a Marker-detectable disease, comprising a kit detecting specific expression of a splice variant as described herein.
Optionally the kit comprises a NAT-based technology. Optionally the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein.
Optionally the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
Optionally the kit comprises an antibody as described herein. Optionally the kit further comprises at least one reagent for performing an ELISA or a Western blot.
According to preferred embodiments of the present invention, there is provided a method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims.
Optionally detecting specific expression is performed with a NAT-based technology.
Optionally detecting specific expression is performed with an immunoassay.
Optionally the immunoassay comprises an antibody as described herein. According to preferred embodiments of the present invention, there is provided a biomarker capable of detecting Marker-detectable disease, comprising any nucleic acid sequence described herein or a fragment thereof, or any amino acid sequence described herein or a fragment thereof.
According to preferred embodiments of the present invention, there is provided a method for screening for variant-detectable disease, comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
According to preferred embodiments of the present invention, there is provided a method for diagnosing a marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
According to preferred embodiments of the present invention, there is provided a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease, comprising detecting cells affected by Marker- detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
According to preferred embodiments of the present invention, there is provided a method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
According to preferred embodiments of the present invention, there is provided, as listed below, optional but preferred embodiments (although provided as a list, this is for the sake of convenience only and is not intended to indicate a closed list or to otherwise be limiting in any way):
An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: HUMAl ACMJPE A 2 T21 (SEQ ID NO:1), HUMA1ACMJPEA_2_T27 (SEQ ID NO:2), or HUMA1ACM_PEA_2_T7 (SEQ ID NO:3).
An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: HUMAlACM_PEA_2_node_23 (SEQ ID NO:4), HUMAl ACM_PEA_2_node_41 (SEQ ID N0:5), HUMA IACM JPE A_2_node_51 (SEQ ID NO:6), HUMAlACM_PEA_2_node_0 (SEQ ID NO:7),
HUMAlACM JPEA_2_node_l (SEQ ID NO:8), HUMAl ACM_PEA_2_node_10 (SEQ ID NO:9), HUMAl ACM_PEA_2_node_l 1 (SEQ ID NO: 10),
HUMAlACM JPEA_2_node_12 (SEQ ID NC-.l l), HUMAlACM_PEA_2_node_13 (SEQ ID NO: 12), HUMAl ACM_PEA_2_node_14 (SEQ ID NO: 13), HUMAlACM_PEA_2_node_15 (SEQ ID NO:14), HUMAlACM_PEA_2_node_16 (SEQ ID NO:15), HUMAlACM_PEA_2_node_17 (SEQ ID NO:16), HUMAlACM_PEA_2_node_18 (SEQ ID NO:17), HUMAlACM_PEA_2_node_19 (SEQ ID NO: 18), HUMAlACM_PEA_2_node_2 (SEQ ID NO: 19), HUMAlACM_PEA_2_node_20 (SEQ ID NO:20), HUMAlACM_PEA_2_node_21 (SEQ ID NO:21), HUMAl ACM_PEA_2_node_22 (SEQ ID NO:22), HUMAlACM_PEA_2_node_26 (SEQ ID NO:23), HUMA IACM JPE A_2_node_27 (SEQ ID NO:24), HUMAl ACM_PEA_2_node_28 (SEQ ID NO:25), HUMAlACM_PEA_2_node_29 (SEQ ID NO:26), HUMAlACM_PEA_2_node_30 (SEQ ID NO:27), HUMAl ACM_PEA_2_node_31 (SEQ ID NO:28), HUMAlACM_PEA_2_node_34 (SEQ ID NO:29), HUMAlACM_PEA_2_node_35 (SEQ ID NO:30), HUMAlACM_PEA_2_node_36 (SEQ ID NO:31), HUMAlACM_PEA_2_node_37 (SEQ ID NO:32), HUMAlACM_PEA_2_node_38 (SEQ ID NO:33), HUMAlACM_PEA_2_node_39 (SEQ ID NO:34), HUMAlACM_PEA_2_node_40 (SEQ ID NO:35), HUMAlACM_PEA_2_node_42 (SEQ ID NO:36), HUMAl ACM_PEA_2_node_43 (SEQ ID NO:37), HUMAlACM_PEA_2_node_44 (SEQ ID NO:38), HUMAl ACM_PEA_2_node_45 (SEQ ID NO:39), HUMAl ACM JPEA_2_node_46 (SEQ ID NO:40), HUMAlACM_PEA_2_node_47 (SEQ ID NO:41), HUMAlACM_PEA_2_node_48 (SEQ ID NO:42), HUMAl ACM_PEA_2_node_49 (SEQ ID NO:43), HUMAl ACM_PEA_2_node_5 (SEQ ID NO:44), HUMAlACM_PEA_2_node_50 (SEQ ID NO:45), HUMAlACM_PEA_2__node_6 (SEQ ID NO:46),
HUMAlACM_PEA_2_node_7 (SEQ ID NO:47), HUMAlACM_PEA_2_node_8 (SEQ ID NO:48), or HUMAl ACM_PEA_2_node_9 (SEQ ID NO:49). An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), or HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3JPEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68), or
HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69).
An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: HUMEGFRBB3_PEA_l_node_0 (SEQ ID NO:70), HUMEGFRBB3_PEA_l_node_13 (SEQ ID NO:71), HUMEGFRBB3_PEA_l_node_14 (SEQ ID NO:72), HUMEGFRBB3_PEA_l_node_l 8 (SEQ ID NO:73), HUMEGFRBB3_PEA_l_node_23 (SEQ ID NO:74), HUMEGFRBB3_PEA_l_node_26 (SEQ ID NO:75), HUMEGFRBB3_PEA_l_node_27 (SEQ ID NO:76), HUMEGFRBB3_PEA_l_node_40 (SEQ ID NO.77), HUMEGFRBB3_PEA_l_node_42 (SEQ ID NO:78), HUMEGFRBB3_PEA_ljnode_46 (SEQ ID NO:79), HUMEGFRBB3_PEA_l_node_49 (SEQ ID NO:80)3 HUMEGFRBB3_PEA_l_node_50 (SEQ ID NO:81), HUMEGFRBB3_PEA_l_node_51 (SEQ ID NO:82), HUMEGFRBB3_PEA_l_node_54 (SEQ ID NO:83), HUMEGFRBB3_PEA_l_node_58 (SEQ ID NO:84), HUMEGFRBB3_PEA_l_node_60 (SEQ ID NO:85), HUMEGFRBB3_PEA_l_node_66 (SEQ ID NO:86), HUMEGFRBB3_PEA_l_node_68 (SEQ ID NO:87), HUMEGFRBB3_PEA_l_node_89 (SEQ ID NO:88), HUMEGFRBB3_PEA_l_node_95 (SEQ ID NO:89), HUMEGFRBB3_PEA_l_node_97 (SEQ ID NO:90), HUMEGFRBB3JPEA_l_node_98 (SEQ ID NO:91), HUMEGFRBB3_PEA_l_node_100 (SEQ ID NO:92), HUMEGFRBB3_PEA_l_node_l (SEQ ID NO:93), HUMEGFRBB3JPEA_l_node_2 (SEQ ID NO:94), HUMEGFRBB3_PEA_l_node_9 (SEQ ID NO:96), HUMEGFRBB3_PEA l_node_10
(SEQ ID NO:97), HUMEGFRBB3_PEA_l_node_l 1 (SEQ ID NO:98),
HUMEGFRBB3_PEA_l_node_16 (SEQ ID NO:99), HUMEGFRBB3JPEAJ_node_17
(SEQ ID NO: 100), HUMEGFRBB3_PEA_l_node_20 (SEQ ID NO: 101), HUMEGFRBB3_PEA_l_node_21 (SEQ ID NO:102),
HUMEGFRBB3_PEA_l_node_22 (SEQ ID NO:103),
HUMEGFRBB3_PEA_l_node_24 (SEQ ID NO:104),
HUMEGFRBB3_PEA_l_node_28 (SEQ ID NO:105),
HUMEGFRBB3_PEA_l_node_30 (SEQ ID NO:106), HUMEGFRBB3JPEA_l_node_31 (SEQ ID NO:107),
HUMEGFRBB3_PEA_l_node_34 (SEQ ID NO:108),
HUMEGFRBB3_PEA_l_node_35 (SEQ ID NO:109),
HUMEGFRBB3_PEA_l_node_37 (SEQ ID NO:110),
HUMEGFRBB3_PEA_l_node_39 (SEQ ID NO:111), HUMEGFRBB3_PEA_l_node_44 (SEQ ID NO:112),
HUMEGFRBB3_PEA_l_node_45 (SEQ ID NO: 113),
HUMEGFRBB3_PEA_l_node_47 (SEQ ID NO:114),
HUMEGFRBB3_PEA_l_node_48 (SEQ ID NO:115),
HUMEGFRBB3_PEA_l_node_52 (SEQ ID NO:116), HUMEGFRBB3_PEA_l_node_53 (SEQ ID NO:117),
HUMEGFRBB3_PEA_l_node_57 (SEQ ID NO:118),
HUMEGFRBB3_PEA_l_node_61 (SEQ ID NO:119),
HUMEGFRBB3_PEA_l_node_62 (SEQ ID NO:120),
HUMEGFRBB3_PEA_l_node_64 (SEQ ID NO:121), HUMEGFRBB3_PEA_l_node_71 (SEQ ID NO:122),
HUMEGFRBB3_PEA_l_node_73 (SEQ ID NO:123),
HUMEGFRBB3_PEA_l_node_74 (SEQ ID NO:124),
HUMEGFRBB3_PEA_l_node_78 (SEQ ID NO:125),
HUMEGFRBB3_PEA_l_node_80 (SEQ ID NO:126), HUMEGFRBB3_PEA_l_node_81 (SEQ ID NO:127),
HUMEGFRBB3_PEA_l_node_82 (SEQ ID NO:128),
HUMEGFRBB3_PEA_l_node_84 (SEQ ID NO:129),
HUMEGFRBB3_PEA_l_node_85 (SEQ ID NO:130),
HUMEGFRBB3_PEA_l_node_90 (SEQ ID NO:131), HUMEGFRBB3_PEA_l_node_91 (SEQ ID NO: 132),
HUMEGFRBB3_PEA_l_node_92 (SEQ ID NO: 133),
HUMEGFRBB3_PEA_l_node_96 (SEQ ID NO: 134), or
HUMEGFRBB3_PEA_l_node_99 (SEQ ID NO: 135). An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of: HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137), HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138), HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), HUMEGFRBB3JPEA_1_P46 (SEQ ID NO:142), HUMEGFRBB3_PEA_l_P50 (SEQ ID NO:143), HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), HUMEGFRBB3JPEAJ_P54 (SEQ ID NO: 145), or HUMEGFRBB3_PEA_1JP55 (SEQ ID NO: 146).
An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS AN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90% homologous to
SRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPDQD KMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFT SKADLSGITGARNLAVSQV corresponding to amino acids 1 - 129 of Q96DW8 (SEQ ID NO:202), which also corresponds to amino acids 229 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence SL corresponding to amino acids 358 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO.51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO:180) OF HUMAL ACM_PEA_2_P36 (SEQ ID NO:51).
An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%,homologous to a polypeptide having the sequence MERMLPLLALGLLAAG corresponding to amino acids 1 - 16 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90% homologous to
FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKK KWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPDQDKMEEVEA MLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGI TGARNLAVSQV corresponding to amino acids 1 - 341 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 357 of HUMA IACMJPE A J2 P36 (SEQ ID NO:51), and a third amino acid sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order. An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P36 (SEQ
ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%,homologous to the sequence MERMLPLLALGLLAAG of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51). An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P36 (SEQ
ID NO:51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVV ELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISR DYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO: 181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2JP36 (SEQ ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVV ELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISR DYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO: 181) IN HUMAL ACM_PEA_2_P36 (SEQ ID NO:51).
An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGN ASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDI LLQLGIEEAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 357 of AACT__HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2JP36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of
HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), second amino acid sequence being at least 90% homologous to
FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDF AFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF ATDFQDSAAAKKLIND
YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17-214 of HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), and a third amino acid sequence ER corresponding to amino acids 215-216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first, second and third amino acid sequences are contiguous and in a sequential order. An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P49 (SEQ
ID NO:52), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2JP49 (SEQ ID NO:52). An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ
ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDWKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO: 52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI
FFKER (SEQ ID NO: 183) corresponding to amino acids 47 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P49 (SEQ
ID NO: 52), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKER (SEQ ID NO: 183) IN HUMA1ACM_PEA_2_P49 (SEQ ID NO:52). An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ
ID NO: 52), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACTJHUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence having the sequence ER corresponding to amino acids 215 - 216 of HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated chimeric polypeptide encoding for HUMAl ACM PEA 2 P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a third amino acid sequence being at least 70% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first, second and third amino acid sequences are contiguous and in a sequential order. An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P59 (SEQ
ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53). An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ
ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53). An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ
ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN
VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order. An isolated polypeptide encoding for a tail of HUMA1ACMJPEA 2 P59 (SEQ
ID NO:53), comprising a polypeptide being at least 70% homologous, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53). An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ
ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAlACM J>EA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P59 (SEQ ID NO: 53), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACT HUM AN, which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEWMGNLEΓVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG PIYKYPDVQNECRPCHENCTQG corresponding to amino acids 1 - 620 of ERB3JHUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 620 of HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence SVMG (SEQ ID NO: 186) corresponding to amino acids 621 - 624 of HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3JPEA 1 P15 (SEQ ID NO: 137), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence SVMG (SEQ ID NO: 186) in HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137).
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGD corresponding to amino acids 1 - 41 of ERB3 HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 41 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138), and a second amino acid sequence being at least 90% homologous to
AEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARD PPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTL GSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPV SLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRH SLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDE DEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPI MPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGH
QAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT corresponding to amino acids 918 - 1342 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 42 - 466 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order. An isolated chimeric polypeptide encoding for an edge portion of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), comprising a polypeptide having a length "n", wherein n is at least about 10, 20, 30, 40 or 50 amino acids in length, wherein at least two amino acids comprise DA, having a structure as follows: a sequence starting from any of amino acid numbers 41-x to 41; and ending at any of amino acid numbers 42+ ((n-2) - x), in which x varies from 0 to n-2.
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1JP31 (SEQ ID NO: 139), comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG PIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIG corresponding to amino acids 1 - 638 of ERB3 HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 638 of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) corresponding to amino acids 639 - 699 of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) in HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139).
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCF corresponding to amino acids 1 - 244 of ERB3JHUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 244 of HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) corresponding to amino acids 245 - 278 of HUMEGFRBB3_PEA_1_P41 (SEQ ID NO:140), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) in HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140). An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P45
(SEQ ID NO: 141), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSC corresponding to amino acids 1 - 183 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 183 of HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), and a second amino acid sequence having the sequence KWP corresponding to amino acids 184 - 186 of HUMEGFRBB3 PEA 1JP45 (SEQ ID NO: 141), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLT corresponding to amino acids 1 - 140 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 140 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) corresponding to amino acids 141 - 183 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3JPEA 1JP46 (SEQ ID NO: 142), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) in HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142).
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDΓVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQT corresponding to amino acids 1 - 204 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 204 of HUMEGFRBB3_PEAJ_P50 (SEQ ID NO: 143), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence
CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) corresponding to amino acids 205 - 254 of HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) in HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143).
An isolated chimeric polypeptide encoding for HUMEGFRBB3JPEA_1_P53 (SEQ ID NO: 144), comprising a first amino acid sequence being at least 90% homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGS corresponding to amino acids 1 - 568 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 568 of HUMEGFRBB3_PEA_1_P53 (SEQ ID NO:144), and a second amino acid sequence having the sequence VY corresponding to amino acids 569 - 570 of HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated chimeric polypeptide encoding for HUMEGFRBB3JPEA_1JP54 (SEQ ID NO: 145), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRD AEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCP corresponding to amino acids 1 - 291 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 291 of HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence RKCLRGRNNDQQ (SEQ ID NO: 191) corresponding to amino acids 292 - 303 of HUMEGFRBB3_PEA_1JP54 (SEQ ID NO: 145), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence RKCLRGRNNDQQ (SEQ ID NO: 191) in HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145).
An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), comprising a first amino acid sequence being at least 90% homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG PIYKYPDVQNECRPCHENCTQG corresponding to amino acids 1 - 620 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 620 of HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), and a second amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to a polypeptide having the sequence SVMG (SEQ ID NO: 186) corresponding to amino acids 621 - 624 of HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P55
(SEQ ID NO: 146), comprising a polypeptide being at least 70%, optionally at least about
80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, homologous to the sequence SVMG (SEQ ID NO: 186) in
HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146).
The isolated oligonucleotide as described above, comprising an amplicon according to any one of the SEQ ID NOs: 56 or 59.
The isolated oligonucleotide as described above, comprising an amplicon according to any one of the SEQ ID NOs: 149 or 152.
A primer pair, comprising a pair of isolated oligonucleotides capable of amplifying one of said amplicons.
The primer pair as described above, comprising a pair of isolated oligonucleotides: SEQ NOs 54 and 55, or 57 and 58; or SEQ NOs 147 and 148, or 150 and 151.
An antibody capable of specifically binding to an epitope of an amino acid sequence as described above.
An antibody capable of specifically binding to an epitope of an amino acid sequence as described above, optionally wherein said amino acid sequence corresponds to a bridge, edge portion, tail, or head as in any of the previous claims, also optionally wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
A kit for detecting a Marker-detectable disease, comprising a kit detecting specific expression of a splice variant as described herein. Optionally, the kit comprises a NAT-based technology; optionally and preferably, this kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein; alternatively and optionally, the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims. Alternatively and optionally, the kit comprises an antibody according to any of the above claims (optionally and preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
A method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant as described herein; optionally the marker-detectable disease is cluster HUMAlACM marker-detectable disease and is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer. Alternatively, the marker-detectable disease is cluster HUMEGFRBB3 marker-detectable disease and is selected from the group consisting of colon cancer, breast cancer and ovarian cancer.
Detecting specific expression is optionally performed with a NAT-based technology, and/or with an immunoassay (optionally comprising an antibody according to any of the above embodiments).
There is also optionally provided a biomarker capable of detecting Marker- detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
There is also optionally provided a method for screening for variant-detectable disease, comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments.
There is also optionally provided a method for diagnosing a marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments. There is also optionally provided a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments.
There is also optionally provided a method of selecting a therapy for a marker- detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above embodiments and selecting a therapy according to said detection. Such a method may optionally be used when the marker-detectable disease is cluster HUMAlACM marker- detectable disease and is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer; and/or when the marker-detectable disease is cluster HUMEGFRBB3 marker- detectable disease and is selected from the group consisting of colon cancer, breast cancer and ovarian cancer. According to preferred embodiments of the present invention, preferably any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
Unless otherwise noted, all experimental data relates to variants of the present invention, named according to the segment being tested (as expression was tested through RT-PCR as described).
All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
According to preferred embodiments, the present invention provides isolated nucleic acid sequences comprising a sequence described herein.
According to preferred embodiments, the present invention provides amino acid sequences comprising a sequence described herein.
According to preferred embodiments, the present invention provides a head, tail, bridge or edge sequence described herein. According to preferred embodiments, the present invention provides an antibody capable of specifically binding to an epitope of an amino acid sequence comprising sequences described herein. The present invention further provides the antibody as above, wherein said amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion as described herein. The present invention further provides the antibody as above, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
The present invention further provides a kit for detecting a Marker-detectable disease, comprising a kit detecting specific expression of a splice variant according to any of the above claims. The present invention further provides the kit as above, wherein said kit comprises a NAT-based technology. The present invention further provides the kit as above, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims. The present invention further provides said kit, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims. The present invention further provides said kit, wherein said kit comprises an antibody according to any of the above claims. The present invention further provides said kit, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot. The present invention further provides a method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims. The present invention further provides the method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims, wherein said detecting specific expression is performed with a NAT-based technology. The present invention further provides the method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims, wherein said detecting specific expression is performed with an immunoassay. The present invention further provides said method, wherein said immunoassay comprises an antibody according to any of the above claims.
The present invention further provides a biomarker capable of detecting Marker- detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
The present invention further provides a method for screening for variant- detectable disease, comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
The present invention further provides a method for diagnosing a marker- detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
The present invention further provides a method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims. The present invention further provides a method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). AU of these are hereby incorporated by reference as if fully set forth herein. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
Figure 1 shows a schematic description of the cancer biomarker selection engine.
Figure 2 shows a schematic illustration, depicting grouping of transcripts of a given cluster based on presence or absence of unique sequence regions.
Figure 3 shows a schematic presentation of the oligonucleotide based microarray fabrication.
Figure 4 shows a schematic summary of the oligonucleotide based microarray experimental flow. Figure 5 shows a schematic summary of quantitative real-time PCR analysis.
Figure 6 shows a graph of cancer and cell-line vs. normal tissue expression for HUMAlACM.
Figure 7 shows HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMA lACMseg26-3 IWT in normal and cancerous ovary tissues.
Figure 8 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous ovary samples relative to the normal samples.
Figure 9 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous lung samples relative to the normal samples. Figure 10 is a histogram showing down regulation of the above-indicated serine
(or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous lung samples relative to the normal samples.
Figure 11 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous colon samples relative to the normal samples.
Figure 12 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous colon samples relative to the normal samples.
Figure 13 shows the expression of HUMAlACM transcripts which are detectable by amplicons as depicted in sequence names HUMAlACMseg23 (SEQ ID NO:59) (Figure 13A) and HUMAl ACMseg26-3 IWT (Figure 13B) in different normal tissues. Figure 14 shows a graph of cancer and cell-line vs. normal tissue expression for
Cluster HUMEGFRBB3.
Figure 15 is a histogram showing over expression of the amplicon for seglδ (SEQ ID NO: 149) (node 18) Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts in cancerous Ovary samples relative to the normal samples. Figure 16 is a histogram showing over expression of the amplicon for seg46 (node 46) Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts in cancerous Ovary samples relative to the normal samples.
DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides variants, which may optionally be used as diagnostic markers.
Preferably these variants are useful as diagnostic markers for marker-detectable (also referred to herein as "variant-detectable") diseases as described herein.
Differential variant markers are collectively described as "variant disease markers".
The markers of the present invention, alone or in combination, can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker-detectable disease. For example, optionally and preferably, these markers may be used for staging the disease in patients (for example if the disease features cancer) and/or monitoring the progression of the disease. Furthermore, the markers of the present invention, alone or in combination, can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer. Also, one or more of the markers may optionally be used in combination with one or more other disease markers (other than those described herein).
Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
These markers are specifically released to the bloodstream under conditions of a particular disease, and/or are otherwise expressed at a much higher level and/or specifically expressed in tissue or cells afflicted with or demonstrating the disease. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of a particular disease and/or a condition that is indicative of a higher risk for a particular disease.
The present invention therefore also relates to diagnostic assays for marker- detectable disease and/or an indicative condition, and methods of use of such markers for detection of marker-detectable disease and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
In another embodiment, the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides. Such bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
As used herein a "tail" refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
As used herein a "head" refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein. As used herein "an edge portion" refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein. An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein. A "bridge" may optionally be an edge portion as described above, but may also include a join between a head and a "known protein" portion of a variant, or a join between a tail and a "known protein" portion of a variant, or a join between an insertion and a "known protein" portion of a variant. Optionally and preferably, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant. Also optionally, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).
It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself.
Furthermore, bridges are described with regard to a sliding window in certain contexts below. For example, certain descriptions of the bridges feature the following format: a bridge between two edges (in which a portion of the known protein is not present in the variant) may optionally be described as follows: a bridge portion of CONTIG-N AME Pl (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-N AME Pl): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for example), in which x varies from 0 to n-2. In this example, it should also be read as including bridges in which n is any number of amino acids between 10-50 amino acids in length. Furthermore, the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1 , nor 50 + ((n-2) - x) (for example) greater than the total sequence length.
In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
In another embodiment, this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
In another embodiment, this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample. According to the present invention, the splice variants described herein are non- limiting examples of markers for diagnosing marker-detectable disease and/or an indicative condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of marker-detectable disease and/or an indicative condition, including a transition from an indicative condition to marker-detectable disease.
According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker. Furthermore, such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
Panels of markers according to the present invention optionally with one or more known marker(s)
The present invention is of methods, uses, devices and assays for diagnosis of a disease or condition. Optionally a plurality of biomarkers (or markers) may be used with the present invention. The plurality of markers may optionally include a plurality of markers described herein, and/or one or more known markers. The plurality of markers is preferably then correlated with the disease or condition. For example, such correlating may optionally comprise determining the concentration of each of the plurality of markers, and individually comparing each marker concentration to a threshold level. Optionally, if the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed), the marker concentration correlates with the disease or condition. Optionally and preferably, a plurality of marker concentrations correlate with the disease or condition. Alternatively, such correlating may optionally comprise determining the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and comparing the index value to a threshold level.
Also alternatively, such correlating may optionally comprise determining a temporal change in at least one of the markers, and wherein the temporal change is used in the correlating step.
Also alternatively, such correlating may optionally comprise determining whether at least "X" number of the plurality of markers has a concentration outside of a predetermined range and/or above or below a threshold (as described above). The value of "X" may optionally be one marker, a plurality of markers or all of the markers; alternatively or additionally, rather than including any marker in the count for "X", one or more specific markers of the plurality of markers may optionally be required to correlate with the disease or condition (according to a range and/or threshold). Also alternatively, such correlating may optionally comprise determining whether a ratio of marker concentrations for two markers is outside a range and/or above or below a threshold. Optionally, if the ratio is above or below the threshold level and/or outside a range, the ratio correlates with the disease or condition. Optionally, a combination of two or more these correlations may be used with a single panel and/or for correlating between a plurality of panels.
Optionally, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects. As used herein, sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected out of the total number of negative samples present. Preferably, the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. More preferably, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects. Also more preferably, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms.
A marker panel may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value, or a value indicating a particular outcome. A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis. The skilled artisan will also understand that diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or condition differentiating markers, etc., may be combined in a single assay or device. Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different puφose(s). Preferred panels comprise markers for the following purposes: diagnosis of a disease; diagnosis of disease and indication if the disease is in an acute phase and/or if an acute attack of the disease has occurred; diagnosis of disease and indication if the disease is in a non-acute phase and/or if a non-acute attack of the disease has occurred; indication whether a combination of acute and non-acute phases or attacks has occurred; diagnosis of a disease and prognosis of a subsequent adverse outcome; diagnosis of a disease and prognosis of a subsequent acute or non-acute phase or attack; disease progression (for example for cancer, such progression may include for example occurrence or recurrence of metastasis). The above diagnoses may also optionally include differential diagnosis of the disease to distinguish it from other diseases, including those diseases that may feature one or more similar or identical symptoms.
In certain embodiments, one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s). In other embodiments, threshold level(s) of a diagnostic or prognostic indicator(s) can be established, and the level of the indicator(s) in a patient sample can simply be compared to the threshold level(s). The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test—they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves, or "ROC" curves, are typically calculated by plotting the value of a variable versus its relative frequency in "normal" and "disease" populations, and/or by comparison of results from a subject before, during and/or after treatment. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.
ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create an ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (say l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art (see for example Hanley et al, Radiology 143: 29-36 (1982), incorporated by reference as if fully set forth herein).
One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis/prognosis. In preferred embodiments, particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of the entire marker profile by plotting ROC curves for the sensitivity of a particular panel of markers versus 1 -(specificity) for the panel at various cutoffs. In these methods, a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) that an individual has had a disease, is at risk for developing such a disease, optionally the type of disease which the individual has had or is at risk for, and so forth etc. In such embodiments, an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient. Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.
In preferred embodiments, markers and/or marker panels are selected to exhibit at least 70% sensitivity, more preferably at least 80% sensitivity, even more preferably at least 85% sensitivity, still more preferably at least 90% sensitivity, and most preferably at least 95% sensitivity, combined with at least 70% specificity, more preferably at least 80% specificity, even more preferably at least 85% specificity, still more preferably at least 90% specificity, and most preferably at least 95% specificity. In particularly preferred embodiments, both the sensitivity and specificity are at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%. Sensitivity and/or specificity may optionally be determined as described above, with regard to the construction of ROC graphs and so forth, for example.
According to preferred embodiments of the present invention, individual markers and/or combinations (panels) of markers may optionally be used for diagnosis of time of onset of a disease or condition. Such diagnosis may optionally be useful for a wide variety of conditions, preferably including those conditions with an abrupt onset.
The phrase "determining the prognosis" as used herein refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome may be about 3%. hi preferred embodiments, a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance. The term "about" in this context refers to +/-1%.
The skilled artisan will understand that associating a prognostic indicator with a predisposition to an adverse outcome is a statistical analysis. For example, a marker level of greater than 80 pg/mL may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to 80 pg/mL, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.
In other embodiments, a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level. A preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. The term "about" in this context refers to +/-10%. In yet other embodiments, a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages. Exemplary, non-limiting methods and systems for identification of suitable biomarkers for marker panels are now described. Methods and systems for the identification of one or more markers for the diagnosis, and in particular for the differential diagnosis, of disease have been described previously. Suitable methods for identifying markers useful for the diagnosis of disease states are described in detail in U.S. patent application no. 2004-0126767, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING MARKER COMBINATIONS, filed Dec. 27, 2002, hereby incorporated by reference in its entirety as if fully set forth herein. One skilled in the art will also recognize that univariate analysis of markers can be performed and the data from the univariate analyses of multiple markers can be combined to form panels of markers to differentiate different disease conditions.
In developing a panel of markers useful in diagnosis, data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers. The group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects. The first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state. For example, this first set of patients may be those that have recently had a disease and/or a particular type of the disease. The confirmation of this condition state may be made through more rigorous and/or expensive testing, preferably according to a previously defined diagnostic standard. Hereinafter, subjects in this first set will be referred to as "diseased".
The second set of subjects are simply those who do not fall within the first set. Subjects in this second set may be "non-diseased;" that is, normal subjects. Alternatively, subjects in this second set may be selected to exhibit one symptom or a constellation of symptoms that mimic those symptoms exhibited by the "diseased" subjects.
The data obtained from subjects in these sets includes levels of a plurality of markers. Preferably, data for the same set of markers is available for each patient. This set of markers may include all candidate markers which may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required. Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition. The levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.
As noted above, a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient. An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve as described above.
As discussed above, the measurement of the level of a single marker may have limited usefulness. The measurement of additional markers provides additional information, but the difficulty lies in properly combining the levels of two potentially unrelated measurements. In the methods and systems according to embodiments of the present invention, data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response. The data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file. The database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased.
Next, an artificial cutoff region may be initially selected for each marker. The location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, the cutoff region is initially centered about the center of the overlap region of the two sets of patients. In one embodiment, the cutoff region may simply be a cutoff point. In other embodiments, the cutoff region may have a length of greater than zero. In this regard, the cutoff region may be defined by a center value and a magnitude of length. In practice, the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range. Each marker value for each patient may then be mapped to an indicator. The indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis. In other embodiments, the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.
The relative importance of the various markers may be indicated by a weighting factor. The weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5. In a preferred embodiment, the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker. Next, a panel response may be calculated for each subject in each of the two sets. The panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker. One advantage of using an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker. Typically, a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state. The use of an indicator which is constant on one side of the cutoff region eliminates this concern.
The panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient. Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response. Having obtained panel responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the selection of an effective panel. The objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.
In a preferred embodiment, the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function. For example, the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers. In other embodiments, other features of the ROC curve may be used to define the objective function. For example, the point at which the slope of the ROC curve is equal to one may be a useful feature. In other embodiments, the point at which the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee," may be used. In an embodiment, the sensitivity at the knee may be maximized. In further embodiments, the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of these ROC- curve features may be used.
It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a "positive" test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.
An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimums. In the optimization process, the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables. In a preferred embodiment, the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.
In addition to the above-described parameters, the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.
The optimization algorithm may be provided with certain constraints as well. For example, the resulting ROC curve may be constrained to provide an area-under-curve of greater than a particular value. ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets. Thus, a minimum acceptable value, such as 0.75, may be used as a constraint, particularly if the objective function does not incorporate the area under the curve. Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.
The iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function. The number of iterations may be limited in the optimization process. Further, the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.
Thus, the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels which require the measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In one embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated.
Individual panel response values may also be used as markers in the methods described herein. For example, a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above). Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested. For a "diseased" and "non-diseased" group of patients, the resulting panel responses may be treated as if they were just levels of another disease marker.
Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003 (hereby incorporated by reference as if fully set forth herein), and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. As discussed above, suitable tests may exhibit one or more of the following results on these various measures: at least 75% sensitivity, combined with at least 75% specificity; ROC curve area of at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; and/or a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (l-sensitivity)/specifϊcity) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1.
According to other preferred embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting marker-detectable disease and/or an indicative condition, such that a biomarker may optionally comprise any of the above. According to still other preferred embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein. Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
Non-limiting examples of methods or assays are described below.
The present invention also relates to kits based upon such diagnostic methods or assays.
Nucleic acid sequences and Oligonucleotides
Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion. The present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion. The present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.
In cases where the polynucleotide sequences of the present invention encode previously unidentified polypeptides, the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove. A "nucleic acid fragment" or an "oligonucleotide" or a "polynucleotide" are used herein interchangeably to refer to a polymer of nucleic acids. A polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase. As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is composed of genomic and cDNA sequences. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
Preferred embodiments of the present invention encompass oligonucleotide probes.
An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein). Alternatively, an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid- phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988) and "Oligonucleotide Synthesis" Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.
Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases. Preferably, the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.
The oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
Preferably used oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non- natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. NOs: 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.
Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374. Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3-deazaguanine and 3-deazaadenine. Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No: 6,303,374.
It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
It will be appreciated that oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element. As used herein, the phrase "cis acting regulatory element" refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. Preferably, the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed. Examples of cell type- specific and/or tissue-specific promoters include promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al, (1988) Adv. Immunol. 43:235- 275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729- 733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). The nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining elements), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct, hi addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
Hybridization assays
Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described).
Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.
Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
Thus, the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and
5 x 106 cpm 32P labeled probe, at 65 0C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCl3 1 % SDS and 5 x 10^ cpm 32P labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 0C.
More generally, hybridization of short nucleic acids (below 200 bp in length, e.g.
17-40 bp in length) can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 niM EDTA
(pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tm, final wash solution of 3 M
TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm; (H) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI3
0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 0C below the Tm, final wash solution of 6 x SSC, and final wash at 22 0C; (Hi) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature.
The detection of hybrid duplexes can be carried out by a number of methods. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample. Probes can be labeled according to numerous well known methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
For example, oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.
Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays.
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
NAT Assays Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR for example (or variations thereof such as real-time PCR for example).
As used herein, a "primer" defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab.
8:14 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction
(LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86,
1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods MoI. Biol., 28:253-260; and Sambrook et al., 1989, supra).
The terminology "amplification pair" (or "primer pair") refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
In one particular embodiment, amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid. In one preferred embodiment, RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA. In another preferred embodiment, the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.
The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods. Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. Optionally, the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al, 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N. Y.). It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail below). The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 0C, most preferably less than 3 0C, ideally between 3 0C and 0 0C.
Polymerase Chain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al, is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification. This technology provides one approach to the problems of low target sequence concentration. PCR can be used to directly increase the concentration of the target to an easily detectable level. This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR- amplified."
Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes referred to as "Ligase Amplification Reaction" (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 Al (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
Self-Sustained Synthetic Reaction (3SR/NASBA): The self-sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs). Q-Beta (Qβ) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Qβ replicase. A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere. A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Qβ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., > 55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.
The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as: (1+X)n =y, where "X" is the mean efficiency (percent copied in each cycle), "n" is the number of cycles, and "y" is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100 %. If 20 cycles of PCR are performed, then the yield will be 2^0, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.85^0, or 220,513 copies of the starting material. In other words, a PCR running at 85 % efficiency will yield only 21 % as much final product, compared to a reaction running at 100 % efficiency. A reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product.
In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield. At 50 % mean efficiency, it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products. Also, many variables can influence the mean efficiency of PCR, including target
DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA (e.g., DNA spilled onto lab surfaces) or cross-contamination is also a major consideration. Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator. The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise temperature cycling.
Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method of the detection of allele-specifϊc variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence. This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
A similar 3 '-mismatch strategy is used with greater effect to prevent ligation in the LCR. Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory. The direct detection method according to various preferred embodiments of the present invention may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, (e.g., as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats. Two examples are the "Cycling Probe Reaction" (CPR), and "Branched DNA" (bDNA). Cycling probe reaction (CPR): The cycling probe reaction (CPR), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
Branched DNA: Branched DNA (bDNA), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
The detection of at least one sequence change according to various preferred embodiments of the present invention may be accomplished by, for example restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single- Strand Conformation Polymorphism (SSCP) analysis or Dideoxy fingerprinting (ddF).
The demand for tests which allow the detection of specific nucleic acid sequences and sequence changes is growing rapidly in clinical diagnostics. As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost-effective, and easy-to-use tests for as yet mutations within specific sequences is rapidly increasing.
A handful of methods have been devised to scan nucleic acid segments for mutations. One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, specialized equipment and highly trained personnel are required, and the method is too labor-intense and expensive to be practical and effective in the clinical setting.
In view of the difficulties associated with sequencing, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs. Restriction fragment length polymorphism (RFLP): For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
Single point mutations have been also detected by the creation or destruction of RFLPs. Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory. RFLP analysis suffers from low sensitivity and requires a large amount of sample. When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
A handful of rare-cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-rich sequences, and cleave at sites that tend to be highly clustered. Recently, endonucleases encoded by group I introns have been discovered that might have greater than 12 base-pair specificity, but again, these are few in number.
Allele specific oligonucleotide (ASO): If the change is not in a recognition sequence, then allele-specific oligonucleotides (ASOs), can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles. The ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
With either of the techniques described above (i.e., RFLP and ASO), the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest.
Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNA:RNA duplexes.
Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of mutations.
A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient. TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
Single-Strand Conformation Polymorphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non- denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions. Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment. SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP is reportedly able to detect 90 % of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50 % for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened. According to a presently preferred embodiment of the present invention the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Qβ-Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy fingerprinting. Detection may also optionally be performed with a chip or other such device. The nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group. This reporter group can be a fluorescent group such as phycoerythrin. The labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station, describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.
Amino acid sequences and peptides
The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide" and "protein" include glycoproteins, as well as non-glycoproteins.
Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N. Y.], after which their composition can be confirmed via amino acid sequencing. In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
The present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein. The present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low-complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50. Preferably, nucleic acid sequence homology/identity is determined by using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11. Finally, the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
It will be appreciated that peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.
Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH- CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor- valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.
Table 1 non-conventional or modified amino acids which can be used with the present invention.
Table 1
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000077_0001
Table 1 Cont. Since the peptides of the present invention are preferably utilized in diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing. In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI. Cell. Biol. 6:559- 565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 and also as described above.
Antibodies
"Antibody" refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)'2 fragments. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.
The functional fragments of antibodies, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages, are described as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Monoclonal antibody development may optionally be performed according to any method that is known in the art. The method described below is provided for the purposes of description only and is not meant to be limiting in any way. Step 1 : Immunization of Mice and Selection of Mouse Donors for Generation of Hvbridoma Cells
Producing mAb requires immunizing an animal, usually a mouse, by injection of an antigen X to stimulate the production of antibodies targeted against X. Antigen X can be the whole protein or any sequence thereof that gives rise to a determinant. According to the present invention, optionally and preferably such antigens may include but are not limited to any variant described herein or a portion thereof, including but not limited to any head, tail, bridge or unique insertion, or a bridge to such head, tail or unique insertion, or any other epitope described herein according to the present invention. Injection of peptides requires peptide design (with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability) and synthesis. The antigen is optionally and preferably prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes optionally used as immunogens. Other immunogens or adjuvants may also optionally be used.
In general, mice are immunized every 2-3 weeks but the immunization protocols are heterogeneous. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.
Step 2: Screening of Mice for Antibody Production
After several weeks of immunization, blood samples are optionally and preferably obtained from mice for measurement of serum antibodies. Several techniques have been developed for collection of small volumes of blood from mice (Loeb and Quimby 1999). Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and/or immunoassays for example (for example a Western blot may optionally be used). If the antibody titer is high, cell fusion can optionally be performed. If the titer is too low, mice can optionally be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.
Step 3: Preparation of Myeloma Cells
Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are optionally and preferably cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine- aminopterin-thymidine (HAT) selection medium used after cell fusion. The selection growth medium contains the inhibitor aminopterin, which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cell line to which the normal antibody-producing cells are fused. Because neither the myeloma nor the antibody- producing cell will grow on its own, only hybrid cells grow. The HAT medium allows only the fused cells to survive in culture. A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth.
The antibody forming cells are isolated from the mouse's spleen and are then fused with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. The resulting cell is called a hybridoma.
Step 4: Fusion of Myeloma Cells with Immune Spleen Cells and antibody screening
Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. Alternatively, the cells are centrifuged, the supernatant is discarded and PEG is then added. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and Kennedy 1994). Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells (Hoffman and others 1996).
Once hybridoma colonies reach a satisfactory cell count, the plates are assayed by an assay, eg ELISA or a regular immunoassay such as RIA for example, to determine which colonies are secreting antibodies to the immunogen. Cells from positive wells are isolated and expanded. Conditioned medium from each colony is retested to verify the stability of the hybridomas (that is, they continue to produce antibody).
Step 5: Cloning of Hybridoma Cell Lines by "Limiting Dilution" or Expansion and Stabilization of Clones by Ascites Production
At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time.
For prolonged stability of the antibody-producing cell lines, it is necessary to clone and then recline the chosen cells. Cloning consists of subcloonng the cells by either limiting dilution at an average of less than one cell in each culture well or by platingout the cells in a thin layer of semisolid agar of methyl cellulose or by single-cell manipulation. At each stage, cultures are assayed for production of the appropriate antibodies.
Step 6: Antibody purification
The secreted antibodies are optionally purified, preferably by one or more column chromatography steps and/or some other purification method, including but not limited to ion exchange, affinity, hydrophobic interaction, and gel permeation chromatography. The operation of the individual chromatography step, their number and their sequence is generally tailored to the specific antibody and the specific application.
Large-scale antibody production may also optionally and preferably be performed according to the present invention. Two non-limiting, illustrative exemplary methods are described below for the purposes of description only and are not meant to be limiting in any way. In vivo production may optionally be performed with ascites fluid in mice. According to this method, hybridoma cell lines are injected into the peritoneal cavity of mice to produce ascitic fluid (ascites) in its abdomen; this fluid contains a high concentration of antibody.
An exemplary in vitro method involves the use of culture flasks. In this method, monoclonal antibodies can optionally be produced from the hybridoma using gas permeable bags or cell culture flasks.
Antibody Engineering in Phage Display Libraries
PCT Application No. WO 94/18219, and its many US equivalents, including US Patent No. 6096551 , all of which are hereby incorporated by reference as if fully set forth herein, describes methods for producing antibody libraries using universal or randomized immunoglobulin light chains, by using phage display libraries. The method involves inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene for the purpose of producing light chain gene libraries for use in combination with heavy chain genes and gene libraries to produce antibody libraries of diverse and novel immunospecificities. The method comprises amplifying a CDR portion of an immunoglobulin light chain gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide. The resultant gene portions are inserted into phagemids for production of a phage display library, wherein the engineered light chains are displayed by the phages, for example for testing their binding specificity.
Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. A scFv antibody fragment is an engineered antibody derivative that includes heavy- and light chain variable regions joined by a peptide linker. The minimal size of antibody molecules are those that still comprise the complete antigen binding site. ScFv antibody fragments are potentially more effective than unmodified IgG antibodies. The reduced size of 27-30 kDa permits them to penetrate tissues and solid tumors more readily. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)]. Optionally, there may be 1, 2 or 3 CDRs of different chains, but preferably there are 3 CDRs of 1 chain. The chain could be the heavy or the light chain.
Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co- workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species, hi practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14[ 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
Preferably, the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
Optionally, a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination. One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.
Immunoassays
In another embodiment of the present invention, an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample. This method comprises: providing an antibody that specifically binds to a marker; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample.
To prepare an antibody that specifically binds to a marker, purified protein markers can be used. Antibodies that specifically bind to a protein marker can be prepared using any suitable methods known in the art.
After the antibody is provided, a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays. Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include but are not limited to glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead. Antibodies can also be attached to a solid support.
After incubating the sample with antibodies, the mixture is washed and the antibody-marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture. Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10 0C to 40 0C.
The immunoassay can be used to determine a test amount of a marker in a sample from a subject. First, a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody-marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above. The amount of an antibody-marker complex can optionally be determined by comparing to a standard. As noted above, the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and/or signal. Preferably used are antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, for example. Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies".
Radioimmunoassay (RIA): In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody
125 and radiolabeled antibody binding protein (e.g., protein A labeled with I ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy. Western blot: This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
Immunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
Radio-imaging Methods
These methods include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non-invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPECT can optionally be used with two labels simultaneously. SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US Patent No. 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
Display Libraries
According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-
17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention. Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young AC, et al, "The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes" J MoI Biol 1997 Dec 12;274(4):622-34; Giebel LB et al "Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities" Biochemistry 1995 Nov 28;34(47): 15430-5; Davies EL et al, "Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes" J Immunol Methods 1995 Oct 12;186(l):125-35; Jones C RT al. "Current trends in molecular recognition and bioseparation" J Chromatogr A 19<95 JuI 14;707(l):3-22; Deng SJ et al. "Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries" Proc Natl Acad Sci U S A 1995 May 23;92(l l):4992-6; and Deng SJ et al. "Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display" J Biol Chem 1994 Apr l;269(13):9533-8, which are incorporated herein by reference.
The following sections relate to Candidate Marker Examples.
CANDIDATE MARKER EXAMPLES SECTION This Section relates to Examples of sequences according to the present invention, including illustrative methods of selection thereof with regard to cancer; other markers were selected as described below for the individual markers.
Description of the methodology undertaken to uncover the biomolecular sequences of the present invention Human ESTs and cDNAs were obtained from GenBank versions 136 (June 15,
2003 ftp.ncbi.nih.gov/genbank/release.notes/gbl36.release.notes); NCBI genome assembly of April 2003; RefSeq sequences from June 2003; Genbank version 139 (December 2003); Human Genome from NCBI (Build 34) (from Oct 2003); and RefSeq sequences from December 2003. With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see www.ncbi.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST database in GenBank, may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced. Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No. 10/426,002, published as US20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into "clusters" that represent genes or partial genes.
These were annotated using the GeneCarta (Compugen, Tel-Aviv, Israel) platform. The GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
A brief explanation is provided with regard to the method of selecting the candidates. However, it should be noted that this explanation is provided for descriptive purposes only, and is not intended to be limiting in any way. The potential markers were identified by a computational process that was designed to find genes and/or their splice variants that are specifically expressed in cardiac tissue, as opposed to other types of tissues and also particularly as opposed to muscle tissue, by using databases of expressed sequences. Various parameters related to the information in the EST libraries, determined according to classification by library annotation, were used to assist in locating genes and/or splice variants thereof that are specifically and/or differentially expressed in heart tissues. The detailed description of the selection method and of these parameters is presented in Example 1 below.
SELECTING CANDIDATES WITH REGARD TO CANCER
A brief explanation is provided with regard to a non-limiting method of selecting the candidates for cancer diagnostics. However, it should noted that this explanation is provided for descriptive purposes only, and is not intended to be limiting in any way. The potential markers were identified by a computational process that was designed to find genes and/or their splice variants that are over-expressed in tumor tissues, by using databases of expressed sequences. Various parameters related to the information in the EST libraries, determined according to a manual classification process, were used to assist in locating genes and/or splice variants thereof that are over-expressed in cancerous tissues. The detailed description of the selection method is presented in Example 1 below. The cancer biomarkers selection engine and the following wet validation stages are schematically summarized in Figure 1.
PART II - Cancer markers EXAMPLE 1
Identification of differentially expressed gene products - Algorithm In order to distinguish between differentially expressed gene products and constitutively expressed genes (i.e., house keeping genes ) an algorithm based on an analysis of frequencies was configured. A specific algorithm for identification of transcripts over expressed in cancer is described hereinbelow.
Dry analysis Library annotation - EST libraries are manually classified according to:
(i) Tissue origin
(ii) Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell-lines; normal tissues; cancer tissues; fetal tissues; and others such as normal cell lines and pools of normal cell-lines, cancer cell-lines and combinations thereof. A specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
(iϋ) Protocol of library construction - various methods are known in the art for library construction including normalized library construction; non-normalized library construction; subtracted libraries; ORESTES and others. It will be appreciated that at times the protocol of library construction is not indicated. The following rules are followed:
EST libraries originating from identical biological samples are considered as a single library.
EST libraries which include above-average levels of DNA contamination are eliminated.
Dry computation - development of engines which are capable of identifying genes and splice variants that are temporally and spacially expressed.
Clusters (genes) having at least five sequences including at least two sequences from the tissue of interest are analyzed.
EXAMPLE 2
Identification of genes over expressed in cancer.
Two different scoring algorithms were developed.
Libraries score -candidate sequences which are supported by a number of cancer libraries, are more likely to serve as specific and effective diagnostic markers.
The basic algorithm - for each cluster the number of cancer and normal libraries contributing sequences to the cluster was counted. Fisher exact test was used to check if cancer libraries are significantly over-represented in the cluster as compared to the total number of cancer and normal libraries. Library counting: Small libraries (e.g., less than 1000 sequences) were excluded from consideration unless they participate in the cluster. For this reason, the total number of libraries is actually adjusted for each cluster.
Clones no. score - Generally, when the number of ESTs is much higher in the cancer libraries relative to the normal libraries it might indicate actual over-expression. The algorithm —
Clone counting: For counting EST clones each library protocol class was given a weight based on an assessment of how much the protocol reflects actual expression levels:
(i) non-normalized : 1
(ii) normalized : 0.2 (iii) all other classes : 0.1
Clones number score - The total weighted number of EST clones from cancer libraries was compared to the EST clones from normal libraries. To avoid cases where one library contributes to the majority of the score, the contribution of the library that gives most clones for a given cluster was limited to 2 clones. The score was computed as
Figure imgf000094_0001
where: c - weighted number of "cancer" clones in the cluster. C- weighted number of clones in all "cancer" libraries, n - weighted number of "normal" clones in the cluster. N- weighted number of clones in all "normal" libraries. Clones number score significance - Fisher exact test was used to check if EST clones from cancer libraries are significantly over-represented in the cluster as compared to the total number of EST clones from cancer and normal libraries.
Two search approaches were used to find either general cancer-specific candidates or tumor specific candidates. • Libraries/sequences originating from tumor tissues are counted as well as libraries originating from cancer cell-lines ("normal" cell- lines were ignored). • Only libraries/sequences originating from tumor tissues are counted
EXAMPLE 3
Identification of tissue specific genes
For detection of tissue specific clusters, tissue libraries/sequences were compared to the total number of libraries/sequences in cluster. Similar statistical tools to those described in above were employed to identify tissue specific genes. Tissue abbreviations are the same as for cancerous tissues, but are indicated with the header "normal tissue".
The algorithm - for each tested tissue T and for each tested cluster the following were examined:
1. Each cluster includes at least 2 libraries from the tissue T. At least 3 clones (weighed - as described above) from tissue T in the cluster; and 2. Clones from the tissue T are at least 40 % from all the clones participating in the tested cluster
Fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant.
EXAMPLE 4
Identification of splice variants over expressed in cancer of clusters which are not over expressed in cancer
Cancer-specific splice variants containing a unique region were identified. Identification of unique sequence regions in splice variants
A Region is defined as a group of adjacent exons that always appear or do not appear together in each splice variant.
A "segment" (sometimes referred also as "seg" or "node") is defined as the shortest contiguous transcribed region without known splicing inside. Only reliable ESTs were considered for region and segment analysis. An EST was defined as unreliable if:
(i) Unspliced;
(ii) Not covered by RNA;
(iii) Not covered by spliced ESTs; and (iv) Alignment to the genome ends in proximity of long poly-A stretch or starts in proximity of long poly-T stretch.
Only reliable regions were selected for further scoring. Unique sequence regions were considered reliable if:
(i) Aligned to the genome; and (ii) Regions supported by more than 2 ESTs.
The algorithm
Each unique sequence region divides the set of transcripts into 2 groups:
(i) Transcripts containing this region (group TA).
(ii) Transcripts not containing this region (group TB). The set of EST clones of every cluster is divided into 3 groups:
(i) Supporting (originating from) transcripts of group TA (Sl).
(ii) Supporting transcripts of group TB (S2).
(iii) Supporting transcripts from both groups (S3).
Library and clones number scores described above were given to Sl group. Fisher Exact Test P-values were used to check if:
S 1 is significantly enriched by cancer EST clones compared to S2; and
Sl is significantly enriched by cancer EST clones compared to cluster background
(S1+S2+S3). Identification of unique sequence regions and division of the group of transcripts accordingly is illustrated in Figure 2. Each of these unique sequence regions corresponds to a segment, also termed herein a "node".
Region 1: common to all transcripts, thus it is not considered; Region 2: specific to Transcript 1 : T_l unique regions (2+6) against T_2+3 unique regions (3+4); Region 3: specific to Transcripts 2+3: T_2+3 unique regions (3+4) against Tl unique regions (2+6); Region 4: specific to Transcript 3: T_3 unique regions (4) against T 1+2 unique regions (2+5+6); Region 5: specific to Transcript 1+2: T_l+2 unique regions (2+5+6) against T3 unique regions (4); Region 6: specific to Transcript 1 : same as region 2.
EXAMPLE 5 Identification of cancer specific splice variants of genes over expressed in cancer
A search for EST supported (no mRNA) regions for genes of:
(i) known cancer markers
(ii) Genes shown to be over-expressed in cancer in published micro-array experiments. Reliable EST supported-regions were defined as supported by minimum of one of the following:
(i) 3 spliced ESTs; or
(ii) 2 spliced ESTs from 2 libraries;
(iii) 10 unspliced ESTs from 2 libraries, or (iv) 3 libraries.
Oligonucleotide-based micro-array experiment protocol-
Microarray fabrication Microarrays (chips) were printed by pin deposition using the MicroGrid II MGII 600 robot from BioRobotics Limited (Cambridge, UK). 50-mer oligonucleotides target sequences were designed by Compugen Ltd (Tel-Aviv, IL) as described by A. Shoshan et al, "Optical technologies and informatics", Proceedings of SPIE. VoI 4266, pp. 86-95 (2001). The designed oligonucleotides were synthesized and purified by desalting with the Sigma-Genosys system (The Woodlands, TX, US) and all of the oligonucleotides were joined to a C6 amino-modified linker at the 5' end, or being attached directly to CodeLink slides (Cat #25-6700-01. Amersham Bioscience, Piscataway, NJ, US). The 50- mer oligonucleotides, forming the target sequences, were first suspended in Ultra-pure DDW (Cat # 01 -866- IA Kibbutz Beit-Haemek, Israel) to a concentration of 50μM. Before printing the slides, the oligonucleotides were resuspended in 30OmM sodium phosphate (pH 8.5) to final concentration of 15OmM and printed at 35-40% relative humidity at 210C.
Each slide contained a total of 9792 features in 32 subarrays. Of these features, 4224 features were sequences of interest according to the present invention and negative controls that were printed in duplicate. An additional 288 features (96 target sequences printed in triplicate) contained housekeeping genes from Human Evaluation Library2, Compugen Ltd, Israel. Another 384 features are E.coli spikes 1-6, which are oligos to E- CoIi genes which are commercially available in the Array Control product (Array control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
Post-coupling processing of printed slides
After the spotting of the oligonucleotides to the glass (CodeLink) slides, the slides were incubated for 24 hours in a sealed saturated NaCl humidification chamber (relative humidity 70-75%).
Slides were treated for blocking of the residual reactive groups by incubating them in blocking solution at 50°C for 15 minutes (lOml/slide of buffer containing 0.1M Tris, 5OmM ethanolamine, 0.1% SDS). The slides were then rinsed twice with Ultra-pure DDW (double distilled water). The slides were then washed with wash solution (10ml/slide. 4X SSC, 0.1% SDS)) at 50°C for 30 minutes on the shaker. The slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 rpm.
Next, in order to assist in automatic operation of the hybridization protocol, the slides were treated with Ventana Discovery hybridization station barcode adhesives. The printed slides were loaded on a Bio-Optica (Milan, Italy) hematology staining device and were incubated for 10 minutes in 50ml of 3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess fluid was dried and slides were then incubated for three hours in 20 mm/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc. Redding CA).
The following protocol was then followed with the Genisphere 900-RP (random primer), with mini elute columns on the Ventana Discovery HybStation™, to perform the microarray experiments. Briefly, the protocol was performed as described with regard to the instructions and information provided with the device itself. The protocol included cDNA synthesis and labeling. cDNA concentration was measured with the TBS-380 (Turner Biosystems. Sunnyvale, CA.) PicoFlour, which is used with the OHGreen ssDNA Quantitation reagent and kit.
Hybridization was performed with the Ventana Hybridization device, according to the provided protocols (Discovery Hybridization Station Tuscon AZ).
The slides were then scanned with GenePix 4000B dual laser scanner from Axon Instruments Inc, and analyzed by GenePix Pro 5.0 software.
Schematic summary of the oligonucleotide based microarray fabrication and the experimental flow is presented in Figures 3 and 4. Briefly, as shown in Figure 3, DNA oligonucleotides at 25uM were deposited
(printed) onto Amersham 'CodeLink' glass slides generating a well defined 'spot'. These slides are covered with a long-chain, hydrophilic polymer chemistry that creates an active 3-D surface that covalently binds the DNA oligonucleotides 5 '-end via the C6-amine modification. This binding ensures that the full length of the DNA oligonucleotides is available for hybridization to the cDNA and also allows lower background, high sensitivity and reproducibility.
Figure 4 shows a schematic method for performing the microarray experiments. It should be noted that stages on the left-hand or right-hand side may optionally be performed in any order, including in parallel, until stage 4 (hybridization). Briefly, on the left-hand side, the target oligonucleotides are being spotted on a glass microscope slide (although optionally other materials could be used) to form a spotted slide (stage 1). On the right hand side, control sample RNA and cancer sample RNA are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled probes. It should be noted that the control and cancer samples come from corresponding tissues (for example, normal prostate tissue and cancerous prostate tissue). Furthermore, the tissue from which the RNA was taken is indicated below in the specific examples of data for particular clusters, with regard to overexpression of an oligonucleotide from a "chip" (microarray), as for example "prostate" for chips in which prostate cancerous tissue and normal tissue were tested as described above. In stage 3, the probes are mixed. In stage 4, hybridization is performed to form a processed slide. In stage 5, the slide is washed and scanned to form an image file, followed by data analysis in stage 6.
Diseases and conditions that may be diagnosed with one or more variant(s) according to the present invention
Ovarian cancer
Certain splice variants described herein are potential markers for ovarian cancer. Other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
1. The identification of a metastasis of unknown origin which originated from a primary ovarian cancer, for example gastric carcinoma (such as Krukenberg tumor), breast cancer, colorectal carcinoma and pancreatic carcinoma.
2. As a marker to distinguish between different types of ovarian cancer, therefore potentially affect treatment choice (e.g. discrimination between epithelial tumors and germ cell tumors).
3. As a tool in the assessment of abdominal mass and in particular in the differential diagnosis between a benign and malignant ovarian cysts.
4. As a tool for the assessment of infertility.
5. Other conditions that may elevate serum levels of ovary related markers. These include but are not limited to: cancers of the endometrium, cervix, fallopian tubes, pancreas, breast, lung and colon; nonmalignant conditions such as pregnancy, endometriosis, pelvic inflammatory disease and uterine fibroids.
6. Conditions which have similar symptoms, signs and complications as ovarian cancer and where the differential diagnosis between them and ovarian cancer is of clinical importance including but not limited to: a. Non-malignant causes of pelvic mass. Including, but not limited to: benign (functional) ovarian cyst, uterine fibroids, endometriosis, benign ovarian neoplasms and inflammatory bowel lesions b. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, skeletal or abdominal pain, paraneoplastic syndrome, c. Ascites.
Lung cancer
Certain splice variants described herein are potential markers for lung cancer. Other conditions that may be diagnosed by these markers or variants of them include but are not limited to the presence, risk and/or extent of the following:
1. The identification of a metastasis of unknown origin which originated from a primary lung cancer.
2. The assessment of a malignant tissue residing in the lung and is from a non-lung origin, including, but not limited to: osteogenic and soft tissue sarcomas; colorectal, uterine, cervix and corpus tumors; head and neck, breast, testis and salivary gland cancers; melanoma; and bladder and kidney tumors.
3. As a marker to distinguish between different types of lung cancer, therefore potentially affect treatment choice (e.g. small cell vs. non small cell tumors).
4. As a tool in the assessment of unexplained dyspnea and/or chronic cough and/or hemoptysis.
5. As a tool in the differential diagnosis of the origin of a pleural effusion.
6. Conditions which have similar symptoms, signs and complications as lung cancer and where the differential diagnosis between them and lung cancer is of clinical importance including but not limited to: a Non-malignant causes of lung symptoms and signs. Symptoms and signs include, but are not limited to: lung lesions and infiltrates, wheeze, stridor.. b. Other symptoms, signs and complications suggestive of lung cancer, such as tracheal obstruction, esophageal compression, dysphagia, recurrent laryngeal nerve paralysis, hoarseness, phrenic nerve paralysis with elevation of the hemidiaphragm and Horner syndrome. c. Any condition suggestive of a malignant tumor including but not limited to anorexia, cachexia, weight loss, fever, hypercalcemia, hypophosphatemia, hyponatremia, syndrome of inappropriate secretion of antidiuretic hormone, elevated ANP, elevated ACTH, hypokalemia, clubbing, neurologic-myopathic syndromes and thrombophlebitis.
RELATED DISEASE MARKERS AND RISK FACTORS FOR DETECTION BY BIOMARKERS In addition to the general clinical factors described above, as well as specific diagnostic aspects of each biomarker described below, there are field-specific disease markers/risk factors which may optionally relate to or present diagnostic applications for biomarkers according to the present invention. These field specific factors, as described below, relate to detection of ovarian cancer (or risk factors thereof) which may also serve as diagnostic markers.
OVARIAN CANCER
Known ovarian cancer markers may be used for a variety of diagnoses and/or detection of risk factors, in addition to those related to ovarian cancer itself. These known markers include but are not limited to CA 125. CA 125 may optionally be used for a number of diagnostic assays, such as detection of sepsis (and/or similar bacterial infections) and/or monitoring of the course of infection (as described with regard to PCT Application No. WO 03/048776, hereby incorporated by reference as if fully set forth herein) for example. Ovarian cancer markers according to the present invention which may also optionally have this utility include but are not limited to: ErbB-2 variants, EGFR variants, BCMP variants, Stromelysin-3 variants, TGFBl variants, and/or Inhibin beta variants. CANDIDATE MARKER EXAMPLES SECTION
This section relates to examples of sequences according to the present invention, including illustrative methods of selection thereof.
The markers of the present invention were tested with regard to their expression in various cancerous and non-cancerous tissue samples. A description of the samples used in the ovarian cancer testing panel is provided in Table 2 below. A description of the samples used in the colon cancer testing panel is provided in Table 3 below. A description of the samples used in the lung cancer testing panel is provided in Table 4 below. A description of the samples used in the breast cancer testing panel is provided in Table 5 below. A description of the samples used in the normal tissue panel is provided in Table 6 below. Tests were then performed as described in the "Materials and Experimental Procedures" section below.
Table 2: Tissue samples in ovarian cancer testing panel
Sample name Lot number Source Pathology Grade age
33-B-Pap Sero Serous papillary
A503175 BioChain 1
CystAde Gl 41 cystadenocarcinoma
Mixed epithelial cystadenocarcinoma with mucinous,
41-G-Mix
98-03-G803 GOG endometrioid, 2 Sero/Muc/Endo G2 38 squamous and papillary serous
(Stage2)
35-G-Endo Adeno Endometrioid _
94-08-7604 GOG G2 adenocarcinoma 39
14-B-Adeno G2 A501111 BioChain Adenocarcinoma 2 41 12-B-Adeno G3 A406023 Biochain Adenocarcinoma 3 45
Papillary serous and
40-G-Mix endometrioid
95-11-G006 GOG Sero/Endo G2 cystadenocarcinoma 49
(Stage3C)
Papillary
ILS-7286 ABS
G2 50 cystadenocarcinoma
Papillary
3-A-Pap Adeno G2 ILS-1431 ABS 52 adenocarcinoma
Papillary
2-A-Pap Adeno G2 ILS-1408 ABS adenocarcinoma 53
5-G-Adeno G3 Adenocarcinoma
99-12-G432 GOG
(Stage3C) ό 46
11 -B- Adeno G3 A407068 Biochain Adenocarcinoma 3 49 Mixed serous and
39--G-Mix
2001-12-G037 GOG endometrioid 3 49 Sero/Endo G3 adenocarcinoma Serous
29-G-Sero Adeno
2001-12-G035 GOG adenocarcinoma 3 50 G3 (Stage3A)
70-G-Pap Sero Papillary serous „
95-08-G069 GOG 50
Adeno G3 adenocarcinoma 6-A-Adeno G3 AO 106 ABS adenocarcinoma 3 51 31-B-Pap Sero Serous papillary „
A503176 BioChain 52
CystAde G3 cystadenocarcinoma Papillary serous
25-A-Pap Sero
N0021 ABS adenocarcinoma 3 55
Adeno G3 (StageT3 CNlMX) Mixed serous and
37-G-Mix
2002-05-G513 GOG endometrioid 3 56 Sero/Endo G3 adenocarcinoma
7-A- Adeno G3 IND-00375 ABS adenocarcinoma 3 59 8-B-Adeno G3 A501113 BioChain adenocarcinoma 3 60 10-B- Adeno G3 A407069 Biochain Adenocarcinoma 3 60 Mixed serous and
38-G-Mix endometrioid „
2002-05-G509 GOG 64 Sero/Endo G3 adenocarcinoma of mullerian (Stage3C) Poorly differentiated
13-G- Adeno G3 94-05-7603 GOG adenocarcinoma from 3 67 primary peritoneal Papillary serous „
2 er°2001-07-G801 GOG 68 Aκdfen~o J G3> ** S adenocarcinoma Papillary
34-G-Pap Endo endometrioid _
95-04-2002 GOG 68 Adeno G3 adenocarcinoma (Stage3C)
30-G-Pap Sero, Papillary serous »
2001-08-G011 GOG 72 Adeno G3 carcinoma (Stage 1C) Papillary „ 1-A-Pap Adeno G3 ILS- 1406 ABS 73 adenocarcinoma Adenocarcinoma „
9-G-Adeno G3 99-06-G901 GOG 84 (maybe serous)
32-G-Pap Sero Serous papillary,
93-09-4901 GOG
CystAde G3 cystadenocarcinoma 67 Papillary serous
66-G-Pap Sero carcinoma (metastais „
2000-01-G413 GOG
Adeno G3 SIV of primary 67 peritoneum) (Stage4)
19- B-Muc Adeno Mucinous „
A504085 G3 BioChain 34 adenocarcinoma Mucinous
21 -G- Muc CystAde
95-10-G020 GOG cystadenocarcinoma 2-3 44 G2-3
(Stage2)
18-B-Muc Adeno Mucinous ~
A504083 BioChain 45 G3 adenocarcinoma
20- A-Pap Muc Papillary mucinous
ABS 46
CystAde cystadenocarcinoma
17-B-Muc AdenoA504()84 Mucinous „
BioChain 51 adenocarcinoma
Mucinous
22-A-Muc CystAde A0139
ABS cystadenocarcinoma 2 72 CxZ
(Stage 1C)
43-G-Clear cell Clear cell _
2001-10-G002 GOG 74 Adeno G3 adenocarcinoma
Clear cell
44-G-Clear cell
2001-07-G084 GOG adenocarcinoma 73 Adeno
(Stage3A)
15-B-Adeno G3 A407065 BioChain Carcinoma 3 27
16-Ct- Adeno 1090387 Clontech Carcinoma NOS NA 58
Mucinous
23-A-Muc CyStAdevNM 00187 ^8 cystadenocarcinoma 3 45 G3 with low malignant
Epithelial
42-G-Adeno adenocarcinoma of
98-08-G001 GOG 46 borderline borderline malignancy
Serous
CysAdenoFibroma of
^"f6™ 2000-10-G620 GOG 71 CysAdenoFibroma borderline malignancy
62-G-Ben Benbin mucinus
Muc99-10-G442 GOG 32
CysAdenoma cysadenoma
60-G- Mucinous
MuC99-01-G043 GOG 40
CysAdenoma Cysadenoma
56-G-Ben Bengin mucinus
MuC99-01-G407 GOG 46 CysAdeno cysadenoma
64-G-Ben Bengin Serous
Sero99-06-G039 GOG 57
CysAdenoma CysAdenoma
61-G- Mucinous
Muc99-07-G011 GOG 63
CysAdenoma Cysadenoma
59-G-Sero 98-12-G401 Serous
GOG 77 CysAdenoFibroma CysAdenoFibroma
Normal (matched
51-G-N M41 98-03-G803N GOG 38 tumor 98-03-G803)
Normal (matched
75-G-N M60 99-01-G043N GOG 40 tumor 99-01-G043)
Normal (matched
49-B-N M14 A501112 BioChain 41 tumor A501111) Normal (matched
52-G-N M42 98-08-G001N GOG 46 tumor 98-08-G001) Normal (matched
68-G-N M56 99-01-G407N GOG 46 bengin 99-01-G407) Normal (matched
50-B-N M8 A501114 BioChain 60 tumor A501113) Normal (matched
67-G-N M38 2002-05-509N GOG 64 tumor 2002-05-G509) Normal (matched
69-G-N M24 2001-07-G801NGOG 68 tumor 2001-07-G801) Normal (matched
73-G-N M59 98-12-G401N GOG 77 tumor 98-12-G401) Normal (matched
72-G-N M66 2000-01-G413N GOG tumor 2000-01-G413)
45-B-N A503274 BioChain Normal PM 41
46-B-N A504086 BioChain Normal PM 41
71 -CG-N CG-188-7 Ichilov Normal PM 49
48-B-N A504087 BioChain Normal PM 51
Table 3: Tissue samples in colon cancer testing panel
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
BLEED)
Table 4: Tissue samples in lung cancer testing panel
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000110_0002
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000112_0002
Figure imgf000113_0001
Figure imgf000114_0001
Materials and Experimental Procedures
RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ USA 07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA www.biochain.com), ABS (Wilmington, DE 19801, USA, www.absbioreagents.com), Ambion (Austin, TX 78744 USA, www.ambion.com), or GOG for ovary samples— Pediatic Cooperative Human Tissue Network, Gynecologic Oncology Group Tissue Bank, Children Hospital of Columbus (Columbus OH 43205 USA). Alternatively, RNA was generated from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion).
RT PCR - Purified RNA (1 μg) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 μM dNTP in a total volume of 15.6 μl. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 μl of 5X SuperscriptII first strand buffer (Invitrogen), 2.4μl 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 °C, followed by further incubation at 42 0C for 2 min. Then, 1 μl (200units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25 μl) was incubated for 50 min at 42 °C and then inactivated at 70 °C for 15min. The resulting cDNA was diluted 1 :20 in TE buffer (10 mM Tris ρH=8, 1 mM EDTA pH=8).
Real-Time RT-PCR analysis- cDNA (5μl), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche). The amplification was effected as follows: 50 0C for 2 min, 95 0C for 10 min, and then 40 cycles of 95 0C for 15sec, followed by 60 0C for 1 min. Detection was performed by using the PE Applied Biosystem SDS 7000. The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and was used to calculate the relative transcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=efficiencyΛ"Ct. The efficiency of the PCR reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes. Schematic summary of quantitative real-time PCR analysis is presented in Figure 5. As shown, the x-axis shows the cycle number. The Cj =
Threshold Cycle point, which is the cycle that the amplification curve crosses the fluorescence threshold that was set in the experiment. This point is a calculated cycle number in which PCR product signal is above the background level (passive dye ROX) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geometrically increasing phase, during which measurements are most accurate, followed by a linear phase and a plateau phase; for quantitative measurements, the latter two phases do not provide accurate measurements). The y-axis shows the normalized reporter fluorescence. It should be noted that this type of analysis provides relative quantification.
The sequences of the housekeeping genes measured in all the examples on ovarian cancer panel were as follows:
SDHA (GenBank Accession No. NM_004168;
SEQ ID NO: 193)
SDHA Forward primer : (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: (SEQ ID NO: 154) CCACCACTGCATCAAATTCATG
SDHA-amplicon (SEQ ID NO: 155): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC
CAGTAGTGGATCATGAATTTGATGCAGTGGTGG
PBGD (GenBank Accession No. BC019323; SEQ ID NO: 194), PBGD Forward primer: (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG PBGD Reverse primer: (SEQ ID NO: 157) CCAGGGTACGAGGCTTTCAAT PBGD-amplicon: (SEQ ID NO: 158)
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGAC GGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG HPRTl (GenBank Accession No. NM_000194, SEQ ID NO: 195), HPRTl Forward primer: (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO: 160) GGTCCTTTTCACCAGCAAGCT HPRTl-amplicon: (SEQ ID NO:161)
TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATC CAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
GAPDH (GenBank Accession No. BC026907, SEQ ID NO: 196) GAPDH Forward primer: (SEQ ID NO: 162) TGCACC ACCAACTGCTTAGC
GAPDH Reverse primer: (SEQ ID NO: 163) CCATC ACGCC ACAGTTTCC
GAPDH-amplicon (SEQ ID NO: 164) :
TGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGG
TATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCCACCCAGAAG ACTGTGGATGG
The sequences of the housekeeping genes measured in all the examples on colon cancer tissue testing panel were as follows:
PBGD (GenBank Accession No. BC019323; SEQ ID NO: 194),
PBGD Forward primer: (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG PBGD Reverse primer: (SEQ ID NO: 157) CCAGGGTACGAGGCTTTCAAT PBGD-amplicon: (SEQ ID NO: 158) TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGAC GGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
HPRTl (GenBank Accession No. NM_000194, SEQ ID NO:195), HPRTl Forward primer: (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO:160) GGTCCTTTTCACCAGCAAGCT HPRTl-amplicon: (SEQ ID NO:161)
TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATC CAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
G6PD (GenBank Accession No. NM_000402, SEQ ID NO: 197) G6PD Forward primer: (SEQ ID NO: 165) gaggccgtcaccaagaacat G6PD Reverse primer: (SEQ ID NO: 166) ggacagccggtcagagctc G6PD-amplicon: (SEQ ID NO: 167) gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcggga gggacctgcagagctctgaccggctgtcc
RPS27A (GenBank Accession No. NM_002954, SEQ ID NO: 198) RPS27A Forward primer: (SEQ ID NO: 168) CTGGC AAGC AGCTGGAAGAT RPS27A Reverse primer: (SEQ ID NO: 169) TTTCTTAGCACCACCACGAAGTC RPS27A-amplicon: (SEQ ID NO: 170)
CTGGCAAGCAGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCAAAA GGAGTCTACTCTTCATCTTGTGTTGAGACTTCGTGGTGGTGCTAAGAAA
The sequences of the housekeeping genes measured in all the examples in the lung panel were as follows:
Ubiquitin (GenBank Accession No. BC000449, SEQ ID NO: 199) Ubiquitin Forward primer: (SEQ ID NO:171) ATTTGGGTCGCGGTTCTTG Ubiquitin Reverse primer: (SEQ ID NO: 172) TGCCTTGACATTCTCGATGGT Ubiquitin-amplicon (SEQ ID NO: 173)
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGC
AGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA
SDHA (GenBank Accession No. NM_004168; SEQ ID NO: 193)
SDHA Forward primer: (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG SDHA Reverse primer: (SEQ ID NO: 154) CCACCACTGCATC AAATTCATG SDHA-amplicon (SEQ ID NO: 155): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC CAGTAGTGGATCATGAATTTGATGCAGTGGTGG
PBGD (GenBank Accession No. BC019323, SEQ ID NO: 194),
PBGD Forward primer: (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG
PBGD Reverse primer: (SEQ ID NO: 157) CCAGGGTACGAGGCTTTCAAT PBGD-amplicon: (SEQ ID NO: 158)
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGAC
GGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG
HPRTl (GenBank Accession No. NM_000194, SEQ ID NO:195),
HPRTl Forward primer: (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO: 160) GGTCCTTTTC ACCAGC AAGCT HPRTl-amplicon: (SEQ ID NO: 161)
TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATC CAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
The sequences of the housekeeping genes measured in all the examples on breast cancer panel were as follows:
G6PD (GenBank Accession No. NM_000402, SEQ ID NO: 197)
G6PD Forward primer: (SEQ ID NO: 165) gaggccgtcaccaagaacat
G6PD Reverse primer: (SEQ ID NO: 166) ggacagccggtcagagctc
G6PD-amplicon (SEQ ID NO: 167) : gaggccgtcaccaagaacattcacgagtcctgcatgagccagataggctggaaccgcatcatcgtggagaagcccttcggga gggacctgcagagctctgaccggctgtcc
SDHA (GenBank Accession No. NM_004168; SEQ ID NO: 193) SDHA Forward primer: (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG SDHA Reverse primer: (SEQ ID NO: 154) CCACCACTGCATCAAATTCATG SDHA-amplicon (SEQ ID NO :155):
TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC CAGTAGTGGATCATGAATTTGATGCAGTGGTGG
PBGD (GenBank Accession No. BC019323; SEQ ID NO: 194), PBGD Forward primer: (SEQ ID NO: 156) TGAGAGTGATTCGCGTGGG
PBGD Reverse primer: (SEQ ID NO: 157) CCAGGGTACGAGGCTTTCAAT PBGD-amplicon: (SEQ ID NO: 158)
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAGAC GGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG HPRTl (GenBank Accession No. NM_000194, SEQ ID NO: 195), HPRTl Forward primer: (SEQ ID NO: 159) TGACACTGGCAAAACAATGCA HPRTl Reverse primer: (SEQ ID NO: 160) GGTCCTTTTCACCAGCAAGCT HPRTl-amplicon: (SEQ ID NO: 161)
TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATC CAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC
The sequences of the housekeeping genes measured in all the examples on normal tissue samples panel were as follows:
RPL 19 (GenBank Accession No. NM_000981, SEQ ID NO:200) RPL19Forward primer: (SEQ ID NO:174) TGGCAAGAAGAAGGTCTGGTTAG RPL19Reverse primer : (SEQ ID NO:175) TGATCAGCCCATCTTTGATGAG RPL19-amplicon (SEQ ID NO:176):
TGGCAAGAAGAAGGTCTGGTTAGACCCCAATGAGACCAATGAAATCGCCAAT GCCAACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATCA
TATA box (GenBank Accession No. NM_003194, SEQ ID NO:201), TATA box Forward primer (SEQ ID NO: 177): CGGTTTGCTGCGGTAATCAT
TATA box Reverse primer: (SEQ ID NO: 178) TTTCTTGCTGCCAGTCTGGAC
TATA box -amplicon: (SEQ ID NO: 179)
CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACTGA
TTTTCAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAACAGTC CAGACTGGCAGCAAGAAA
Ubiquitin (GenBank Accession No. BC000449, SEQ ID NO: 199) Ubiquitin Forward primer: (SEQ ID NO:171) ATTTGGGTCGCGGTTCTTG Ubiquitin Reverse primer: (SEQ ID NO: 172) TGCCTTGACATTCTCGATGGT Ubiquitin-amplicon (SEQ ID NO: 173)
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGC
AGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA SDHA (GenBank Accession No. NM 004168; SEQ ID NO: 193)
SDHA Forward primer: (SEQ ID NO: 153) TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: (SEQ ID NO: 154) CCACC ACTGCATC AAATTC ATG
SDHA-amplicon (SEQ ID NO :155) :
TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATC
CAGTAGTGGATCATGAATTTGATGCAGTGGTGG
Actual Marker Examples
The following examples relate to specific actual marker examples.
DESCRIPTION FOR CLUSTER HUMAlACM
Cluster HUMAlACM features 3 transcript(s) and 46 segment(s) of interest, the names for which are given in Tables 7 and 8, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 9.
Table 7 - Transcripts of interest
Figure imgf000120_0001
Table 8 - Segments of interest
Segment Hame1
HUMAlACM PEA 2 node 23 (SEQ ID NO:4)
HUMAlACM PEA 2 node 41 (SEQ ID NO:5)
HUMAlACM PEA 2 node 51 (SEQ ID NO:6)
HUMAl ACM_PEA_2_node 0 (SEQ ID NO:7)
HUMAl ACM_PEA_2_ node 1 (SEQ ID NO:8)
HUMAlACM PEA 2 node 10 (SEQ ID NO:9)
HUMAlACM PEA 2 node H(SEQ ID NO: 10)
HUMAlACM PEA 2 node 12 (SEQ ID NO: U)
HUMAlACM PEA 2 node 13 (SEQ ID NO:12)
HUMAlACM PEA 2 node 14 (SEQ ID NO: 13)
HUMAlACM PEA 2 node 15 (SEQ ID NO: 14)
HUMAlACM. PEA 2 node lό (SEQ ID NO:15)
HUMAlACM PEA 2 node_17 (SEQ ID NO:16)
HUMAlACM PEA 2 node 18 (SEQ ID NO: 17)
HUMAlACM PEA 2 node 19 (SEQ ID NO: 18)
HUMAlACM PEA 2 node 2 (SEQ ID NO: 19) HUMAl ACM_PEA 2 node_2 0 (SEQ ID NO:20)
HUMA1ACM_PEA 2 node_21 (SEQ ID NO:21)
HUMA1ACM_PEA 2 node_22 (SEQ ID NO:22)
HUMAl ACM PEA 2_node_2 6 (SEQ ID NO:23)
HUMA1ACM_PEA 2 node_27 (SEQ ID NO:24)
HUMAIACM-PEA 2 node_28 (SEQ ID NO:25)
HUMAl ACM_PEA 2 node_29 (SEQ ID NO:26)
HUMAlACM_PEA__2_node_30 (SEQ ID NO:27)
HUMAl ACM_PEA 2_node_31 (SEQ ID NO:28)
HUMAIACM-PEA _2_node_34 (SEQ ID NO:29)
HUMAlACM_PEA_2_node_35 (SEQ ID NO:30)
HUMAl ACMJPEA _2_node_36 (SEQ ID NO:31)
HUMA1ACMJPEA_2_ node_37 (SEQ ID NO:32)
HUMAIACMJPEA 2_node_38 (SEQ ID NO:33)
HUMAlACM_PEA_2_node_39 (SEQ ID NO:34)
HUMAlACM_PEA_2_node_40 (SEQ ID NO:35)
HUMAlACM JPEA_2_node_42 (SEQ ID NO:36)
HUMA1ACM_PEA 2 _node_43 (SEQ ID NO:37)
HUMA1ACM_PEA 2_node_44 (SEQ ID NO:38)
HUMAlACM_PEA_2_node_45 (SEQ ID NO:39)
HUMAl ACM-PEA _2_node_46 (SEQ ID NO:40)
HUMAlACM_PEA_2_node_47 (SEQ ID NO:41)
HUMAlACM_PEA_2__node_48 (SEQ ID NO:42)
HUMAl ACM-PEA _2_node_49 (SEQ ID NO:43)
HUMAlACM JPEA_2_node_5 (SEQ ID NO:44)
HUMAlACM J>EA_2_node_50 (SEQ ID NO:45)
HUMAl ACM-PEA _2_node_6 (SEQ ID NQ:46)
HUMAlACM J>EA_2_node_7 (SEQ ID NO:47)
HUMAl ACM_PEA_2_node_8 (SEQ ID NO:48)
HUMAlACM_PEA_2_node_9 (SEQ ID NO:49)
Table 9 - Proteins of interest
Protein Nome '
HUMAl ACM-PEA, 2_P36 (SEQ ID NO:51)
HUMAl ACM_PEA_2_P49 (SEQ ID NO:52)
HUMAl ACM_PEA._2_P59 (SEQ ID NO:53)
These sequences are variants of the known protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO:50) (SwissProt accession identifier AACTJHUMAN; known also according to the synonyms ACT), referred to herein as the previously known protein. The variant proteins according to the present invention are variants of a known diagnostic marker, called Alpha 1 antichymotrypsin (Enzymes) for Chronic lung diseases and HISTOMARKER a-1 antichymotrypsin Histiocytic marker. Protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO:50) is known or believed to have the following function(s): Although its physiological function is unclear, it can inhibit neutrophil cathepsin G and mast cell chymase, both of which can convert angiotensin I to the active angiotensin II. The sequence for protein Alpha- 1- antichymotrypsin precursor is given at the end of the application, as "Alpha- 1- antichymotrypsin precursor amino acid sequence". Known polymorphisms for this sequence are as shown in Table 10.
Table 10 - Amino acid mutations for Known Protein
Figure imgf000122_0001
Protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO:50) localization is believed to be Extracellular.
The following GO Annotation(s) apply to the previously known protein. The following annotation(s) were found: acute-phase response; inflammatory response; regulation of lipid metabolism, which are annotation(s) related to Biological Process; DNA binding; serine protease inhibitor; protein binding, which are annotation(s) related to Molecular Function; and extracellular; intracellular, which are annotation(s) related to
Cellular Component.
The GO assignment relies on information from one or more of the
SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from
<http://www.ncbi.nlm.nih.gov/projects/LocusLink/>. Alpha- 1 -antichymotrypsin is the major plasma inhibitor of neutrophil leukocyte elastase (NE) in the serum. Alpha- 1 -antichymotrypsin is a marker related to blood pressure regulation, and marker for myocardial necrosis. Therefore, cluster HUMAlACM can be used as a diagnostic marker for diseases such as chronic lung diseases, pulmunary embolysm and stroke.
Alpha- 1 -antichymotrypsin is an extracellular protein synthesized in the liver. Its concentration increases in the acute phase of inflammation or infection. In the serum, Alpha- 1 -antichymotrypsin can be found bound to prostate specific antigen (PSA). Therefore, cluster HUMAlACM can optionally be used as a diagnostic marker for prostate cancer.
Cluster HUMAlACM can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term "number" in the left hand column of the table and the numbers on the y-axis of the figure below refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histograms in Figure 6 and Table 11. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: skin malignancies.
Table 11 - Normal tissue distribution
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000124_0002
As noted above, cluster HUMAlACM features 3 transcript(s), which were listed in Table 7 above. These transcript(s) encode for protein(s) which are variant(s) of protein Alpha- 1-antichymotrypsin precursor (SEQ ID NO: 50). A description of each variant protein according to the present invention is now provided.
Variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMAl ACMJPE A_2_T7 (SEQ ID NO:3). An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows: Comparison report between HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) and Q96DW8 (SEQ ID NO:202):
LAn isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDS AAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90 % homologous to
SRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPDQD KMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFT SKADLSGITGARNLAVSQV corresponding to amino acids 1 - 129 of Q96DW8 (SEQ ID NO:202), which also corresponds to amino acids 229 - 357 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS AN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQ (SEQ ID NO: 180) OF HUMA1ACM_PEA_2JP36 (SEQ ID NO:51).
Comparison report between HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) and Q9UNU9 (SEQ ID NO:203): 1.An isolated chimeric polypeptide encoding for HUMA1ACM PEA 2 P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) corresponding to amino acids 1 - 16 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90 % homologous to
FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKK KWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPDQDKMEEVEA MLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGI
TGARNLAVSQV corresponding to amino acids 1 - 341 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order. 2.An isolated polypeptide encoding for a head of HUMAl ACMJPEA_2_P36 (SEQ
ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51). Comparison report between HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) and
AAA51559 (SEQ ID NO.204):
1.An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVV ELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISR DYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO: 181) corresponding to amino acids 47 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order. 2.An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P36 (SEQ
ID NO:51), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVV ELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISR DYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO: 181) IN HUMAL ACM_PEA_2_P36 (SEQ ID NO:51).
Comparison report between HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) and AACT_HUMAN (SEQ ID NO:205):
1.An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO: 51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWE MPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGN ASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDI
LLQLGIEEAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 357 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 357 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SL corresponding to amino acids 358 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans¬ membrane region..
Variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 13 - Amino acid mutations
Figure imgf000128_0001
Figure imgf000129_0001
Variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) is encoded by the following transcript(s): HUMAl ACMJ5E A_2_T7 (SEQ ID NO:3), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMAl ACM_PEA_2_T7 (SEQ ID NO:3) is shown in bold; this coding portion starts at position 84 and ends at position 1160. The transcript also has the following SNPs as listed in Table 14 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 14 - Nucleic acid SNPs
Figure imgf000129_0002
Figure imgf000130_0001
Variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMA1ACM_PEA_2_T21 (SEQ ID NO:1). An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMAl ACMJPE A_2_P49 (SEQ ID NO:52) and Q9UNU9 (SEQ ID NO:203):
1.An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPD
KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
Comparison report between HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) and Q8N177 (SEQ ID NO:206): 1.An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDBGMVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACMJPE A_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
Comparison report between HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) and AAA51559 (SEQ ID NO:204):
1.An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKER (SEQ ID NO:183) corresponding to amino acids 47 - 216 of HUMA1ACM_PEA__2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order. 2.An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P49 (SEQ
ID NO:52), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI
FFKER in HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
Comparison report between HUMAl ACMJPE A_2_P49 (SEQ ID NO:52) and AACT_HUMAN (SEQ ID NO:205):
LAn isolated chimeric polypeptide encoding for HUMAl ACM J>EA_2_P49 (SEQ ID NO: 52), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACM PEA 2JP49 (SEQ ID NO:52), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ER corresponding to amino acids 215 - 216 of HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans¬ membrane region- Variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 15, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 15 - Amino acid mutations
Figure imgf000134_0001
Variant protein HUMA1ACM_PEA_2JP49 (SEQ ID NO:52) is encoded by the following transcript(s): HUMAl ACMJPE A_2_T21 (SEQ ID NO: I)5 for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMAl ACMJPE A_2_T21 (SEQ ID NO:1) is shown in bold; this coding portion starts at position 84 and ends at position 731. The transcript also has the following SNPs as listed in Table 16 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 16 - Nucleic acid SNPs
Figure imgf000134_0002
Figure imgf000135_0001
Variant protein HUMA1ACMJPEA_2_P59 (SEQ ID NO:53) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMA1ACM_PEA_2_T27 (SEQ ID NO:2). An alignment is given to the known protein (Alpha- 1-antichymotrypsin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) and Q9UNU9 (SEQ ID NO:203):
1.An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO:182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), second amino acid sequence being at least 90 % homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDF AFSLYKQLVLKAPD KNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQ SSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLIND
YVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 214 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACMJPE A_2_P59 (SEQ ID NO:53).
3.An isolated polypeptide encoding for a tail of HUMAl ACMJPEA 2 P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) in HUMAl ACMJPE A_2_P59 (SEQ ID NO:53).
Comparison report between HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) and Q8N177 (SEQ ID NO:206): 1.An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P59 (SEQ
ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMAl ACMJPEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
Comparison report between HUMA1ACM_PEA_2_P59 (SEQ ID NO:53) and AAA51559 (SEQ ID NO:204):
LAn isolated chimeric polypeptide encoding for HUMA1ACMJPEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI
FFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKG LKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA KRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) IN HUMA1ACM_PEA_2_P59
(SEQ ID NO:53). Comparison report between HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) and
AACT_HUMAN (SEQ ID NO:205):
1.An isolated chimeric polypeptide encoding for HUMAl ACMJPEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTET SEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 214 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO.53), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans¬ membrane region..
Variant protein HUMAl ACM JPEA_2_P59 (SEQ ID NO:53) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 17, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMAl ACM JPEA_2_P59 (SEQ ID NO:53) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 17 - Amino acid mutations
Figure imgf000139_0001
157 A -> P No
Variant protein HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) is encoded by the following transcript(s). HUMAl ACM_PEA_2_T27 (SEQ ID NO:2), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) is shown in bold; this coding portion starts at position 84 and ends at position 782. The transcript also has the following SNPs as listed in Table 18 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMA1ACM_PEA_2_P59 (SEQ ID NO: 53) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 18 - Nucleic acid SNPs
Figure imgf000140_0001
Figure imgf000141_0001
As noted above, cluster HUMAlACM features 46 segment(s), which were listed in Table 8 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster HUMAl ACM_PEA_2_node_23 (SEQ ID NO:4) according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T27 (SEQ ID NO:2). Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts
Figure imgf000141_0002
Segment cluster HUMAl ACMJPE A_2_node_41 (SEQ ID NO:5) according to the present invention is supported by 13 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segment location on transcripts
Figure imgf000141_0003
Segment cluster HUMAl ACMJPE A_2_node_51 (SEQ ID NO:6) according to the present invention is supported by 203 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcπpt(s): HUMAl ACMJPEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts
Figure imgf000142_0001
According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
Segment cluster HUMAlACM_PEA_2_node_0 (SEQ ID NO:7) according to the present invention is supported by 199 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM JPEA 2JT27 (SEQ ID NO:2) and HUMA IACM J3E A_2_T7 (SEQ ID NO.3). Table 22 below describes the starting and ending position of this segment on each transcript.
Table 22 - Segment location on transcripts
Figure imgf000142_0002
Segment cluster HUMAlACM_PEA_2_node_l (SEQ ID NO:8) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACMJPE A_2_T27 (SEQ ID NO:2) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segment location on transcripts
Figure imgf000143_0001
Segment cluster HUMAl ACM_PEA_2_node_10 (SEQ ID NO:9) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and
HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location on transcripts
Figure imgf000143_0002
Segment cluster HUMAlACM_PEA_2_node_ll (SEQ ID NO:10) according to the present invention is supported by 244 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location on transcripts
Figure imgf000143_0003
Figure imgf000144_0003
Segment cluster HUMAlACM_PEA_2_node_12 (SEQ ID NO:11) according to the present invention is supported by 230 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segment location on transcripts
Figure imgf000144_0001
Segment cluster HUMAlACM_PEA_2_node_13 (SEQ ID NO:12) according to the present invention is supported by 209 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 27 below describes the starting and ending position of this segment on each transcript.
Table 27 - Segment location on transcripts
Figure imgf000144_0002
Segment cluster HUMAlACM_PEA_2_node_14 (SEQ ID NO: 13) according to the present invention is supported by 216 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 28 below describes the starting and ending position of this segment on each transcript.
Table 28 - Segment location on transcripts
Figure imgf000145_0001
Segment cluster HUMAlACM_PEA_2_node_15 (SEQ ID NO:14) according to the present invention is supported by 211 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACMJPEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 29 below describes the starting and ending position of this segment on each transcript.
Table 29 - Segment location on transcripts
Figure imgf000145_0002
Segment cluster HUMAlACM_PEA_2_node_16 (SEQ ID NO: 15) according to the present invention is supported by 222 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - Segment location on transcripts
Figure imgf000146_0001
Segment cluster HUMAlACMJPEA_2_node_17 (SEQ ID NO: 16) according to the present invention is supported by 224 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segment location on transcripts
Figure imgf000146_0002
Segment cluster HUMAlACM_PEA_2_node_18 (SEQ ID NO: 17) according to the present invention can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 32 below describes the starting and ending position of this segment on each transcript. Table 32 - Segment location on transcripts
Figure imgf000146_0003
Figure imgf000147_0001
Segment cluster HUMAlACM_PEA_2_node_19 (SEQ ID NO: 18) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment location on transcripts
Figure imgf000147_0002
Segment cluster HUMAlACM_PEA_2_node_2 (SEQ ID NO: 19) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACMJPEA_2_T27 (SEQ ID NO:2) and
HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segment location on transcripts
Figure imgf000147_0003
Segment cluster HUMAlACM_PEA_2_node_20 (SEQ ID NO:20) according to the present invention is supported by 216 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripts): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM JPEA_2_T27 (SEQ ID NO.2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segment location on transcripts
Figure imgf000148_0001
Segment cluster HUMAlACM_PEA_2_node_21 (SEQ ID NO:21) according to the present invention is supported by 218 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segment location on transcripts
Figure imgf000148_0002
Segment cluster HUMAlACM_PEA_2_node_22 (SEQ ID NO:22) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACMJPEA_2_T7 (SEQ ID NO:3). Table 37 below describes the starting and ending position of this segment on each transcript. Table 37 - Segment location on transcripts
Figure imgf000148_0003
Figure imgf000149_0001
Segment cluster HUMAlACM_PEA_2_node_26 (SEQ ID NO:23) according to the present invention is supported by 231 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segment location on transcripts
Figure imgf000149_0002
Segment cluster HUMAlACM_PEA_2_node_27 (SEQ ID NO:24) according to the present invention is supported by 238 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 39 below describes the starting and ending position of this segment on each transcript.
Table 39 - Segment location on transcripts
Figure imgf000149_0003
Segment cluster HUMAlACM_PEA_2_node_28 (SEQ ID NO:25) according to the present invention is supported by 248 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 40 below describes the starting and ending position of this segment on each transcript.
Table 40 - Segment location on transcripts
Figure imgf000149_0004
Segment cluster HUMAlACM_PEA_2_node_29 (SEQ ID NO:26) according to the present invention is supported by 257 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 41 below describes the starting and ending position of this segment on each transcript.
Table 41 - Segment location on transcripts
Figure imgf000150_0001
Segment cluster HUMAlACM_PEA_2_node_30 (SEQ ID NO:27) according to the present invention can be found in the following transcript(s): HUMA1ACMJPEA_2_T7 (SEQ ID NO:3). Table 42 below describes the starting and ending position of this segment on each transcript.
Table 42 - Segment location on transcripts
Figure imgf000150_0002
Segment cluster HUMAlACM_PEA_2_node_31 (SEQ ID NO:28) according to the present invention is supported by 256 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 43 below describes the starting and ending position of this segment on each transcript.
Table 43 - Segment location on transcripts
Figure imgf000150_0003
Segment cluster HUMAlACM_PEA_2_node_34 (SEQ ID NO:29) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 44 below describes the starting and ending position of this segment on each transcript.
Table 44 - Segment location on transcripts
Figure imgf000151_0001
Segment cluster HUMAlACM_PEA_2_node_35 (SEQ ID NO:30) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMAl ACMJPE A_2_T7 (SEQ ID NO:3). Table 45 below describes the starting and ending position of this segment on each transcript.
Table 45 - Segment location on transcripts
Figure imgf000151_0002
Segment cluster HUMAlACMJPEA_2_node_36 (SEQ ID NO:31) according to the present invention is supported by 271 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 46 below describes the starting and ending position of this segment on each transcript.
Table 46 - Segment location on transcripts
Figure imgf000151_0003
Segment cluster HUMAlACM_PEA_2_node_37 (SEQ ID NO:32) according to the present invention is supported by 275 libraries The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 47 below describes the starting and ending position of this segment on each transcript.
Table 47 - Segment location on transcripts
Figure imgf000152_0001
Segment cluster HUMAlACM_PEA_2_node_38 (SEQ ID NO.33) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 48 below describes the starting and ending position of this segment on each transcript. Table 48 - Segment location on transcripts
Figure imgf000152_0002
Segment cluster HUMAlACM_PEA_2_node_39 (SEQ ID NO:34) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID
NO:3). Table 49 below describes the starting and ending position of this segment on each transcript.
Table 49 - Segment location on transcripts
Figure imgf000152_0003
Figure imgf000153_0002
Segment cluster HUMAlACM_PEA_2_node_40 (SEQ ID NO:35) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO.3). Table 50 below describes the starting and ending position of this segment on each transcript.
Table 50 - Segment location on transcripts
Figure imgf000153_0003
Segment cluster HUMAlACM_PEA_2_node_42 (SEQ ID NO:36) according to the present invention can be found in the following transcript(s): HUMAlACM J>EA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 51 below describes the starting and ending position of this segment on each transcript.
Table 51 - Segment location on transcripts
Figure imgf000153_0001
Segment cluster HUMAlACM_PEA_2_node_43 (SEQ ID NO:37) according to the present invention is supported by 274 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMAlACM JPEA_2_T7 (SEQ ID NO:3). Table 52 below describes the starting and ending position of this segment on each transcript. Table 52 - Segment location on transcripts
Figure imgf000154_0001
Segment cluster HUMAlACM_PEA_2_node_44 (SEQ ID NO:38) according to the present invention is supported by 271 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 53 below describes the starting and ending position of this segment on each transcript.
Table 53 - Segment location on transcripts
Figure imgf000154_0002
Segment cluster HUMAlACM_PEA_2_node_45 (SEQ ID NO:39) according to the present invention is supported by 255 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMAl ACM_PEA_2_T7 (SEQ ID NO:3). Table 54 below describes the starting and ending position of this segment on each transcript.
Table 54 - Segment location on transcripts
Figure imgf000154_0003
Segment cluster HUMAlACM_PEA_2_node_46 (SEQ ID NO:40) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 55 below describes the starting and ending position of this segment on each transcript.
Table 55 - Segment location on transcripts
Figure imgf000155_0001
Segment cluster HUMAlACM_PEA_2_node_47 (SEQ ID NO:41) according to the present invention is supported by 233 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripts): HUMAl ACMJPE A_2_T21 (SEQ ID NO:1) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 56 below describes the starting and ending position of this segment on each transcript.
Table 56 - Segment location on transcripts
Figure imgf000155_0002
Segment cluster HUMAlACM_PEA_2_node_48 (SEQ ID NO:42) according to the present invention is supported by 222 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 57 below describes the starting and ending position of this segment on each transcript.
Table 57 - Segment location on transcripts
Figure imgf000155_0003
Segment cluster HUMAlACM_PEA_2_node_49 (SEQ ID NO:43) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 58 below describes the starting and ending position of this segment on each transcript.
Table 58 - Segment location on transcripts
Figure imgf000156_0001
Segment cluster HUMAl ACMJPE A_2_node_5 (SEQ ID NO:44) according to the present invention is supported by 249 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 59 below describes the starting and ending position of this segment on each transcript. Table 59 - Segment location on transcripts
Figure imgf000156_0002
Segment cluster HUMAl ACMJPE A_2_node_50 (SEQ ID NO:45) according to the present invention can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1) and HUMAl ACM_PEA_2_T7 (SEQ ID
NO:3). Table 60 below describes the starting and ending position of this segment on each transcript.
Table 60 - Segment location on transcripts
Figure imgf000156_0003
Figure imgf000157_0002
Segment cluster HUMAl ACM_PEA_2_node_6 (SEQ ID NO:46) according to the present invention can be found in the following transcript(s): HUMAl ACMJPE A_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and
HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 61 below describes the starting and ending position of this segment on each transcript.
Table 61 - Segment location on transcripts
Figure imgf000157_0001
Segment cluster HUMAl ACM_PEA_2_node_7 (SEQ ID NO:47) according to the present invention is supported by 269 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA IACM JPE A_2_T7 (SEQ ID NO:3). Table 62 below describes the starting and ending position of this segment on each transcript.
Table 62 - Segment location on transcripts
Figure imgf000157_0003
Segment cluster HUMAl ACM_PEA_2_node_8 (SEQ ID NO:48) according to the present invention is supported by 267 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMA1ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 63 below describes the starting and ending position of this segment on each transcript.
Table 63 - Segment location on transcripts
Figure imgf000158_0001
Segment cluster HUMAl ACM_PEA_2_node_9 (SEQ ID NO:49) according to the present invention is supported by 260 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMAl ACM_PEA_2_T21 (SEQ ID NO:1), HUMA1ACM_PEA_2_T27 (SEQ ID NO:2) and HUMA1ACM_PEA_2_T7 (SEQ ID NO:3). Table 64 below describes the starting and ending position of this segment on each transcript.
Table 64 - Segment location on transcripts
Figure imgf000158_0002
Variant protein alignment to the previously known protein:
Sequence name: /tmp/zO3ClXLvio/046Tkajl5w:Q96DW8 (SEQ ID NO:202)
Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) x Q96DW8 (SEQ ID NO:202) ..
Alignment segment 1/1:
Quality: 1255.00 Escore: 0 Matching length: 129 Total length: 129 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
229 SRFYLSKKKWVMVPMMSLHHLTI PYFRDEELSCTVVELKYTGNASALFIL 278
I I l I I I I I I I I I I I I I I I I I I I I I I I 1 I I i I 1 I I I I I I I I I I I I I I l I I I
1 SRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFIL 50 . . . . .
279 PDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDIL 328
1 I I I 1 I Il I 1 I I I Il I Il 1 I I I Il I I I I Il I I I I Il Il I Il Il I I Il Il I 51 PDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDIL 100 329 LQLGIEEAFTSKADLSGITGARNLAVSQV 357
I I Il I I I I I I I I Il Il Il I Il I Il I I I I I 101 LQLGIEEAFTSKADLSGITGARNLAVSQV 129
Sequence name: /tmp/zO3ClXLvio/046Tkajl5w:Q9UNU9 (SEQ ID NO:203)
Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) x Q9UNU9 (SEQ ID NO:203)..
Alignment segment 1/1:
Quality: 3313.00 Escore: 0
Matching length: 341 Total length: 341 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
17 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 66
I I I I I I I I l I I l I I I l I I 1 1 I I l I I I I I I l 1 1 I I l I I l I l I l I I l I I I I I
1 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 50 67 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 116
II I I I I Il N I I I I I M I 1111 I 11 I I I I 111 I Il I 11 I Il I I I I Il I I I
51 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 100 117 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 166 I I I I I I I M I I M I I I I I I I I I I I I I I M I I I I M I I I I I M I I M I I M
101 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 150
167 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAK 216
I I I I I Il I I I I I Il I I Ii I I I Il Il Il I Il I Il I I Il I I I I Il I Il I M I 151 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAK 200 217 WEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVEL 266
1111111 ! 111111111111!1111111I MI I II I II II II 201 WEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVEL 250 . . . . .
267 KYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKF 316
1111111111111 ! !1111111!1 IMIMIIIIIMIIIIM I MM M 251 KYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKF 300 317 SISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQV 357 1!111!!1!11111111111111!1111111!IMMIMI
301 SISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQV 341
Sequence name: /tmp/zO3ClXLvio/046Tkajl5w:AAA51559
Sequence documentation:
Alignment of: HUMAl ACM_PEA_2_P36 (SEQ ID NO:51) x AAA51559 (SEQ ID NO:204)..
Alignment segment 1/1:
Quality: 456.00 Escore: 0
Matching length: 46 Total length: 46 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment: . . . .
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD 46
MII MMMMMIIIMMMMMIMMMMMMM I IM
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD 46
Sequence name: /tmp/zO3ClXLvio/046Tkajl5w:AACT_HUMAN Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P36 (SEQ ID NO:51) x AACTJHUMAN (SEQ ID NO-.205)..
Alignment segment 1/1: Quality: 3459.00 Escore: 0
Matching length: 357 Total length: 357
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50 I I I M I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I i I I 1
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 11 ! ! 11 !11111111111111111111!!1 !111IMII II MI II II II 51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100
101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
I I I I! Il 11 I I I 1 I I 1 I I 1 I Il I I I I 1 Il Il I Il 1 Il I Il Il Il Il I Il I 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150 . . . . .
151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
M I M I M I I I I M I I I I 1 M I I I I I I M M M I I 1 M I I I M M M M I
151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200 201 SQTMMVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLT 250
Il Il Il Il I Il I III 1 I I I Il Il Il Il Il I Il I I I I I M I Il I I Il I Il I 201 SQTMMVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLT 250
251 IPYFRDEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWR 300 M Il Il Il I I Il I 1 I 1 I I I I I Il I I I I Il I Il I I Il I Il I Il I I Il Il I I
251 IPYFRDEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWR 300 301 DSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGAR 350
I I M Il Il I I M I 11 Il I I I I M M I I Il M M I M I Il I I Il I M Il I I 301 DSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGAR 350 351 NLAVSQV 357
II I Il I I
351 NLAVSQV 357
Sequence name: /tmp/rbFeImuTHa/8ucpPZb4R0:Q9UNU9
Sequence documentation: Alignment of: HUMAl ACMJPE A_2_P49 (SEQ ID NO:52) x Q9UNU9 (SEQ ID NO:203)..
Alignment segment 1/1: Quality: 1902.00 Escore: 0
Matching length: 198 Total length: 198 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
17 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 66
1 I Il I 1 I I I I I I I I I I I I I I I I I I I I Il I I I Il I I i I I I I I I I Il I I I I I 1 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 50 . . . . .
67 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 116
I I I I I I Il Il I I I I I I I I I I 1 I I I I Il I I I I I Il Il I Il I Il I Il 1 I I I I 51 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 100 117 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 166
I I Il I I I I I Il I I 1 I I I I I I Il I I I I I I I I I I Il I I I I Il I Il Il Il Il I 101 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 150
167 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK 214 I I I I I M I I I M I I I M I Il I I I I I I Il I I I I I Il I Il 1 I I I I I I I Il
151 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK 198
Sequence name: /tmp/rbFeImuTHa/8ucpPZb4R0:Q8N177 Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) x Q8N177 (SEQ ID NO:206) .. Alignment segment 1 / 1 :
Quality: 2048.00 Escore: 0
Matching length: 214 Total length: 214
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment: 1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
M M M I I I I I M I M I M I I I I I I M I I I I M I I I M I I I I I I M I MI
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50 51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 Il I I I Il Il I I I I M I M I M I I I I I M I I I I I I I I I I M M Il I I Il I I
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
M M I M M M I M M M I I M I M I I l I M M I I I l I I M M M M Ml 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150 151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
I I I I I I I I I I 1 I I I I i i I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I 151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
201 SQTMMVLVNYI FFK 214
I I I I I I 1 I I I I I I I 201 SQTMMVLVNYIFFK 214
Sequence name: /tmp/rbFeImuTHa/8ucpPZb4R0: AAA51559 Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) x AAA51559 (SEQ ID NO:204)..
Alignment segment 1/1:
Quality: 456.00 Escore: 0 Matching length: 46 Total length: 46
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD 46
I I I 1 I I I I I I I I I I i i I I I I i I I I I I I I I I I I I I I I I I I I I I I I I I
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD 46
Sequence name: /tmp/rbFeImuTHa/8ucpPZb4R0:AACT_HUMAN
Sequence documentation: Alignment of: HUMA1ACM_PEA_2_P49 (SEQ ID NO:52) x AACT_HUMAN (SEQ ID NO:205)..
Alignment segment 1/1 : Quality: 2048.00 Escore: 0
Matching length: 214 Total length: 214
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment: 1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
I 11 I I I I I I 1 I I I I I I I 1 I I I I I I 1 I 11 I 1 I I 1 I I I I I 1 I I I I 1 I I I I I I
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 I I I I I I I I I M I I I I I I I I I I I M I I I I I I I I I I I I I I I M 1 I I I I I I I I
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100
101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
I I I I I II I I Il I I 1 I I I I 1 I I Il I I I I I I I I I I I Il I Il I I I I I I I I II I 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150 151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200 MII MMIII II MIIiI I I II M IIII IIIIIMI
151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
201 SQTMMVLVNYIFFK 214
I M I I I I Il Il I M
201 SQTMMVLVNYIFFK 214
Sequence name: /tmp/NkFvfSdFNO/YvqLBjirnL:Q9UNU9 Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P59 (SEQ ID NO:53) x Q9UNU9 (SEQ ID NO:203)..
Alignment segment 1/1:
Quality: 1902.00 Escore: 0 Matching length: 198 Total length: 198
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
17 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 66
Il M I I M Il Il M I M I I M I M M M M I I I I Il M M I I I Ml M I I
1 FCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDFAFSLYKQLVL 50 . . . . .
67 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 116
I I I M I M Il Il I M M M I I Il I Il I M I M I Il I Il I M I M I M M I
51 KAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIH 100 117 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 166 ' I I i I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 1 I I I I I I I I I I I I
101 QSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAF 150 167 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK 214
I I 1 1 I I I I I I I I I I I I I I l I I I I I I I I I ! I I I I I I I I I I I I I I I I I I I
151 ATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYI FFK 198
Sequence name: /tmp/NkFvfSdFNO/YvqLBjirnL:Q8N177 Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P59 (SEQ ID NO:53) x Q8N177 (SEQ ID NO:206).. Alignment segment 1/1:
Quality: 2048.00 Escore: 0
Matching length: 214 Total length: 214
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00
Gaps: 0
Alignment: 1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
I I I I I I Il I Il I I I Il I Il I I I I I I I I I I Il Il I Il I I I I I I I I I I I I I I 1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 111111111111111111 ! 11 ! 11 I M I I I I I I M I M I I I I I I I I M I I
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100
101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
I I Il Il Il I I Il I I Il Il Il I I Il Il I I Il I Il 1 Il I Il I I I I Il Il Il I 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
I I I I Il I Il I I I I I I I Il I Il I Il Il I I I Il Il I I Il I Il I I Il I I I I I I 151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
201 SQTMMVLVNYIFFK 214
M I I M I M Il M I
201 SQTMMVLVNYIFFK 214
Sequence name: /tmp/NkFvfSdFNO/YvqLBjirnL:AAA51559 Sequence documentation:
Alignment of: HUMAl ACM_PEA_2_P59 (SEQ ID NO:53) x AAA51559 (SEQ ID NO:204) ..
Alignment segment 1/1 :
Quality: 456.00 Escore: 0
Matching length: 46 Total length: 46 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment: . . . .
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD 46
1 I I I I I I I I I I I I I I i I I l I I I I I I I I I I I I I l I I l I I I 1 1 1 I I 1 I
1 MERMLPLLALGLLAAGFCPAVLCHPNS PLDEENLTQENQDRGTHVD 46
Sequence name: /tmp/NkFvfSdFNOArvqLBjirnL:AACT_HUMAN Sequence documentation:
Alignment of: HUMA1ACM_PEA_2_P59 (SEQ ID NO:53) x AACT_HUMAN (SEQ ID NO:205) ..
Alignment segment 1/1:
Quality: 2048.00 Escore: 0 Matching length: 214 Total length: 214
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50
I I I I I l I I I I I I l I I I I l I I I I I I I l I I I I I l I I I l I I I l I I I l I I l I I l
1 MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLA 50 . . . . .
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100
I I I l I I I I I l I I I I l 1 1 I I l I I I I 1 1 I I l I I I 1 I I I 1 1 1 1 I I l I I I I I l I
51 SANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL 100 101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150
II I I Il I Il I Il I I I Il I I Il I Il I Il I I Il I I I Il Il I Il I 1111 I I 11
101 KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLL 150 151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
I I I 1 1 I I I I I I I 1 1 I I I I I i I I I I 1 1 I I I I I I 1 1 1 1 1 1 I I I I I I I I I I i I
151 DRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLD 200
201 SQTMMVLVNYIFFK 214
I I I I I I I I I I I I I I 201 SQTMMVLVNYIFFK 214
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMA lACMseg;26-3 IWT in normal and cancerous ovary tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by or according to seg26-31WT, HUMAl ACMseg26-3 IWT amplicon and primers HUMAlACMseg26-31WTF and HUMA lACMseg26-3 IWTR was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BC019323, (SEQ ID NO:194); amplicon - PBGD-amplicon (SEQ ID NO: 158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO: 195); amplicon - HPRTl- amplicon, (SEQ ID NO: 161)), SDHA (GenBank Accession No. NM_004168, (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO: 155)) and GAPDH (GenBank Accession No. BC026907 (SEQ ID NO:196); GAPDH amplicon (SEQ ID NO: 164)) were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 45, 45, 48, 71 Table 2, above, "Tissue samples in ovarian cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples.
Figure 7 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts in cancerous ovary samples relative to the normal samples.
As is evident from Figure 7, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 45, 45, 48, 71 Table 2, "Tissue samples in ovarian cancer testing panel"). Notably down regulation of at least 5 fold was found in 21 out of 40 adenocarcinoma samples. Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymo trypsin), member 3 (AACT_HUMAN) transcripts detectable by the above amplicon in ovary cancer samples versus the normal tissue samples was determined by T test as 1.28E-02.
Threshold of 5 fold down regulation was found to differentiate between cancer and normal samples with P value of 6.52E-02 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg26- 3 IWTF forward primer; and HUMAl ACMseg26-3 IWTR reverse primers.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMAl ACMseg26-3 IWT. Primers:
Forward primer HUMAl ACMseg26-3 IWTF (SEQ ID NO:54) : TGGAGCTGAAGTACACAGGCAA
Reverse primer HUMA lACMseg26-3 IWTR (SEQ ID NO:55) : CTTCAGGGTCTCTGGGAGCA
Amplicon HUMA lACMseg26-3 IWT (SEQ ID NO:56) : TGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCATCCTCCCTGATCA AGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMAlACMseg23 (SEQ ID NO: 59) in normal and cancerous ovary tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by or according to seg23, HUMAl ACMseg23 amplicon (SEQ ID NO:59) and primers HUMAlACMseg23F (SEQ ID NO:57) and HUMAlACMseg23R (SEQ ID NO:58) was measured by real time PCR. In parallel the expression of four housekeeping genes - PBGD (GenBank Accession No. BCO 19323 (SEQ ID NO: 194); amplicon - PBGD- amplicon (SEQ ID NO.158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO:195); amplicon - HPRTl -amplicon, (SEQ ID NO:161)), SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon, (SEQ ID NO: 155)) and GAPDH (GenBank Accession No. BC026907 (SEQ ID NO: 196); GAPDH amplicon (SEQ ID NO: 164)) were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 45, 45, 48, 71 Table 2, above, "Tissue samples in ovarian cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples. Figure 8 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous ovary samples relative to the normal samples.
As is evident from Figure 8, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 45, 45, 48, 71 Table 2, "Tissue samples in ovarian cancer testing panel"). Notably down regulation of at least 5 fold was found in 24 out of 40 adenocarcinoma samples. Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by the above amplicon in ovary cancer samples versus the normal tissue samples was determined by T test as 2.18E-02.
Threshold of 5 fold down regulation was found to differentiate between cancer and normal samples with P value of 3.57E-02 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg23F forward primer (SEQ ID NO:57); and HUMAl ACMseg23R reverse primers (SEQ ID NO:58).
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMAl ACMseg23 (SEQ ID NO:59). Forward primer HUMAlACMseg23F (SEQ ID NO:57) :
ATCTCAGGGCCATTTCTGTCC
Reverse primer HUMAl ACMseg23R (SEQ ID NO:58) : GGCCTTGATGCTTATGCTGC
Amplicon HUMAlACMseg23 (SEQ ID NO:59) : ATCTCAGGGCCATTTCTGTCCTGACTCAACAATGGTGTTATTAGGCAAACCAC TGTAGAGGAGAGTGCCCACAGCATGGGGGCAGCATAAGCATCAAGGCC
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichvmotrvpsin), member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMAl ACMseg26-3 IWT (SEQ ID NO:56) in normal and cancerous lung tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by or according to seg26-31WT, HUMAl ACMseg26-31 amplicon (SEQ ID NO:56) and primers HUMAlACMseg26-31F (SEQ ID NO:54) and HUMAlACMseg26-31R (SEQ ID NO:55) was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BCO 19323 (SEQ ID NO: 194); amplicon - PBGD-amplicon (SEQ ID NO: 158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO:195); amplicon - HPRTl -amplicon, (SEQ ID NO:161)), Ubiquitin (GenBank Accession No. BC000449 (SEQ ID NO: 199); amplicon - Ubiquitin- amplicon (SEQ ID NO:173)) and SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO:155)) were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 47-50, 90-93, 96-99, Table 4, above, "Tissue samples in lung cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples. Figure 9 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous lung samples relative to the normal samples.
As is evident from Figure 9, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 47-50, 90-93, 96-99 Table 4, "Tissue samples in lung cancer testing panel"). Notably down regulation of at least 5 fold was found in 7 out of 8 small cell carcinoma samples. Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts detectable by the above amplicon in lung small cell cancer samples versus the normal tissue samples was determined by T test as 4.28E-03.
Threshold of 5 fold down regulation was found to differentiate between cancer and normal samples with P value of 1.03E-04 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg26- 3 IWTF forward primer (SEQ ID NO:54); and HUMAl ACMseg26-3 IWTR reverse primers (SEQ ID NO:55). The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMA lACMseg26-3 IWT (SEQ ID NO:56). Primers:
Forward primer HUMA lACMseg26-3 IWTF (SEQ ID NO:54) : TGGAGCTGAAGTACACAGGCAA
Reverse primer HUMA lACMseg26-3 IWTR (SEQ ID NO:55) : CTTCAGGGTCTCTGGGAGCA Amplicon HUMA lACMseg26-3 IWT (SEQ ID NO:56) :
TGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCATCCTCCCTGATCA AGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMAlACMseg23 in normal and cancerous lung tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by or according to seg23, HUMAlACMseg23 amplicon and primers HUMAlACMseg23F and HUMAlACMseg23R was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BCO 19323 (SEQ ID NO: 194); amplicon - PBGD-amplicon (SEQ ID NO: 158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO:195); amplicon - HPRTl -amplicon, (SEQ ID NO:161)), Ubiquitin (GenBank Accession No. BC000449 (SEQ ID NO: 199); amplicon - Ubiquitin- amplicon (SEQ ID NO: 173)) and SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO: 155)) were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 47-50, 90-93, 96-99, Table 4, above, "Tissue samples in lung cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples. Figure 10 is a histogram showing down regulation of the above-indicated serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts in cancerous lung samples relative to the normal samples. As is evident from Figure 10, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts detectable by the above amplicon was lower in several cancer samples, most of them small-cell carcinoma samples, than in the non-cancerous samples (Sample Nos. 47-50, 90-93, 96-99 Table 4, "Tissue samples in lung cancer testing panel"). Notably down regulation of at least 5 fold was found in 4 out of 8 small cell carcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
Threshold of 5 fold down regulation was found to differentiate between small-cell carcinoma and normal samples with P value of 1.44E-02 as checked by exact fisher test.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg23F forward primer; and HUMAl ACMseg23R reverse primers. The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMAlACMseg23.
Forward primer HUMAl ACMseg23F (SEQ ID NO:57) :
ATCTCAGGGCCATTTCTGTCC
Reverse primer HUMAlACMseg23R (SEQ ID NO:58) : GGCCTTGATGCTTATβCTGC
Amplicon HUMAl ACMseg23 (SEQ ID NO:59) : ATCTCAGGGCCATTTCTGTCCTGACTCAACAATGGTGTTATTAGGCAAACCAC TGTAGAGGAGAGTGCCCACAGCATGGGGGCAGCATAAGCATCAAGGCC
Expression of serine (or cysteine) proteinase inhibitor, clade A ("alpha- 1 antiproteinase, antichvmotrvpsin). member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMAl ACMseg26-31 (SEQ ID NO:56) in normal and cancerous colon tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts detectable by or according to seg26-31, HUMAlACMseg26-31 amplicon (SEQ ID NO:56) and primers HUMAl ACMseg26-3 IF (SEQ ID NO:54) and HUMAlACMseg26-31R (SEQ ID NO:55) was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BC019323 (SEQ ID NO:194); amplicon - PBGD-amplicon (SEQ ID NO:158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO: 195); amplicon - HPRTl -amplicon, (SEQ ID NO: 161)), G6PD (GenBank Accession No. NM_000402 (SEQ ID NO: 197); G6PD amplicon (SEQ ID NO: 167)), RPS27A (GenBank Accession No. NM_002954 (SEQ ID NO: 198); RPS27A amplicon (SEQ ID NO: 170)), were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 41-45, 49-52, 62-67, 69-71, Table 3, above, "Tissue samples in colon cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples. Figure 11 is a histogram showing down regulation of the above-indicated serine
(or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN) transcripts in cancerous colon samples relative to the normal samples.
As is evident from Figure 11, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 41-45, 49-52, 62-67, 69-71, Table 3, "Tissue samples in colon cancer testing panel"). Notably down regulation of at least 5 fold was found in 18 out of 36 adenocarcinoma samples. Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by the above amplicon in colon cancer samples versus the normal tissue samples was determined by T test as 2.24E-03.
Threshold of 5 fold down regulation was found to differentiate between cancer and normal samples with P value of 1.99E-03 as checked by exact fisher test. The above values demonstrate statistical significance of the results.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg26-
3 IWTF forward primer (SEQ ID NO:54); and HUMA lACMseg26-3 IWTF reverse primer (SEQ ID NO:55).
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMA lACMseg26-3 IWT (SEQ ID NO:56). Forward primer HUMA lACMseg26-3 IWTF (SEQ ID NO:54) :
TGGAGCTGAAGTACACAGGCAA
Reverse primer HUMA lACMseg26-3 IWTR (SEQ ID NO:55) : CTTCAGGGTCTCTGGGAGCA
Amplicon HUMA lACMseg26-3 IWT (SEQ ID NO:56) : TGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCATCCTCCCTGATCA AGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichvmotrvpsin), member 3 (AACT HUMAN) HUMAlACM transcripts which are detectable by amplicons as depicted in sequence name HUMAlACMseg23 (SEQ ID NO:59) in normal and cancerous colon tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by or according to seg23, HUMAlACMseg23 amplicon (SEQ ID NO:59) and primers HUMAlACMseg23F (SEQ ID NO:57) and HUMAl ACMseg23R (SEQ ID NO:58) was measured by real time PCR. hi parallel the expression of four housekeeping genes - PBGD (GenBank Accession No. BCO 19323 (SEQ ID NO: 194); amplicon - PBGD- amplicon (SEQ ID NO: 158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO:195); amplicon - HPRTl -amplicon, (SEQ ID NO:161)), G6PD (GenBank Accession No. NM_000402 (SEQ ID NO: 197); G6PD amplicon (SEQ ID NO: 167)), RPS27A (GenBank Accession No. NM_002954 (SEQ ID NO: 198); RPS27A amplicon (SEQ ID NO: 170)), were measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 41-45, 49-52, 62-67, 69-71, Table 3, above, "Tissue samples in colon cancer testing panel"). Then the reciprocal of this ratio was calculated, to obtain a value of fold down-regulation for each sample relative to median of the normal PM samples. Figure 12 is a histogram showing down regulation of the above-indicated serine
(or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts in cancerous colon samples relative to the normal samples.
As is evident from Figure 12, the expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by the above amplicon in cancer samples was significantly lower than in the non-cancerous samples (Sample Nos. 41-45, 49-52, 62-67, 69-71, Table 3 , "Tissue samples in colon cancer testing panel"). Notably down regulation of at least 5 fold was found in 22 out of 36 adenocarcinoma samples. Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by the above amplicon in colon cancer samples versus the normal tissue samples was determined by T test as 1.39E-04.
Threshold of 5 fold down regulation was found to differentiate between cancer and normal samples with P value of 2.72E-03 as checked by exact fisher test. The above values demonstrate statistical significance of the results. Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg23F forward primer (SEQ ID NO:57); and HUMAlACMseg23R reverse primers (SEQ ID NO:58).
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMAl ACMseg23 (SEQ ID NO:59).
Forward primer HUMAlACMseg23F (SEQ ID NO:57) :
ATCTCAGGGCCATTTCTGTCC
Reverse primer HUMAlACMseg23R (SEQ ID NO:58) : GGCCTTGATGCTTATGCTGC Amplicon HUMAlACMseg23 (SEQ ID NO:59) :
ATCTCAGGGCCATTTCTGTCCTGACTCAACAATGGTGTTATTAGGCAAACCAC TGTAGAGGAGAGTGCCCACAGCATGGGGGCAGCATAAGCATCAAGGCC
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT HUMAN*) HUMAlACM transcripts which are detectable by amplicons as depicted in sequences names HUMAlACMseg23 (SEQ ID NO:59) and HUMA !ACMseg26-3 IWT (SEO ID NO:56) in different normal tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACTJHUMAN) transcripts detectable by or according to HUMAl ACMseg23 (SEQ ID NO:59) and HUMA lACMseg26-3 IWT amplicons (SEQ ID NO:56) and primers: HUMAlACMseg23F (SEQ ID NO:57), HUMAl ACMseg23R (SEQ ID NO:58), HUMAl ACMseg26-3 IWTF (SEQ ID NO:54) and HUMAl ACMseg26-3 IWTR (SEQ ID NO:55) was measured by real time PCR. In parallel the expression of four housekeeping genes -RPL 19 (GenBank Accession No. NM_000981 (SEQ ID NO:200); RPL19 amplicon SEQ ID NO: 176), TATA box (GenBank Accession No. NM_003194 SEO ID NO:201: TATA amplicon SEO ID NO: 179). Ubiquitin (GenBank Accession No. BC000449 (SEQ ID NO: 199); amplicon - Ubiquitin-amplicon (SEQ ID NO: 173)) and SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO:155)) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the colon samples (Sample Nos. 1-3 Table 6 above, "Tissue samples in normal panel"), to obtain a value of relative expression of each sample relative to median of the colon samples.
Figure 13 A shows the expression of HUMAlACM transcripts which are detectable by amplicons as depicted in sequence names HUMAlACMseg23 (SEQ ID
NO:59) and Figure 13B shows the expression of HUMAlACM transcripts which are detectable by amplicons as depicted in sequence names HUMAl ACMseg26-3 IWT (SEQ
ID NO:56) in different normal tissues.
Forward primer HUMAlACMseg23F (SEQ ID NO:57) : ATCTCAGGGCCATTTCTGTCC
Reverse primer HUMAl ACMseg23R (SEQ ID NO:58) : GGCCTTGATGCTTATGCTGC
Amplicon HUMAlACMseg23 (SEQ ID NO:59) :
ATCTCAGGGCCATTTCTGTCCTGACTCAACAATGGTGTTATTAGGCAAACCAC TGTAGAGGAGAGTGCCCACAGCATGGGGGCAGCATAAGCATCAAGGCC
Forward primer HUMA lACMseg26-3 IWTF (SEQ ID NO:54) : TGGAGCTGAAGTACACAGGCAA
Reverse primer HUMAlACMseg26-31WTR (SEQ ID NO:55) : CTTCAGGGTCTCTGGGAGCA
Amplicon HUMAl ACMseg26-3 IWT (SEQ ID NO:56) TGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCATCCTCCCTGATCA AGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichvmotrvpsin), member 3 (AACT HUMAN") HUMAlACM transcripts which are detectable by amplicons as depicted in sequence names HUMAlACMseg23 (SEO ID NO:59) and HUMA lACMseg26-3 IWT (SEO ID NO:56) in normal and cancerous breast tissues.
Expression of serine (or cysteine) proteinase inhibitor, clade A (alpha- 1 antiproteinase, antichymotrypsin), member 3 (AACT_HUMAN) transcripts detectable by or according to seg23 and seg26-31, HUMAl ACMseg23 (SEQ ID NO:59) and HUMAl ACMseg26-3 IWT amplicons (SEQ ID NO:56) and primers HUMAlACMseg23F (SEQ ID NO:57), HUMAlACMseg23R (SEQ ID NO:58), HUMAl ACMseg26-3 IWTF (SEQ ID NO:54) and HUMA lACMseg26-3 IRWT (SEQ ID NO:55) was measured by real time PCR. In parallel the expression of four housekeeping genes -PBGD (GenBank Accession No. BCO 19323 (SEQ ID NO: 194); amplicon - PBGD-amplicon (SEQ ID NO: 158)), HPRTl (GenBank Accession No. NM_000194, (SEQ ID NO:195); amplicon - HPRTl -amplicon, (SEQ ID NO:161)), SDHA (GenBank Accession No. NM 004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO: 155)) and G6PD (GenBank Accession No. NM_000402 (SEQ ID NO: 197); G6PD amplicon (SEQ ID NO: 167)), RPS27A (GenBank Accession No. NM_002954 (SEQ ID NO: 198); RPS27A amplicon (SEQ ID NO: 170)) were measured similarly. For each RT sample, the expression of the above amplicons were normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (Sample Nos. 56-60, 63-67, Table 5, above, "Tissue samples in breast cancer testing panel"), to obtain a value of fold differential expression for each sample relative to median of the normal PM samples.
In one experiment that was carried out with each of the above amplicons, no differential expression in the cancerous samples relative to the normal PM samples was observed.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMAlACMseg23F (SEQ ID NO:57) and HUMA lACMseg26-3 IWTF (SEQ ID NO:54) forward primers; and HUMAl ACMseg23R (SEQ ID NO:58) and HUMAlACMseg26-31WT R (SEQ ID NO:55) reverse primers.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMAl ACMseg23 (SEQ ID NO:59) and HUMA lACMseg26-3 IWT (SEQ ID NO:56). Primers:
Forward primer HUMAlACMseg23F (SEQ ID NO:57) : ATCTCAGGGCCATTTCTGTCC Reverse primer HUMAlACMseg23R (SEQ ID NO:58) :
GGCCTTGATGCTTATGCTGC
Atnplicon HUMAlACMseg23 (SEQ ID NO.59) :
ATCTCAGGGCCATTTCTGTCCTGACTCAACAATGGTGTTATTAGGCAAACCAC TGTAGAGGAGAGTGCCCACAGCATGGGGGCAGCATAAGCATCAAGGCC
Primers:
Forward primer HUMAl ACMseg26-3 IWTF (SEQ ID NO:54) : TGGAGCTGAAGTACACAGGCAA
Reverse primer HUMA lACMseg26-3 IWTR (SEQ ID NO:55) : CTTCAGGGTCTCTGGGAGCA
Amplicon HUMA lACMseg26-3 IWT (SEQ ID NO:56) : TGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCATCCTCCCTGATCA AGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG
DESCRIPTION FOR CLUSTER HUMEGFRBB3
Cluster HUMEGFRBB3 features 10 transcript(s) and 66 segment(s) of interest, the names for which are given in Tables 65 and 66, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 67. Table 65 - Transcripts of interest yiansciipt JName
HUMEGFRBB3 jPEA_l_T2 (SEQ ID NO:60)
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61)
HUMEGFRBB3_PEA_1 JT9 (SEQ ID NO:62)
HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63)
HUMEGFRBB3 PEA 1_T2 (SEQ ID NO:60) 0
HUMEGFRBB3 PEA 1 T35 (SEQ ID NO:65)
HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66)
HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67)
HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68)
HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69) Table 66 - Segments of interest
HUMEGFRBB3 PEA 1 _node_0 (SEQ ID NO:70)
HUMEGFRBB3 PEA 1 node l 3 (SEQ ID NO:71)
HUMEGFRBB3_PEA l_node_14 (SEQ ID NO:72)
HUMEGFRBB3 PEA 1 node_18 (SEQ ID NO:73)
HUMEGFRBB3_PEA 1 node_23 (SEQ ID NO:74)
HUMEGFRBB3_PEA_l_node_26 (SEQ ID NQ:75)
HUMEGFRBB3_PEA_l_node_27 (SEQ ID NO:76)
HUMEGFRBB3_PEA l_node_40 (SEQ ID NO:77)
HUMEGFRBB3 PEA 1 node_42 (SEQ ID NO:78)
HUMEGFRBB3_PEA_l_node_46 (SEQ ID NO:79)
HUMEGFRBB3_PEA l_node_49 (SEQ ID NO:80)
HUMEGFRBB3_PEA l_node_50 (SEQ ID NO:81)
HUMEGFRBB3_PEA 1 node_51 (SEQ ID NO:82)
HUMEGFRBB3JPEA_l_node_54 (SEQ ID NO:83)
HUMEGFRBB3_PEA 1 node_58 (SEQ ID NO:84)
HUMEGFRBB3_PEA_l_node_60 (SEQ ID NO:85)
HUMEGFRBB3_PEA 1 node_66 (SEQ ID NO:86)
HUMEGFRBB3JPEA 1 node_68 (SEQ ID NO:87)
HUMEGFRBB3_PEA_l_node_8 9 (SEQ ID NO:88)
HUMEGFRBB3_PEA 1 node_95 (SEQ ID NO:89)
HUMEGFRBB3_PEA 1 node_97 (SEQ ID NO:90)
HUMEGFRBB3_PEA_l_node_98 (SEQ ID NO:91)
HUMEGFRBB3_PEA 1 node_100 (SEQ ID NO:92)
HUMEGFRBB3__PEA 1 node l (SEQ ID NQ:93)
HUMEGFRBB3_PEA_l_node_2 (SEQ ID NQ:94)
HUMEGFRBB3_PEA_l_node_8 (SEQ ID NO:95)
HUMEGFRBB3 PEA 1 node_9 (SEQ ID NO:96)
HUMEGFRBB3_PEA 1 _node_10 (SEQ ID NO:97)
HUMEGFRBB3 PEA 1 node_l 1 (SEQ ID NO:98)
HUMEGFRBB3_PEA 1 node lβ (SEQ ID NO:99)
HUMEGFRBB3_PEA_l_node_17 (SEQ ID NO: 100)
HUMEGFRBB3 PEA 1 node_20 (SEQ ID NO:101)
HUMEGFRBB3__PEA 1 node_21 (SEQ ID NO: 102)
HUMEGFRBB3._PEA 1 node_22 (SEQ ID NO: 103)
HUMEGFRBB3_PEA 1 node_24 (SEQ ID NO: 104)
HUMEGFRBB3JPEA _1_ node_28 (SEQ ID NO: 105)
HUMEGFRBB3 PEA 1 node_30 (SEQ ID NO:106)
HUMEGFRBB3_PEA_l_node_31 (SEQ ID NO: 107)
HUMEGFRBB3 PEA 1 node_34 (SEQ ID NO: 108)
HUMEGFRBB3 PEA l_node_35 (SEQ ID NO: 109)
HUMEGFRBB3 _PEA_l_node_37 (SEQ ID NO:110)
HUMEGFRBB3_PEA .!_ node_39 (SEQ ID NO:111)
HUMEGFRBB3_PEA_l_node_44 (SEQ ID NO:112)
HUMEGFRBB3_PEA_l_node_45 (SEQ ID NO: 113)
HUMEGFRBB3 PEA 1 node_47 (SEQ ID NO: 114) HUMEGFRBB3 PEA 1 node_48 (SEQ ID NO:1 15)
HUMEGFRBB3 PEA l_node_52 (SEQ ID NO: 116)
HUMEGFRBB3, PEA 1 _node_53 (SEQ ID NO: 117)
HUMEGFRBB3 PEA l_node_57 (SEQ ID NO: 118)
HUMEGFRBB3_PEA_l_node_61 (SEQ ID NO: 119)
HUMEGFRBB3_PEA l node 62 (SEQ ID NO: 120)
HUMEGFRBB3J>EA_l_node_64 (SEQ ID NO: 121)
HUMEGFRBB3 _PEA_l_node_71 (SEQ ID NO: 122)
HUMEGFRBB3_PEA_l_node_73 (SEQ ID NO: 123)
HUMEGFRBB3_PEA_l_node_74 (SEQ ID NO: 124)
HUMEGFRBB3 PEA_l_node_78 (SEQ ID NO: 125)
HUMEGFRBB3_PEA_l_node_8 Q (SEQ ID NO: 126)
HUMEGFRBB3 PEA l_node_81 (SEQ ID NO:127)
HUMEGFRBB3_PEA_l_node_8 2 (SEQ ID NO: 128)
HUMEGFRBB3_PEA_l_node_84 (SEQ ID NO: 129)
HUMEGFRBB3_PEA_l_node_85 (SEQ ID NO: 130)
HUMEGFRBB3_PEA_l_node_90 (SEQ ID NO: 131)
HUMEGFRBB3 PEA l_node_9 1 (SEQ ID NO:132)
HUMEGFRBB3 PEA l_node_9 2 (SEQ ID NO: 133)
HUMEGFRBB3 PEA l_node_96 (SEQ ID NO: 134)
HUMEGFRBB3_PEA_l_node_99 (SEQ ID NO: 135)
Table 67 - Proteins of interest
Figure imgf000182_0001
These sequences are variants of the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136) (SwissProt accession identifier ERBB3JHUMAN; known also according to the synonyms EC 2.7.1.112, c-erbB3, Tyrosine kinase-type cell surface receptor HER3), referred to herein as the previously known protein.
Protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO" 136) is known or believed to have the following function(s): Binds and is activated by neuregulins and NTAK. The sequence for protein Receptor protein-tyrosine kinase erbB- 3 precursor is given at the end of the application, as "Receptor protein-tyrosine kinase erbB-3 precursor amino acid sequence". Known polymorphisms for this sequence are as shown in Table 68.
Table 68 - Amino acid mutations for Known Protein
Figure imgf000183_0001
Protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136) localization is believed to be Type I membrane protein (long form) and secreted (short form).
It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows: ErbB-3 inhibitor. A therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Anticancer; antibody; Diagnostic. In particular, it lias been considered as a diagnostic marker for detection of cancer in_ humans. Differential expression of ErbB-3 mRNA^leyels were demonstrated in certain human breast cancer cell lines, with -the level of expression increased in the cancer cells, implicating the protein as potentially playing a role Jn at least certain human cancers. Differential expression of ErbB-3 ttiRNX levels were demonsttated using microarray analysis in certain human ovarian cancers and colon cancers (Lu KH5 et al., Clin Cancer Res. 2004 May 10:3291-300) with the level ojf expression, increased in the cancer cells, implicating the protein as potentially playing a role in at least certain human cancers, Cluster HUMEΘFRBB3 can be used as a
Figure imgf000184_0001
humans,, especially, epithelial cells cancers, such as colon cancer, ovarian cancer, breast cancer.
The following GO Annotation(s) apply to the previously known protein. The following annotation(s) were found: protein amino acid phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway, which are annotation(s) related to
Biological Process; receptor; epidermal growth factor receptor; ATP binding; transferase, which are annotation(s) related to Molecular Function; and integral plasma membrane protein, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from
<http://www.expasy.ch/sprot/>; or Locuslink, available from
<http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
Cluster HUMEGFRBB3 can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods. The term
"number" in the left hand column of the table and the numbers on the y-axis of the figure below refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million). Overall, the following results were obtained as shown with regard to the histogram in Figure 14 and Table 69. This cluster is overexpressed (at least at a minimum level) in the following pathological conditions: epithelial malignant tumors and a mixture of malignant tumors from different tissues.
Table 69 - Normal tissue distribution
Figure imgf000184_0002
Figure imgf000185_0001
Figure imgf000185_0002
As noted above, cluster HUMEGFRBB3 features 10 transcript(s), which were listed in Table 65 above. These transcript(s) encode for protein(s) which are variant(s) of protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136). A description of each variant protein according to the present invention is now provided.
Variant protein HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137) and ERBB3 HUMAN (SEQ ID NO:207): 1.An isolated chimeric polypeptide encoding for HUMEGFRBB3 PEA 1 P15 (SEQ ID NO: 137), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDP WHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG PIYKYPDVQNECRPCHENCTQG corresponding to amino acids 1 - 620 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 620 of HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SVMG (SEQ ID NO: 186) corresponding to amino acids 621 - 624 of HUMEGFRBB3JPEA 1JP15 (SEQ ID NO: 137), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SVMG (SEQ ID NO: 186) in HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 71 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 71 - Glycosylation site(s)
Figure imgf000187_0001
Variant protein HUMEGFRBB3JPEA_1_P15 (SEQ ID NO.137) is encoded by the following transcript(s): HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) is shown in bold; this coding portion starts at position 199 and ends at position 2070.
Variant protein HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 72, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 72 - Amino acid mutations
Figure imgf000187_0002
Figure imgf000188_0001
The transcript also has the following SNPs as listed in Table 73 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P15 (SEQ ID NO:137) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 73 - Nucleic acid SNPs
Figure imgf000188_0002
Figure imgf000189_0001
Variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3JPEA_1_T35 (SEQ ID NO:65). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138) and ERB3_HUMAN (SEQ ID NO:207): LAn isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P28
(SEQ ID NO: 138), comprising a first amino acid sequence being at least 90 % homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGD corresponding to amino acids 1 - 41 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 41 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), and a second amino acid sequence being at least 90 % homologous to AEVPDLLEKGERLAQPQICTID VYMVMVKCWMIDENIRPTFKELANEFTRMARD
PPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTL GSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPV SLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRH SLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDE DEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPI MPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGH
QAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT corresponding to amino acids 918 - 1342 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 42 - 466 of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated chimeric polypeptide encoding for an edge portion of HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise DA, having a structure as follows: a sequence starting from any of amino acid numbers 41-x to 41; and ending at any of amino acid numbers 42+ ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylate sites of variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 74 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 74 - Glycosylation site(s)
Figure imgf000191_0001
Variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138) is encoded by the following transcript(s): HUMEGFRBB3^PEA_1_T35 (SEQ ID NO:65), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65) is shown in bold; this coding portion starts at position 199 and ends at position 1596.
Variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 75, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 75 - Amino acid mutations
Figure imgf000192_0001
The transcript also has the following SNPs as listed in Table 76 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P28 (SEQ ID NO: 138) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 76 - Nucleic acid SNPs
Figure imgf000192_0002
Figure imgf000193_0001
Variant protein HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3 PEA 1 T38 (SEQ ID NO:66). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) and ERB3_HUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG PIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIG corresponding to amino acids 1 - 638 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 638 of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) corresponding to amino acids 639 - 699 of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence YDGVGDSGNWGYLGVGREVVTWREEGGCLHSGLLCMQSTITGHLGLKNAGFW TSLPKINFQ (SEQ ID NO: 187) in HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB 3_PEA_1JP31 (SEQ ID NO: 139), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 77 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 77 - Glycosylation site(s)
Figure imgf000194_0001
Figure imgf000195_0001
Variant protein HUMEGFRBB3_PEA_1_P31 (SEQ ID NO.139) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66) is shown in bold; this coding portion starts at position 199 and ends at position 2295.
Variant protein HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 78, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 78 - Amino acid mutations
Figure imgf000195_0002
The transcript also has the following SNPs as listed in Table 79 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 79 - Nucleic acid SNPs
Figure imgf000196_0001
Variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO:140) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140) and ERB3 HUMAN (SEQ ID NO:207): 1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P41
(SEQ ID NO: 140), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCF corresponding to amino acids 1 - 244 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 244 of HUMEGFRBB3 PEAJ P41 (SEQ ID NO: 140), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) corresponding to amino acids 245 - 278 of HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TLPLITLITGIAGFSPRLMPRERNSCSLWHSGSI (SEQ ID NO: 188) in HUMEGFRBB3_PEA_1_P41 (SEQ ID NO:140).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 80 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 80 - Glycosylation site(s)
Figure imgf000198_0001
Variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140) is encoded by the following transcript(s): HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEAJ_T50 (SEQ ID NO:67) is shown in bold; this coding portion starts at position 199 and ends at position 1032.
Variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 81, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 81 - Amino acid mutations
Figure imgf000198_0002
Figure imgf000199_0002
The transcript also has the following SNPs as listed in Table 82 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P41 (SEQ ID NO:140) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 82 - Nucleic acid SNPs
Figure imgf000199_0001
Variant protein HUMEGFRBB3_PEA_1_P45 (SEQ ID NO:141) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_ 1_T54 (SEQ ID NO:68). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141) and ERB3_HUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDIVRDRDAEIVVKDNGRSC corresponding to amino acids 1 - 183 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 183 of HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), and a second amino- acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence KWP corresponding to amino acids 184 - 186 of HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 83 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 83 - Glycosylation site(s)
Figure imgf000201_0001
Variant protein HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) is shown in bold; this coding portion starts at position 199 and ends at position 756. Variant protein HUMEGFRBB3_PEA_1_P45 (SEQ ID NO.141) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 84, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 84 - Amino acid mutations
Figure imgf000201_0002
The transcript also has the following SNPs as listed in Table 85 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3 PEAJJP45 (SEQ ID NO: 141) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 85 - Nucleic acid SNPs
Figure imgf000202_0001
Variant protein HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142) and ERB3 HUMAN (SEQ ID NO:207): 1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P46
(SEQ ID NO: 142), comprising a first amino acid sequence being at least 90 % homologous to MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLT corresponding to amino acids 1 - 140 of ERB3 HUMAN (SEQ ID NO.207), which also corresponds to amino acids 1 - 140 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) corresponding to amino acids 141 - 183 of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV (SEQ ID NO: 189) in HUMEGFRBB3JPEA_1 JP46 (SEQ ID NO: 142).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3_PEA_1_P46 (SEQ ID
NO: 142), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 86 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 86 - Glycosylation site(s)
Figure imgf000203_0001
Figure imgf000204_0001
Variant protein HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_ 1_T55 (SEQ ID NO:69) is shown in bold; this coding portion starts at position 199 and ends at position 747.
Variant protein HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 87, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1JP46 (SEQ ID NO: 142) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 87 - Amino acid mutations
Figure imgf000204_0002
The transcript also has the following SNPs as listed in Table 88 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1JP46 (SEQ ID NO: 142) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 88 - Nucleic acid SNPs
Figure imgf000205_0001
Variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) and ERB3JHUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDΓVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQT corresponding to amino acids 1 - 204 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 204 of HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) corresponding to amino acids 205 - 254 of HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3J>EA_1_P5O (SEQ ID NO: 143), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CGFEIPSKNFTHTLSYPFLPKPGSTLWGRHEQWPQNSVLGALTAMLSLLP (SEQ ID NO: 190) in HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 89 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein). Table 89 - Glycosylation site(s)
Figure imgf000206_0001
408 no
Variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1__T2 (SEQ ID NO:60), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60) is shown in bold; this coding portion starts at position 199 and ends at position 960.
Variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 90, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 90 - Amino acid mutations
Figure imgf000207_0001
The transcript also has the following SNPs as listed in Table 91 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 91 - Nucleic acid SNPs
Figure imgf000207_0002
Figure imgf000209_0001
Variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO:144) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) and ERB3 JIUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1JP53
(SEQ ID NO: 144), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDΓVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA
HEAECFSCHPECQPMEGTATCNGS corresponding to amino acids 1 - 568 of ERB3JHUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 568 of HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VY corresponding to amino acids 569 - 570 of HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylate sites of variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 92 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein). Table 92 - Glycosylation site(s)
Figure imgf000210_0001
Variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T8 (SEQ ID NO.61) is shown in bold; this coding portion starts at position 199 and ends at position 1908.
Variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 93, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 93 - Amino acid mutations
Figure imgf000211_0001
The transcript also has the following SNPs as listed in Table 94 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 94 - Nucleic acid SNPs
Figure imgf000211_0002
Figure imgf000212_0001
Figure imgf000213_0001
Variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62). An alignment is given to the known protein (Receptor protein-tyro sine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) and ERB3_HUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P54
(SEQ ID NO: 145), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRG TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDΓVRDRDAEΓVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF
QLEPNPHTKYQYGGVCVASCP corresponding to amino acids 1 - 291 of ERB3_HUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 291 of HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RKCLRGRNNDQQ (SEQ ID NO:191) corresponding to amino acids 292 - 303 of HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P54
(SEQ ID NO: 145), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence RKCLRGRNNDQQ (SEQ ID
NO:191) in HUMEGFRBB3_PEA_1_P54 (SEQ ID NO:145).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylate sites of variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 95 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein). Table 95 - Glycosylation site(s)
Figure imgf000214_0001
Variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) is encoded by the following transcript(s): HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62) is shown in bold; this coding portion starts at position 199 and ends at position 1107.
Variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 96, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 96 - Amino acid mutations
Figure imgf000215_0001
The transcript also has the following SNPs as listed in Table 97 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 97 - Nucleic acid SNPs
Figure imgf000215_0002
Figure imgf000216_0001
Figure imgf000217_0001
Variant protein HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63). An alignment is given to the known protein (Receptor protein-tyrosine kinase erbB-3 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) and ERB3_HUMAN (SEQ ID NO:207):
1.An isolated chimeric polypeptide encoding for HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), comprising a first amino acid sequence being at least 90 % homologous to
MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLY ERCEVVMGNLEIVLTGHNADLSFLQ WIREVTGYVLVAMNEFSTLPLPNLRVVRG
TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTID WRDΓVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNG HCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTF QLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPC GGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPAL DPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKN LNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRR DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFA HEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKG
PIYKYPDVQNECRPCHENCTQG corresponding to amino acids 1 - 620 of ERB3JHUMAN (SEQ ID NO:207), which also corresponds to amino acids 1 - 620 of HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SVMG (SEQ ID NO: 186) corresponding to amino acids 621 - 624 of HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SVMG (SEQ ID NO: 186) in HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146).
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted.
The glycosylation sites of variant protein HUMEGFRBB3 PEA 1JP55 (SEQ ID NO: 146), as compared to the known protein Receptor protein-tyrosine kinase erbB-3 precursor (SEQ ID NO: 136), are described in Table 98 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).
Table 98 - Glycosylation site(s)
Figure imgf000218_0001
Variant protein HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) is encoded by the following transcript(s): HUMEGFRBB3_PEA_ IJTlO (SEQ ID NO:63), for which the sequence(s) is/are given at the end of the application. The coding portion of transcript HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) is shown in bold; this coding portion starts at position 199 and ends at position 2070.
Variant protein HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 99, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 99 - Amino acid mutations
Figure imgf000219_0001
The transcript also has the following SNPs as listed in Table 100 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 100 - Nucleic acid SNPs
Figure imgf000220_0001
Figure imgf000221_0001
As noted above, cluster HUMEGFRBB3 features 66 segment(s), which were listed in Table 66 above and for which the sequence(s) are given at the end of the application.
These segment(s) are portions of nucleic acid sequence(s) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster HUMEGFRBB3_PEA_l_node_0 (SEQ ID NO:70) according to the present invention is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3JPEA_1_T35 (SEQ ID NO:65), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and HUMEGFRBB3_PEA_1_T55 (SEQ ID NO.69). Table 101 below describes the starting and ending position of this segment on each transcript. Table 101 - Segment location on transcripts
Figure imgf000222_0001
Segment cluster HUMEGFRBB3_PEA_l_node_13 (SEQ ID NO:71) according to the present invention is supported by 48 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3JPEA_1_T54 (SEQ ID NO:68) and
HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 102 below describes the starting and ending position of this segment on each transcript.
Table 102 - Segment location on transcripts
Figure imgf000222_0002
Figure imgf000223_0001
Segment cluster HUMEGFRBB3_PEA_l_node_14 (SEQ ID NO:72) according to the present invention is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 103 below describes the starting and ending position of this segment on each transcript.
Table 103 - Segment location on transcripts
Figure imgf000223_0002
Segment cluster HUMEGFRBB3_PEA_l_node_18 (SEQ ID NO:73) according to the present invention is supported by 7 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68). Table 104 below describes the starting and ending position of this segment on each transcript.
Table 104 - Segment location on transcripts
Figure imgf000223_0003
Segment cluster HUMEGFRBB3_PEA_l_node_23 (SEQ ID NO:74) according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60). Table 105 below describes the starting and ending position of this segment on each transcript.
Table 105 - Segment location on transcripts
Figure imgf000224_0001
Segment cluster HUMEGFRBB3_PEA_l_node_26 (SEQ ID NO:75) according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3JPEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 106 below describes the starting and ending position of this segment on each transcript.
Table 106 - Segment location on transcripts
Figure imgf000224_0002
Segment cluster HUMEGFRBB3_PEA_l_node_27 (SEQ ID NO:76) according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62). Table 107 below describes the starting and ending position of this segment on each transcript.
Table 107 - Segment location on transcripts
Figure imgf000225_0001
Segment cluster HUMEGFRBB3_PEA_l_node_40 (SEQ ID NO:77) according to the present invention is supported by 52 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3JPEAJ T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 108 below describes the starting and ending position of this segment on each transcript. Table 108 - Segment location on transcripts
Figure imgf000225_0002
Segment cluster HUMEGFRBB3JPEA_l_node_42 (SEQ ID NO:78) according to the present invention is supported by 50 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3 JPEAJJTl 0 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 109 below describes the starting and ending position of this segment on each transcript.
Table 109 - Segment location on transcripts
Figure imgf000226_0001
Segment cluster HUMEGFRBB3_PEA_l_node_46 (SEQ ID NO:79) according to the present invention is supported by 10 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3 PEAJJT8 (SEQ ID NO:61). Table 110 below describes the starting and ending position of this segment on each transcript.
Table 110 - Segment location on transcripts
Figure imgf000226_0002
Segment cluster HUMEGFRBB3_PEA_l_node_49 (SEQ ID NO:80) according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3JPEAJJT20 (SEQ ID NO:64). Table 111 below describes the starting and ending position of this segment on each transcript. Table 111 - Segment location on transcripts
Figure imgf000227_0001
Segment cluster HUMEGFRBB3_PEA_l_node_50 (SEQ ID NO:81) according to the present invention is supported by 2 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) Table 112 below describes the starting and ending position of this segment on each transcript.
Table 112 - Segment location on transcripts
Figure imgf000227_0002
Segment cluster HUMEGFRBB3_PEA_l_node_51 (SEQ ID NO:82) according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 113 below describes the starting and ending position of this segment on each transcript.
Table 113 - Segment location on transcripts
Figure imgf000227_0003
Segment cluster HUMEGFRBB3_PEA_l_node_54 (SEQ ID NO:83) according to the present invention is supported by 2 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 114 below describes the starting and ending position of this segment on each transcript.
Table 114 - Segment location on transcripts
Figure imgf000228_0001
Segment cluster HUMEGFRBB3_PEA_l_node_58 (SEQ ID NO:84) according to the present invention is supported by 46 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s):
HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID
NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10
(SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 115 below describes the starting and ending position of this segment on each transcript.
Table 115 - Segment location on transcripts
Figure imgf000228_0002
Segment cluster HUMEGFRBB3_PEA_l_node_60 (SEQ ID NO:85) according to the present invention is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3J>EA_l_T20 (SEQ ID NO:64). Table 116 below describes the starting and ending position of this segment on each transcript.
Table 116 - Segment location on transcripts
Figure imgf000229_0001
Segment cluster HUMEGFRBB3_PEA_l_node_66 (SEQ ID NO: 86) according to the present invention is supported by 46 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3JPEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 117 below describes the starting and ending position of this segment on each transcript.
Table 117 - Segment location on transcripts
Figure imgf000229_0002
Segment cluster HUMEGFRBB3_PEA_l_node_68 (SEQ ID NO:87) according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 118 below describes the starting and ending position of this segment on each transcript Table 118 - Segment location on transcripts
Figure imgf000230_0001
Segment cluster HUMEGFRBB3_PEA_l_node_89 (SEQ ID NO:88) according to the present invention is supported by 63 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NC-.60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 119 below describes the starting and ending position of this segment on each transcript.
Table 119 - Segment location on transcripts
Figure imgf000230_0002
Segment cluster HUMEGFRBB3_PEA_l_node_95 (SEQ ID NO:89) according to the present invention is supported by 72 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3JPEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HLJMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 120 below describes the starting and ending position of this segment on each transcript.
Table 120 - Segment location on transcripts
Figure imgf000231_0001
Segment cluster HUMEGFRBB3_PEA_l_node_97 (SEQ ID NO:90) according to the present invention is supported by 60 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 121 below describes the starting and ending position of this segment on each transcript.
Table 121 - Segment location on transcripts
Figure imgf000231_0002
Figure imgf000232_0001
Segment cluster HUMEGFRBB3_PEA_l_node_98 (SEQ ID NO:91) according to the present invention is supported by 131 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO.62), HUMEGFRBB3_PEA_lJT10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 122 below describes the starting and ending position of this segment on each transcript.
Table 122 - Segment location on transcripts
Figure imgf000232_0002
Segment cluster HUMEGFRBB3_PEA_l_node_100 (SEQ ID NO:92) according to the present invention is supported by 13 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripts): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3JPEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3JPEA 1 T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 123 below describes the starting and ending position of this segment on each transcript.
Table 123 - Segment location on transcripts
Figure imgf000233_0001
According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
Segment cluster HUMEGFRBB3_PEA_l_node_l (SEQ ID NO:93) according to the present invention is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s):
HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID
NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10
(SEQ ID NO.63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1 JT35 (SEQ ID NO:65), HUMEGFRBB3_PEA_1_T38 (SEQ ID
NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 124 below describes the starting and ending position of this segment on each transcript.
Table 124 - Segment location on transcripts
Figure imgf000234_0001
Segment cluster HUMEGFRBB3_PEA_l_node_2 (SEQ ID NO:94) according to the present invention is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO.60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 125 below describes the starting and ending position of this segment on each transcript.
Table 125 - Segment location on transcripts
Figure imgf000234_0002
Figure imgf000235_0001
Segment cluster HUMEGFRBB3_PEA_l_node_8 (SEQ ID NO:95) according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 126 below describes the starting and ending position of this segment on each transcript.
Table 126 - Segment location on transcripts
Figure imgf000235_0002
Figure imgf000236_0001
Segment cluster HUMEGFRBB3_PEA_l_node_9 (SEQ ID NO:96) according to the present invention can be found in the following transcript(s)" HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3JPEA_1_T38 (SEQ ID NO: 66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and
HUMEGFRBB3JPEA_1_T55 (SEQ ID NO:69). Table 127 below describes the starting and ending position of this segment on each transcript.
Table 127 - Segment location on transcripts
Figure imgf000236_0002
Figure imgf000237_0001
Segment cluster HUMEGFRBB 3_PEA_l_node_ 10 (SEQ ID NO:97) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and
HUMEGFRBB3_PEA_1_T55 (SEQ ID NO:69). Table 128 below describes the starting and ending position of this segment on each transcript.
Table 128 - Segment location on transcripts
Figure imgf000237_0002
Segment cluster HUMEGFRBB3_PEA_l_node_l 1 (SEQ ID NO:98) according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA__1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67), HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68) and
HUMEGFRBB3JPEA_1_T55 (SEQ ID NO:69). Table 129 below describes the starting and ending position of this segment on each transcript.
Table 129 - Segment location on transcripts
Figure imgf000238_0001
Segment cluster HUMEGFRBB3_PEA_l_node_16 (SEQ ID NO:99) according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s):
HUMEGFRBB3JPEA_1_T2 (SEQ ID NO.60), HUMEGFRBB3_PEA_1_T8 (SEQ ID
NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10
(SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID
NO:67) and HUMEGFRBB3JPEAJ T54 (SEQ ID NO:68). Table 130 below describes the starting and ending position of this segment on each transcript.
Table 130 - Segment location on transcripts
Figure imgf000239_0002
Segment cluster HUMEGFRBB3_PEA_l_node_17 (SEQ ID NO: 100) according to the present invention is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66), HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67) and HUMEGFRBB3_PEA_1_T54 (SEQ ID NO:68). Table 131 below describes the starting and ending position of this segment on each transcript.
Table 131 - Segment location on transcripts
Figure imgf000239_0001
Figure imgf000240_0001
Segment cluster HUMEGFRBB3_PEA_l_node_20 (SEQ ID NO: 101) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3JPEA_1_T38 (SEQ ID NO:66) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 132 below describes the starting and ending position of this segment on each transcript.
Table 132 - Segment location on transcripts
Figure imgf000240_0002
Segment cluster HUMEGFRBB3_PEA_l_node_21 (SEQ ID NO: 102) according to the present invention is supported by 35 libraries. The number of libiaries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 133 below describes the starting and ending position of this segment on each transcript.
Table 133 - Segment location on transcripts
Figure imgf000241_0001
Segment cluster HUMEGFRBB3_PEA_l_node_22 (SEQ ID NO: 103) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 134 below describes the starting and ending position of this segment on each transcript.
Table 134 - Segment location on transcripts
Figure imgf000241_0002
Figure imgf000242_0001
Segment cluster HUMEGFRBB3_PEA_l_node_24 (SEQ ID NO: 104) according to the present invention is supported by 39 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3JPEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEAJ_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 135 below describes the starting and ending position of this segment on each transcript.
Table 135 - Segment location on transcripts
Figure imgf000242_0002
Segment cluster HUMEGFRBB3_PEA_l_node_28 (SEQ ID NO:105) according to the present invention is supported by 41 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3JPEAJJT2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3JPEA_l_T10 (SEQ ID NO.63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3JPEA_1_T38 (SEQ ID NO:66) Table 136 below describes the starting and ending position of this segment on each transcript.
Table 136 - Segment location on transcripts
Figure imgf000243_0001
Segment cluster HUMEGFRBB3_PEA_l_node_30 (SEQ ID NO: 106) according to the present invention is supported by 38 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID
NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 137 below describes the starting and ending position of this segment on each transcript.
Table 137 - Segment location on transcripts
Figure imgf000243_0002
Figure imgf000244_0001
Segment cluster HUMEGFRBB3_PEA_l_node_31 (SEQ ID NO: 107) according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 138 below describes the starting and ending position of this segment on each transcript.
Table 138 - Segment location on transcripts
Figure imgf000244_0002
Segment cluster HUMEGFRBB3_PEA_l_node_34 (SEQ ID NO: 108) according to the present invention can be found in the following transcript(s):
HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID
NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10
(SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and
HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 139 below describes the starting and ending position of this segment on each transcript. Table 139 - Segment location on transcripts
Figure imgf000245_0001
Segment cluster HUMEGFRBB3_PEA_l_node_35 (SEQ ID NO: 109) according to the present invention is supported by 41 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3JPEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 140 below describes the starting and ending position of this segment on each transcript.
Table 140 - Segment location on transcripts
Figure imgf000245_0002
Segment cluster HUMEGFRBB3JPEA_l_node_37 (SEQ ID NO.110) according to the present invention is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s)" HUMEGFRBB3_PEA_1_T2 (SEQ ID NO.60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l__T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3 PEA 1 T38 (SEQ ID NO:66). Table 141 below describes the starting and ending position of this segment on each transcript.
Table 141 - Segment location on transcripts
Figure imgf000246_0001
Segment cluster HUMEGFRBB3_PEA_l_node_39 (SEQ ID NO: 111) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 142 below describes the starting and ending position of this segment on each transcript.
Table 142 - Segment location on transcripts
Figure imgf000246_0002
Figure imgf000247_0001
Segment cluster HUMEGFRBB3_PEA_l_node_44 (SEQ ID NO: 112) according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3JPEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 143 below describes the starting and ending position of this segment on each transcript.
Table 143 - Segment location on transcripts
Figure imgf000247_0002
Segment cluster HUMEGFRBB3_PEA_l_node_45 (SEQ ID NO: 113) according to the present invention is supported by 38 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEAJ_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO.66). Table 144 below describes the starting and ending position of this segment on each transcript.
Table 144 - Segment location on transcripts
Figure imgf000248_0001
Segment cluster HUMEGFRBB3JPEA_l_node_47 (SEQ ID NO: 114) according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 145 below describes the starting and ending position of this segment on each transcript.
Table 145 - Segment location on transcripts
Figure imgf000248_0002
Figure imgf000249_0001
Segment cluster HUMEGFRBB3_PEA_l_node_48 (SEQ ID NO: 115) according to the present invention is supported by 45 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 146 below describes the starting and ending position of this segment on each transcript.
Table 146 - Segment location on transcripts
Figure imgf000249_0002
Segment cluster HUMEGFRBB3_PEA_l_node_52 (SEQ ID NO: 116) according to the present invention can be found in the following transcript(s):
HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID
NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10
(SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and
HUMEGFRBB3_PEA_1_T38 (SEQ ID NO:66). Table 147 below describes the starting and ending position of this segment on each transcript.
Table 147 - Segment location on transcripts
Figure imgf000249_0003
Figure imgf000250_0001
Segment cluster HUMEGFRBB3_PEA_l_node_53 (SEQ ID NO: 117) according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO.63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1 JT38 (SEQ ID NO:66). Table 148 below describes the starting and ending position of this segment on each transcript.
Table 148 - Segment location on transcripts
Figure imgf000250_0002
Segment cluster HUMEGFRBB3_PEA_l_node_57 (SEQ ID NO: 118) according to the present invention can be found in the following transcript(s):
HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 149 below describes the starting and ending position of this segment on each transcript.
Table 149 - Segment location on transcripts
Figure imgf000251_0001
Segment cluster HUMEGFRBB3_PEA_l_node_61 (SEQ ID NO: 119) according to the present invention is supported by 47 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and
HUMEGFRBB3_PEA_l_T20 (SEQ ID NC-.64). Table 150 below describes the starting and ending position of this segment on each transcript. Table 150 - Segment location on transcripts
Figure imgf000251_0002
Segment cluster HUMEGFRBB3_PEA_l_node_62 (SEQ ID NO: 120) according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and
HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 151 below describes the starting and ending position of this segment on each transcript.
Table 151 - Segment location on transcripts
Figure imgf000252_0001
Segment cluster HUMEGFRBB3_PEA_l_node_64 (SEQ ID NO:121) according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3 PEA 1 T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and
HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 152 below describes the starting and ending position of this segment on each transcript.
Table 152 - Segment location on transcripts
Figure imgf000252_0002
Figure imgf000253_0002
Segment cluster HUMEGFRBB3_PEA_l_node_71 (SEQ ID NO: 122) according to the present invention is supported by 47 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and
HUMEGFRBB3 PEAJ JT20 (SEQ ID NO:64). Table 153 below describes the starting and ending position of this segment on each transcript.
Table 153 - Segment location on transcripts
Figure imgf000253_0001
Segment cluster HUMEGFRBB3_PEA_l_node_73 (SEQ ID NO: 123) according to the present invention is supported by 51 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID
NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63) and HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64). Table 154 below describes the starting and ending position of this segment on each transcript. Table 154 - Segment location on transcripts
Figure imgf000254_0001
Segment cluster HUMEGFRBB3JPEA_l_node_74 (SEQ ID NO: 124) according to the present invention is supported by 58 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 155 below describes the starting and ending position of this segment on each transcript.
Table 155 - Segment location on transcripts
Figure imgf000254_0002
Segment cluster HUMEGFRBB3_PEA_l_node_78 (SEQ ID NO: 125) according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 156 below describes the starting and ending position of this segment on each transcript.
Table 156 - Segment location on transcripts
Figure imgf000255_0001
Segment cluster HUMEGFRBB3_PEA_l_node_80 (SEQ ID NO: 126) according to the present invention is supported by 59 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3JPEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 157 below describes the starting and ending position of this segment on each transcript.
Table 157 - Segment location on transcripts
Figure imgf000255_0002
Figure imgf000256_0001
Segment cluster HUMEGFRBB3_PEA_l_node_81 (SEQ ID NO: 127) according to the present invention is supported by 55 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3JPEAJ T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 158 below describes the starting and ending position of this segment on each transcript.
Table 158 - Segment location on transcripts
Figure imgf000256_0002
Segment cluster HUMEGFRBB3_PEA_l_node_82 (SEQ ID NO: 128) according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60)3
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61)3 HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_ l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 159 below describes the starting and ending position of this segment on each transcript.
Table 159 - Segment location on transcripts
Figure imgf000257_0001
Segment cluster HUMEGFRBB3_PEA_l_node_84 (SEQ ID NO: 129) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 160 below describes the starting and ending position of this segment on each transcript.
Table 160 - Segment location on transcripts
Figure imgf000257_0002
Segment cluster HUMEGFRBB3_PEA_l_node_85 (SEQ ID NO: 130) according to the present invention is supported by 56 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 161 below describes the starting and ending position of this segment on each transcript.
Table 161 - Segment location on transcripts
Figure imgf000258_0001
Segment cluster HUMEGFRBB3_PEA_l_node_90 (SEQ ID NO: 131) according to the present invention is supported by 57 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 162 below describes the starting and ending position of this segment on each transcript.
Table 162 - Segment location on transcripts
Figure imgf000258_0002
Figure imgf000259_0001
Segment cluster HUMEGFRBB3_PEA_l_node_91 (SEQ ID NO: 132) according to the present invention is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_ 1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3JPEA__l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 163 below describes the starting and ending position of this segment on each transcript.
Table 163 - Segment location on transcripts
Figure imgf000259_0002
Segment cluster HUMEGFRBB3_PEA_l_node_92 (SEQ ID NO: 133) according to the present invention is supported by 67 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO.61), HUMEGFRBB3_PEA_1JI9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 164 below describes the starting and ending position of this segment on each transcript.
Table 164 - Segment location on transcripts
Figure imgf000260_0001
Segment cluster HUMEGFRBB3_PEA_l_node_96 (SEQ ID NO: 134) according to the present invention can be found in the following transcript(s): HUMEGFRBB3_PEA_1_T2 (SEQ ID NO:60), HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO.63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64) and HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65). Table 165 below describes the starting and ending position of this segment on each transcript. Table 165 - Segment location on transcripts
Figure imgf000260_0002
Figure imgf000261_0001
Segment cluster HUMEGFRBB3_PEA_l_node_99 (SEQ ID NO: 135) according to the present invention is supported by 49 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripts): HUMEGFRBB 3_PEA_1_T2 (SEQ ID NO:60),
HUMEGFRBB3_PEA_1_T8 (SEQ ID NO:61), HUMEGFRBB3_PEA_1_T9 (SEQ ID NO:62), HUMEGFRBB3_PEA_l_T10 (SEQ ID NO:63), HUMEGFRBB3_PEA_l_T20 (SEQ ID NO:64), HUMEGFRBB3_PEA_1_T35 (SEQ ID NO:65) and HUMEGFRBB3_PEA_l_T50 (SEQ ID NO:67). Table 166 below describes the starting and ending position of this segment on each transcript.
Table 166 - Segment location on transcripts
Figure imgf000261_0002
Variant protein alignment to the previously known protein: Sequence name: ERB3_HUMAN Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P15 (SEQ ID NO: 137) x ERB3_HUMAN (SEQ ID NO:207).. Alignment segment 1/1:
Quality: 6309.00 Escore: 0 Matching length: 620 Total length: 620
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
I I I I Il I I I I I I 1 I 1 I 1 I I I I I I I I I I I I I I I I I I I Il I 1 I I I I I I I I 1 I 1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 . . . . .
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
I I I I Il I 1 I I I I Il I I I Il I I I I Il I I I Il I I I I I I I Il Il Il ! I I I I I I
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
I I I I I I I I Il I I I I Il Il I I I I i I I I Il Il I I I 1 Il I Il 1 I I I I I I I I Il 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200 I I I I I I M I M M M M I I I I I M M I M I I I I I I I M I I I I I I M I I M
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
I 1 Il I 1 I I I I I Il 1 I I I I I I Il I Il 1 I Il Il I I I Il I I I I I I I Il Il I Il 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTS 300
I I Ii I I I I I Il I I I I 1 Il I I Il I I I I Il I I I Il I I Il I I Il I I I M Il i I
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTS 300 . . . . .
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350
I 1 I I 1 Il I I Il I Il I I Il I I Il I I I I Il I Il I I Il I Il I I I I I I I I I I I I 301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
I I Il I I I I Il I I I Il I Il Il 1 I I Il Il Ii I I I Il I I Il I I I Il I I I I I Il 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 I 1 I M I I I I M I I I I I M I I I I I I I I M I M I M I M I I I I I I I I I I I I I
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500
IMII I IIIII MMI MMIII MIM M I I I MIMMIiIII IIiM 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500 501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
I I M I M I M Il Il I I M I M M Il M M I I M Il M I I I I I I M I Il I I
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550 . . . . .
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600
IM MMM MMMMMMMMM MIII MMMIIMMMMM
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600 601 YKYPDVQNECRPCHENCTQG 620
I I I I I I I I I I I I I I I I I I I I
601 YKYPDVQNECRPCHENCTQG 620
Sequence name: ERB3_HUMAN Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P28 (SEQ ID NO:138) x ERB3JHUMAN (SEQ ID NO:207)..
Alignment segment 1/1:
Quality: 4507.00 Escore: 0 Matching length: 466 Total length: 1342
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 34.72 Total Percent Identity: 34.72 Gaps: 1 Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGD 41
I I I I l I I I I I I I I I I I I I I I I I I I I I I I l I I I I I I I 1 i I I I
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 . . . . .
41 41
51 KLYERCEWMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 41 41
101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
41 41
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200
41 41 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250 41 41
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTS 300 . . . . .
41 41
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350 41 41
351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400 41 41
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 . . . . .
41 41
451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500 41 41
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
41 41
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600
41 41 601 YKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIA 650
41 41
651 GLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVL 700 . . . . .
41 41
701 ARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGR 750 41 41
751 QSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVR 800
41 41
801 QHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQV 850
41 41 851 ADFGVADLLPPDDKQLLYSEAKTPIKWMALES IHFGKYTHQSDVWSYGVT 900
42 AEVPDLLEKGERLAQPQICTIDVYMVMVKCWMI 74
I I I I I I I I I I I I I I Il Il I I I I I I I I I I I I I I I
901 VWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMI 950 . . . . .
75 DENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKL 124
I I Il I I I I I I I I I I I Il I I I I I I I I Il I Il I I Il I I Il Il I I I I I I Il Il
951 DENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKL 1000 125 EEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSP 174
I I Il I Il I I I I I I I I Il I I I I I I I I I Il Il I I Il I I I Il I I I I I I I Il Il
1001 EEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSP 1050
175 SSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGH 224 M I I M I I I I I I I I I I I M I I I I I I I I M I I I M I I 1 I I I I I I I I I I I I I
1051 SSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGH 1100
225 VTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPG 274
I I I I I I Il I I I I I Il I I Il I I I I I I Il I I I I Il I I I I I I I I I I M I I I I I 1101 VTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPG 1150 275 LEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYM 324
I I I I I I I I I I I I I I I I I I i I I I I l I I I I I I I I I I I I I I I I I I I I 1 I I I 1 !
1151 LEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYM 1200 325 NRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMP 374
! I Il I Il I Il I I I I I I Il I Ii Il I I I I I 1 I I I Il I I I Il I I I Il I I I I I I
1201 NRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMP 1250
375 TAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGH 424
1251 TAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGH 1300
425 QAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 466
II Il I I I Il M I I I Il I Il I I I I Il Il M Il I I I M I M M I 1301 QAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 1342
Sequence name: ERB3 HUMAN
Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P31 (SEQ ID NO: 139) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1 :
Quality: 6485.00 Escore: 0
Matching length: 638 Total length: 638
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
1 MRANDALQ VLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 M M I I l M I I l I M M I M I M I I I l M M I I M M I M M I M I l I M
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
I I I M I I I I M Il I Il Il M M M I I M M M Il I I Il I M M Il I Il I I 51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
M I M I I M M I I I l M I M M I I M I M M I I I I I M I l I M I l I M M
101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150 . . . . .
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200
I M I I I I M M I M Il Il M I I Il M I Il I I I I I I M I M I I Il I I M Il
151 KNDKLCHMDTI DWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
I I M I I I M I I I I M M I I l M M I I M M M M M I l I I M M I I l I I I
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250 251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTS 300
I 1 I I I I l I I I I l I I l I I I I I I l I I I I I I I I I I I I I I l I I I I I I I I I I I I I
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTS 300 . . . . .
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350
II Il Il I I Il I I I I I I I I I I Il I I I I Il I I I I I I Il Il I Il Il I I I I I Il
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
MIIIMIIMMMIIIIMMMIIIMMIIIIMIIIMIIMIM 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 I Il Il M I M M I M I I I I M M M I I I I M M I I I M I M M I I M M I
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500
I I I I I M M I M I I M I I I I M I M I I Il M M I I I M I Il M I M M M 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500 501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
M I I l M M I M I I M I I I I M I M I l M I M I l I M I I I M I l M M M
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550 . . . . .
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600
M I I l M I l M M I M I I I I M I I I l I I M M I I I M I l M M M M M I
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600 601 YKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIG 638
M I I I M I M I M I I I I M I I I I M I M I I I I I I I M I
601 YKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIG 638
Sequence name: ERB3JHUMAN
Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P41 (SEQ ID NO: 140) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1:
Quality: 2443.00 Escore: 0
Matching length: 244 Total length: 244 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 I I I I I I i I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I 1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I 1 I I I I I I
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
I Il I I I I I I I I I Il I I I I I I I I I I I I 11111 Il I I I 1 I 1 I I I Il I I 11 I I 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200
II I I I I I I Il I I I 1 I I I I I Il I I l I I I I I I I I I I Il I I I I I I I I I I I I I I
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200 . . . .
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCF 244
I I I I I I I I I I I I Il Il I I I I I I I I I I I I I I I Il I I I Il I I I I Il 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCF 244
Sequence name: ERB3_HUMAN Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P45 (SEQ ID NO: 141) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1:
Quality: 1770.00 Escore: 0 Matching length: 183 Total length: 183
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQ YQTLY 50
MIMIII I IMMI I IMMMMMIMIIII III IMIIM
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 . . . . .
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
I M M M M I I I M M I I l M M M Ml IM Il I M M M M Il I Il I M
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
I Il I M M M I I l M I I M I l M M Il M I I M M M M I I M Il M Il I
101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150 151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSC 183 M I M Il I l Il M M I M I I Il I l Il I M I I Il
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSC 183 Sequence name: ERB3JHUMAN
Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P46 (SEQ ID NO: 142) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1:
Quality: 1346.00 Escore: 0
Matching length: 140 Total length: 140
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 I I M I I 1 I I I ! I I I I I I I I ! I I 1 I I I I I I I I I I I M I I I I I I I ! 1 I I M I
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
I Il I I I I I i Il Il I Il I I I Il I I I Il Il I I Il I I I I I 1 I I Il I I I I Il I I 51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLT 140
I I I 1 I I Il I I Il I I I I Il I I I I I I I 1 I I I I I I Il I I I I Il 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLT 140
Sequence name: ERB3_HUMAN
Sequence documentation: Alignment of: HUMEGFRBB3_PEA_l_P50 (SEQ ID NO: 143) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1: Quality: 2006.00 Escore: 0
Matching length: 204 Total length: 204
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment: 1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
1 I II I I 1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 I M Il Il I M M I I M I M M I M I I I M Il Il I I M M I M M M M I I
51 KLYERCEWMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
I M I I l I l M M I I 1 M M I M M I I l I I l I M I I M M M M I I M I I I 101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200
I M I l I M M M I I M M I I M M I I l M I I I I I M I l M M M I I l I I I
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200
201 DCQT 204
II I I
201 DCQT 204
Sequence name: ERB3JHUMAN Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P53 (SEQ ID NO: 144) x ERB3JHUMAN (SEQ ID NO:207)..
Alignment segment 1/1 :
Quality: 5756.00 Escore: 0 Matching length: 568 Total length: 568
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
M M M M I M M I M M M M M I M M M M M M M M M M I M M
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 . . . . .
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
I M I I M I M Il M I Il I I M I M Il M M M M M I M M M Il I M I I
51 KLYERCEWMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150 I I I I I I I 1 I I I I I I I I 1 I i I I I I I I I I I I I I I I I I I I 1 I I I I I I I I I I I I 101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTI DWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
I I I I I I I Il II I Ii I I I I I I I Il I I I I I I I I I I I I I I I I I I I I I I Il I Il 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTS 300
I I I I I I I Ii Il I 11 I I I I I IiI I I I I I I I I I I I I I I I I I I I I I I I I I Il I I 251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTS 300 . . . . .
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350
I I I I I i I I I I i I I I I I Il I I I I Ii I I I I I I I I I I I I I I I I I I I I I I I I I I
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
I I I I I i I I I I I I I I I I I I I I I I I I 1 I I I i I I I I I I I I I Il I I i I I I I i I I
351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450 I I I I I I I I I M I I I I I I I I I I I I I I I I I I Il M I M I I I I I I I I I I I i I I
401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450
451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500
I I I I I I I Il Il I I I I I i Il I I I I I I I I I I I I Ii I I I I I I I I I I I I I I I I I 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
I Il I Il I I I i I I I I I I I i I I Il i I i I Il I I Il I Il I I I I I I I I I I Il I 1 I
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
551 SCHPECQPMEGTATCNGS 568
II I I I I I I I I I I I I I I I I
551 SCHPECQPMEGTATCNGS 568
Sequence name: ERB3_HUMAN Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P54 (SEQ ID NO: 145) x ERB3_HUMAN (SEQ ID NO:207)..
Alignment segment 1/1:
Quality: 2938.00 Escore: 0 Matching length: 291 Total length: 291
Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: lOOlOO Total Percent Identity: 100.00 Gaps: 0 Alignment: 1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
Il I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 I I I I I I M I I I I I I I I I I I I I I I M M M I I M M I I I M M I I I I I I I I
51 KLYERCEWMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
- I I I I I I I I I I I I Il I I I I I Il I I I I I I I I I I I I I I I I Il I I I I I I I I Il I 101 NLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200
I I I Il I I I I I Il Il I I I I I I I I I I I I I Il I I I I Il I I I I I Il I I I I Il Il
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200 . . . . .
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
II I 1 I I I l I I l Il I I I Il 111 I I I I I I I I I I I I I I I I Il I I I I l I I I I I I
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250 251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCP 291
I MIII I I I II M MIMIIIMMIIIM M MII IIII I
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCP 291
Sequence name: ERB3_HUMAN
Sequence documentation:
Alignment of: HUMEGFRBB3_PEA_1_P55 (SEQ ID NO: 146) x ERB3 HUMAN (SEQIDNO:207) ..
Alignment segment 1/1:
Quality: 6309.00 Escore: 0
Matching length: 620 Total length: 620 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0
Alignment:
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50
I I I M I I I M I I M I I M I I I M I I M Il I 1 I I I I I M I Il M I I M I M
1 MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLY 50 51 KLYERCEWMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100 11 I M I I I I I I I I I I I ] I I I I I I I M M I I I I I M I I I I ] I I I I I I I I M 51 KLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLP 100
101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150 I I Il M M I I I I I I I l I I Il M Il M Il I Il Il I M I I Il I M M I l I I I
101 NLRWRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE 150
151 KNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSCPPCHEVCKGRCWGPGSE 200
I I I I M M I I I I I I Il I I M I Il M M I I M I M M M M M M I I M M 151 KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSE 200 201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250
M I I M I I I I I I I I Il I I I 11 M I I I I I I I I I Il I I I I 1 Il I I I I I I I I I
201 DCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFN 250 . . . . .
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTS 300
I I I M I M I I Il I I Il I I I Il Il Il I I I I Il Il I Il I Il I I I I I I I I I I I
251 DSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTS 300 301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350
I 1 Il Il Il I I Il I I Il I 1111 Il I I Il 1 I Il I Il I Il I 11 I I I I I I I Il I
301 CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDG 350 351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400 I M I I M Il I I I M I Il I I 1 I I Il Il I M M I M I I I I M I I I I I I I I M
351 FVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQ 400 401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450
I I M I M I M M I I M I I M I Il I I Il M I 1 I M Il I Il M I M I I I I I I 401 SWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEIS 450
451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500
I I I M Il Il Il Il I I I I I Il I Il I I I I Il Il II I I Il I Il I I Il I I I I I I 451 AGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVC 500 . . . . .
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550
I I I M I I I M I M I M M I I I I I I I I M I I M I M I I M Il I M I I I I M
501 DPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECF 550 551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600
I I M Il I M M M 1 M I I M M Il M M I M M M I I M I Il M I I I I M
551 SCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI 600 601 YKYPDVQNECRPCHENCTQG 620 I I I I I I I I I I I Il I M I I M
601 YKYPDVQNECRPCHENCTQG 620
Expression of Receptor tyrosine-protein kinase erbB-3 precursor Cc- erbB3*) HUMEGFRBB3 transcripts which are detectable by amplicon as depicted in sequence name HUMEGFRBB3 seal 8 (SEQ ID NO: 149) in normal and cancerous Ovary tissues. Expression of Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts detectable by or according to seglδ - HUMEGFRBB3 segl8 amplicon (SEQ ID NO: 149) and primers HUMEGFRBB3_segl8F (SEQ ID NO: 147) and HUMEGFRBB3_segl8R (SEQ ID NO: 148) was measured by real time PCR. In parallel the expression of four housekeeping genes - SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO: 155)), HPRTl (GenBank Accession No. NM_000194 (SEQ ID NO:195); amplicon - HPRTl- amplicon (SEQ ID NO: 161)), PBGD (GenBank Accession No. BC019323 (SEQ ID NO:194); amplicon - PBGD-amplicon (SEQ ID NO:158)) and GAPDH (GenBank Accession No. BC026907 (SEQ ID NO:196); GAPDH amplicon (SEQ ID NO: 164)) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (sample numbers 45, 46, 71 and 48, Table 2 above, "Tissue samples in ovarian cancer panel"), to obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure 15 is a histogram showing over expression of the above-indicated Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts in cancerous Ovary samples relative to the normal samples.
As is evident from Figure 15, the expression of Receptor tyrosine-protein kinase erbB-3 precursor (c-erbB3) transcripts detectable by the above amplicon in adenocarcinoma samples was significantly higher than in the non-cancerous samples (sample numbers 45, 46, 71 and 48, Table 2 above, "Tissue samples in ovarian cancer panel"). Notably an over-expression of at least 5 fold was found in 37 out of 43 adenocarcinoma samples, specifically in 24 out of 30 serous carcinoma samples and in 6 out of 6 mucinous carcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below.
The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in all
Ovary adenocarcinoma samples versus the normal tissue samples was determined by T test as 2.16e-008. The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in Ovary serous carcinoma samples versus the normal tissue samples was determined by T test as 1.89e-005. The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in Ovary mucinous carcinoma samples versus the normal tissue samples was determined by T test as 4.96e-003. Threshold of 5 fold over expression was found to differentiate between adenocarcinoma and normal samples with P value of 1.18e-OO3 as checked by exact Fisher test. Threshold of 5 fold over expression was found to differentiate between serous carcinoma and normal samples with P value of 4.53e-003 as checked by exact Fisher test. Threshold of 5 fold over expression was found to differentiate between mucinous carcinoma and normal samples with P value of 4.76e- 003 as checked by exact Fisher test.
The above values demonstrate statistical significance of the results. By dissecting the patient's population into three age groups (indicated in the graph of Figure 15), the transcripts detected by the above amplicon were shown to be highly expressed mainly in patients over the age of 55.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMEGFRBB3_segl8F forward primer (SEQ ID NO: 147); and HUMEGFRBB3_segl8R reverse primer (SEQ
ID NO: 148).
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMEGFRBB3_segl 8 (SEQ ID NO: 149).
Primers:
Forward Primer (HUMEGFRBB3_segl8F) (SEQ ID NO: 147): GCAGACCATTCCAGTCTCCTG
Reverse Primer (HUMEGFRBB3_segl8R) (SEQ ID NO: 148): GATCCTCCCACCTTGGCTTC
Amplicon (HUMEGFRBB3_segl8) (SEQ ID NO: 149): GCAGACCATTCCAGTCTCCTGGAATCTAAACCACAGAGGAGGTGTTTC AAGAAAAGGAGCAGGCCAAGCATGGTGGCTCATGCCTATAATCCTGGCACTT TGGGAAGCCAAGGTGGGAGGATC
Expression of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) HUMEGFRBB3 transcripts which are detectable by amplicon as depicted in sequence name HUMEGFRBB3 seg46 (SEQ ID NO: 152) in normal and cancerous Ovary tissues.
Expression of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by or according to seg46 - HUMEGFRBB3_seg46 amplicon (SEQ ID NO: 152) and primers HUMEGFRBB3_seg46F (SEQ ID NO: 150) and HUMEGFRBB3_seg46R (SEQ ID NO: 151) was measured by real time PCR. In parallel the expression of four housekeeping genes - SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 193); amplicon - SDHA-amplicon (SEQ ID NO:155)), HPRTl (GenBank Accession No. NM_000194 (SEQ ID NO:195); amplicon - HPRTl- amplicon (SEQ ID NO: 161)), PBGD (GenBank Accession No. BC019323 (SEQ ID NO: 194); amplicon - PBGD-amplicon (SEQ ID NO:158)) and GAPDH (GenBank Accession No. BC026907 (SEQ ID NO: 196); GAPDH amplicon (SEQ ID NO: 164)) was measured similarly. For each RT sample, the expression of the above amplicon was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the normal post-mortem (PM) samples (sample numbers 45, 46, 71 and 48, Table 2 above, "'Tissue samples in ovarian cancer panel"), to obtain a value of fold up-regulation for each sample relative to median of the normal PM samples.
Figure 16 is a histogram showing over expression of the above-indicated Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts in cancerous Ovary samples relative to the normal samples.
As is evident from Figure 16, the expression of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in adenocarcinoma samples was significantly higher than in the non-cancerous samples (sample numbers 45, 46, 71 and 48, Table 2 above, "Tissue samples in ovarian cancer panel"). Notably an over-expression of at least 5 fold was found in 35 out of 43 adenocarcinoma samples, and specifically in 22 out of 30 serous carcinoma samples and in 6 out of 6 mucinous carcinoma samples.
Statistical analysis was applied to verify the significance of these results, as described below. The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in all Ovary adenocarcinoma samples versus the normal tissue samples was determined by T test as 2.45e-007. The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in Ovary serous carcinoma samples versus the normal tissue samples was determined by T test as 1.47e-004. The P value for the difference in the expression levels of Receptor tyrosine-protein kinase erbB-3 precursor (c- erbB3) transcripts detectable by the above amplicon in Ovary mucinous carcinoma samples versus the normal tissue samples was determined by T test as 8.83e-003, Threshold of 5 fold over expression was found to differentiate between adenocarcinoma and normal samples with P value of 2.78e-003 as checked by exact Fisher test. Threshold of 5 fold over expression was found to differentiate between serous carcinoma and normal samples with P value of 1.07e-002 as checked by exact Fisher test. Threshold of 5 fold over expression was found to differentiate between mucinous carcinoma and normal samples with P value of 4.76e-003 as checked by exact Fisher test.
The above values demonstrate statistical significance of the results. By dissecting the patient's population into three age groups (indicated in the graph of Figure 16), it was shown that the transcripts detected by the above amplicon are highly expressed mainly in patients who are at least 55 years old.
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMEGFRBB3_seg46F forward primer (SEQ ID NO: 150); and HUMEGFRBB3_seg46R reverse primer (SEQ ID NO: 151). The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: HUMEGFRBB3_seg46 (SEQ ID NO: 152). Primers:
Forward Primer (HUMEGFRBB3__seg46F) (SEQ ID NO: 150): CACCAGGATCTCCAAGGGAG
Reverse Primer (HUMEGFRBB3_seg46R) (SEQ ID NO: 151): CAGGCCCTAGCATTATCTGGC
Amplicon (HUMEGFRBB3_seg46) (SEQ ID NO: 152):
CACCAGGATCTCCAAGGGAGACAGAGAAGGGGCAATACTTGGAGCAT CTGGGGAATGATATGGCTAAGGATAGCACAGAGAGGCCAGATAATGCTAGG GCCTG
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. AU publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:
1. An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: HUMAl ACM JPEA_2_T21 (SEQ ID NO:1), HUMAl ACM_PEA_2_T27 (SEQ ID NO:2), or HUMA1ACM_PEA_2_T7 (SEQ ID NO:3).
2. An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: HUMAlACM_PEA_2_node_23 (SEQ ID NO:4), HUMAl ACM_PEA_2_node_41 (SEQ ID NO:5), HUMAlACM_PEA_2_node_51 (SEQ ID NO:6), HUMAlACM_PEA_2_node_0 (SEQ ID NO:7), HUMAlACM_PEA_2_node_l (SEQ ID NO:8), HUMAlACM_PEA_2_node_10 (SEQ ID NO:9), HUMAl ACM J>EA_2_node_l 1 (SEQ ID NO: 10), HUMAl ACM_PEA_2_node_12 (SEQ ID NO: 11), HUMAlACM_PEA_2_node_13 (SEQ ID NO:12), HUMAlACM_PEA_2_node_14 (SEQ ID NO:13), HUMAlACM_PEA_2_node_15 (SEQ ID NO:14), HUMAlACM_PEA_2_node_16 (SEQ ID NO:15), HUMAlACM_PEA_2_node_17 (SEQ ID NO:16), HUMAlACM_PEA_2_node_18 (SEQ ID NO:17), HUMAlACM_PEA_2_node_19 (SEQ ID NO: 18), HUMAlACM_PEA_2_node_2 (SEQ ID NO: 19), HUMAlACM_PEA_2_node_20 (SEQ ID NO:20), HUMAlACM_PEA_2_node_J21 (SEQ ID NO:21), HUMAlACM_PEA_2_node_22 (SEQ ID NO:22), HUMAlACM_PEA_2_node_26 (SEQ ID NO:23), HUMAlACM_PEA_2_node_27 (SEQ ID NO:24), HUMAlACM_PEA_2_node_28 (SEQ ID NO:25), HUMAl ACM_PEA_2_node_29 (SEQ ID NO:26), HUMAlACMJPEA_2_node_30 (SEQ ID NO:27), HUMAlACM_PEA_2_node_31 (SEQ ID NO:28), HUMAlACM_PEA_2_node_34 (SEQ ID NO:29), HUMAl ACMJPE A_2_node_35 (SEQ ID NO:30), HUMAl ACM_PEA_2_node_36 (SEQ ID NO:31), HUMAlACM_PEA_2_node_37 (SEQ ID NO:32), HUMAlACM_PEA_2_node_38 (SEQ ID NO:33), HUMAlACM_PEA_2_node_39 (SEQ ID NO:34), HUMAlACM_PEA_2_node_40 (SEQ ID NO:35), HUMAlACM_PEA_2_node_42 (SEQ ID NO:36), HUMAlACM_PEA_2_node_43 (SEQ ID NO:37), HUMAlACM_PEA_2_node_44 (SEQ ID NO:38), HUMAlACM_PEA_2_node_45 (SEQ ID NO:39), HUMAlACM_PEA_2_node_46 (SEQ ID NO:40), HUMAlACM_PEA_2_node_47 (SEQ ID NO:41), HUMAlACM_PEA_2_node__48 (SEQ ID NO:42), HUMAlACM_PEA_2_node_49 (SEQ ID NO:43), HUMAl ACM_PEA_2_node_5 (SEQ ID NO:44), HUMAl ACMJPE A_2_node_50 (SEQ ID NO:45), HUMAlACMJPEA_2_node_6 (SEQ ID NO:46), HUMAlACM_PEA_2_node_7 (SEQ ID NO:47), HUMAlACM_PEA_2_node_8 (SEQ ID NO:48), or HUMAl ACMJPE A_2_node_9 (SEQ ID NO:49).
3. An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), or HUMA1ACM_PEA_2_P59 (SEQ ID NO.53).
4. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS
ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF
NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK
RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90% homologous to
SRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPD QDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIE EAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 129 of Q96DW8 (SEQ ID NO:202), which also corresponds to amino acids 229 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence SL corresponding to amino acids 358 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
5. The polypeptide of claim 4, wherein said first amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
6. The polypeptide of claim 4, wherein said first amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51).
7. The polypeptide of claim 4, wherein said first amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
8. The polypeptide of claim 4, wherein said first amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) corresponding to amino acids 1 - 228 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51).
9. An isolated polypeptide encoding for a head of HUMA1ACM_PEA_2JP36 (SEQ ID NO:51), comprising a polypeptide being at least 70%, homologous to the sequence
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) OF HUMAL ACM_PEA_2_P36
(SEQ ID NO:51).
10. The polypeptide of claim 9, wherein said head is at least about 80% homologous to the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDICNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN YIFFKAKWEMPFDPQDTHQ (SEQ ID NO:180) OF HUMA1ACM_PEA_2_P36
(SEQ ID NO:51).
11. The polypeptide of claim 9, wherein said head is at least about 85% homologous to the sequence
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAF ATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVN YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) OF HUMA1ACM_PEA_2_P36
(SEQ ID NO:51).
12. The polypeptide of claim 9, wherein said head is at least about 90% homologous to the sequence MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN YIFFKAKWEMPFDPQDTHQ (SEQ ID NO:180) of HUMAl ACMJPE A_2_P36 (SEQ ID NO:51).
13. The polypeptide of claim 9, wherein said head is at least about 95% homologous to the sequence
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RL YGSEAFATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTMMVLVN YIFFKAKWEMPFDPQDTHQ (SEQ ID NO: 180) OF HUMAL ACM_PEA_2_P36
(SEQ ID NO:51).
14. An isolated chimeric polypeptide encoding for HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 70% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), second amino acid sequence being at least 90% homologous to
FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDF AFSLYKQLVLKA
PDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLL
RTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAA
AKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWEMPFDPQDT
HQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFIL
PDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQL
GIEEAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 341 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 357 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), and a third amino acid sequence SL corresponding to amino acids 358 - 359 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
15. The polypeptide of claim 14, wherein said first amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
16. The polypeptide of claim 14, wherein said first amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
17. The polypeptide of claim 14, wherein said first amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
18. The polypeptide of claim 14, wherein said first amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
19. An isolated polypeptide encoding for a head of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a polypeptide being at least 70%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P36 (SEQ ID NO-.51).
20. The polypeptide of claim 19, wherein said head is at least about 80% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACMJPEA_2_P36 (SEQ ID NO:51).
21. The polypeptide of claim 19, wherein said head is at least about 85% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
22. The polypeptide of claim 19, wherein said head is at least about 90% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
23. The polypeptide of claim 19, wherein said head is at least about 95% homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
24. An isolated chimeric polypeptide encoding for HUMAl ACMJPE A_2_P36 (SEQ ID NO:51), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence being at least 70%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR
DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI
GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO: 181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
25. The polypeptide of claim 24, wherein said second amino acid sequence is at least about 80% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO:181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
26. The polypeptide of claim 24, wherein said second amino acid sequence is at least about 85% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR
DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI
GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO: 181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
27. The polypeptide of claim 24, wherein said second amino acid sequence is at least about 90% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR
DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI
GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO:181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
28. The polypeptide of claim 24, wherein said second amino acid sequence is at least about 95% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR
DEELSCTWELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI
GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO:181) corresponding to amino acids 47 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
29. An isolated polypeptide encoding for a tail of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), comprising a polypeptide being at least 70% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRL YGSEAF ATDFQDSAAAKKLΓND YVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO: 181) in HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
30. The polypeptide of claim 29, wherein said tail is at least about 80% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAF ATDFQDSAAAKKLIND YVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO: 181) in HUMAl ACMJPE A_2_P36 (SEQ ID NO:51).
31. The polypeptide of claim 29, wherein said tail is at least about 85% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR DEELSCTVVELKYTGNAS ALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO:181) in HUMAl ACMJPEA_2_P36 (SEQ ID NO:51).
32. The polypeptide of claim 29, wherein said tail is at least about 90% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL (SEQ ID NO:181) in HUMAl ACMJPE A_2JP36 (SEQ ID NO:51).
33. The polypeptide of claim 29, wherein said tail is at least about 95% homologous to the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFR
DEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREI
GELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVSL
(SEQ ID NO:181) in HUMAl ACM_PEA_2_P36 (SEQ ID NO:51).
34. An isolated chimeric polypeptide encoding for HUMA1ACM PEA 2 P36 (SEQ ID NO: 51), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS
ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF
NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK
RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELS
CTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELY
LPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQV corresponding to amino acids 1 - 357 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 357 of HUMA1ACM_PEA_2_P36 (SEQ ID NO:51), and a second amino acid sequence SL corresponding to amino acids 358 - 359 of HUMAl ACM_PEA_2_P36 (SEQ ID NO:51), wherein said first and second amino acid sequences are contiguous and in a sequential order.
35. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 70% homologous to a polypeptide having the sequence MERMLPLL ALGLL AAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), second amino acid sequence being at least 90% homologous to FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDF AFSLYKQL VLKA PDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLL RTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAA AKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17-214 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a third amino acid sequence ER corresponding to amino acids 215-216 of HUMAl ACMNPE A_2_P49 (SEQ ID NO:52), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
36. The polypeptide of claim 35, wherein said first amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence MERMLPLL ALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
37. The polypeptide of claim 35, wherein said first amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACMJPEA_2_P49 (SEQ ID NO:52).
38. The polypeptide of claim 35, wherein said first amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
39. The polypeptide of claim 35, wherein said first amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
40. An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P49 (SEQ ID NO: 52), comprising a polypeptide being at least 70%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMA1ACM_PEA_2_P49 (SEQ ID NO:52).
41. The polypeptide of claim 40, wherein said head is at least about 80%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
42. The polypeptide of claim 40, wherein said head is at least about 85%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM JPE A_2_P49 (SEQ ID NO:52).
43. The polypeptide of claim 40, wherein said head is at least about 90%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACMJ?EA_2_P49 (SEQ ID NO:52).
44. The polypeptide of claim 40, wherein said head is at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
45. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P49 (SEQ ID NO: 52), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMA1ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
46. An isolated chimeric polypeptide encoding for HUMAl ACMJPE A_2_P49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), and a second amino acid sequence being at least 70% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKER (SEQ ID NO: 183) corresponding to amino acids 47 - 216 of HUMA1ACM_PEA__2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
47. The polypeptide of claim 46, wherein said second amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO:183) corresponding to amino acids 47 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
48. The polypeptide of claim 46, wherein said second amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO: 183) corresponding to amino acids 47 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
49. The polypeptide of claim 46, wherein said second amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKER (SEQ ID NO:183) corresponding to amino acids 47 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
50. The polypeptide of claim 46, wherein said second amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKER (SEQ ID NO:183) corresponding to amino acids 47 - 216 of HUMAl ACMJPEA_2_P49 (SEQ ID NO:52).
51. An isolated polypeptide encoding for a tail of HUMAl ACMJPE A_2_P49 (SEQ ID NO: 52), comprising a polypeptide being at least 70% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO: 183) in HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
52. The polypeptide of claim 51, wherein said tail is at least about 80%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO:183) in HUMA1ACM_PEA_2_P49 (SEQ ID NO:52).
53. The polypeptide of claim 51, wherein said tail is at least about 85%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO: 183) IN HUMA1ACM_PEA_2_P49 (SEQ ID
NO:52).
54. The polypeptide of claim 51 , wherein said tail is at least about 90%, homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO: 183) in HUMAl ACM_PEA_2_P49 (SEQ ID NO:52).
55. The polypeptide of claim 51, wherein said tail is at least about 95%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKER (SEQ ID NO: 183) IN HUMA1ACM_PEA_2_P49 (SEQ ID
NO:52).
56. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2JP49 (SEQ ID NO:52), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN YIFFK corresponding to amino acids 1 - 214 of AACT-HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACMJPEA 2JP49 (SEQ ID NO:52), and a second amino acid sequence having the sequence ER corresponding to amino acids 215 - 216 of HUMAl ACM_PEA_2_P49 (SEQ ID NO:52), wherein said first and second amino acid sequences are contiguous and in a sequential order.
57. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2J>59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 70% homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), second amino acid sequence being at least 90 % homologous to
FCP AVLCHPNSPLDEENLTQENQDRGTHVDLGLASANVDF AFSLYKQLVLKA
PDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLL
RTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAA
AKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFK corresponding to amino acids 1 - 198 of Q9UNU9 (SEQ ID NO:203), which also corresponds to amino acids 17 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a third amino acid sequence being at least 70% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
58. The polypeptide of claim 57, wherein said first amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
59. The polypeptide of claim 57, wherein said first amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
60. The polypeptide of claim 57, wherein said first amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
61. The polypeptide of claim 57, wherein said first amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
62. The polypeptide of claim 57, wherein said third amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM__PEA_2_P59 (SEQ ID NO:53).
63. The polypeptide of claim 57, wherein said third amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
64. The polypeptide of claim 57, wherein said third amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
65. The polypeptide of claim 57, wherein said third amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
66. An isolated polypeptide encoding for a head of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70%, homologous to the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
67. The polypeptide of claim 66, wherein said head is at least about 80%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACMJPE A_2_P59 (SEQ ID NO:53).
68. The polypeptide of claim 66, wherein said head is at least about 85%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
69. The polypeptide of claim 66, wherein said head is at least about 90%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
70. The polypeptide of claim 66, wherein said head is at least about 95%, homologous to a polypeptide having the sequence MERMLPLLALGLLAAG (SEQ ID NO: 182) corresponding to amino acids 1 - 16 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
71. An isolated polypeptide encoding for a tail of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
72. The polypeptide of claim 71, wherein said tail is at least about 80%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
73. The polypeptide of claim 71, wherein said tail is at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
74. The polypeptide of claim 71, wherein said tail is at least about 90%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
75. The polypeptide of claim 71, wherein said tail is at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
76. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ ID NO: 53), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN YIFFK corresponding to amino acids 1 - 214 of Q8N177 (SEQ ID NO:206), which also corresponds to amino acids 1 - 214 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
77. The polypeptide of claim 76, wherein said second amino acid sequence is at least about 80%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
78. The polypeptide of claim 76, wherein said second amino acid sequence is at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
79. The polypeptide of claim 76, wherein said second amino acid sequence is at least about 90%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
80. The polypeptide of claim 76, wherein said second amino acid sequence is at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
81. An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), comprising a polypeptide being at least 70% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
82. The polypeptide of claim 81, wherein said tail is at least about 80%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
83. The polypeptide of claim 81, wherein said tail is at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
84. The polypeptide of claim 81, wherein said tail is at least about 90%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
85. The polypeptide of claim 81, wherein said tail is at least about 95%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
86. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVD corresponding to amino acids 1 - 46 of AAA51559 (SEQ ID NO:204), which also corresponds to amino acids 1 - 46 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACMJPE A_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
87. The polypeptide of claim 86, wherein said second amino acid sequence being at least about 80% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM JPEA_2_P59 (SEQ ID NO:53).
88. The polypeptide of claim 86 wherein said second amino acid sequence being at least about 85%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
89. The polypeptide of claim 86, wherein said second amino acid sequence being at least about 90% homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
90. The polypeptide of claim 86, wherein said second amino acid sequence being at least about 95% homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
91. An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2JP59 (SEQ ID NO: 53), comprising a polypeptide being at least 70% homologous to the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
92. The polypeptide of claim 91, wherein said tail is at least about 80% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53).
93. The polypeptide of claim 91 wherein said tail is at least about 85%, homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO.208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
94. The polypeptide of claim 91, wherein said tail is at least about 90% homologous to a polypeptide having the sequence
LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL
KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF
TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM
MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
95. The polypeptide of claim 91, wherein said tail is at least about 95% homologous to a polypeptide having the sequence LGLASANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEIL KGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRF TEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTM MVLVNYIFFKGECAWLGVQKRWISGPFLS (SEQ ID NO:208) corresponding to amino acids 47 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
96. An isolated chimeric polypeptide encoding for HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), comprising a first amino acid sequence being at least 90 % homologous to
MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLAS ANVDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKF NLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAK RLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVN
YIFFK corresponding to amino acids 1 - 214 of AACT_HUMAN (SEQ ID NO:205), which also corresponds to amino acids 1 - 214 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53), and a second amino acid sequence being at least 70% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53), wherein said first and second amino acid sequences are contiguous and in a sequential order.
97. The polypeptide of claim 96, wherein said second amino acid sequence being at least about 80% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
98. The polypeptide of claim 96 wherein said second amino acid sequence being at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215
- 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
99. The polypeptide of claim 96, wherein said second amino acid sequence being at least about 90% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO:185) corresponding to amino acids 215
- 233 of HUMA1ACM_PEA_2_P59 (SEQ ID NO:53).
100. The polypeptide of claim 96, wherein said second amino acid sequence being at least about 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) corresponding to amino acids 215 - 233 of HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
101. An isolated polypeptide encoding for a tail of HUMAl ACM_PEA_2JP59 (SEQ ID NO:53), comprising a polypeptide being at least 70% homologous to the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
102. The polypeptide of claim 101, wherein said tail is at least about 80% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
103. The polypeptide of claim 101 wherein said tail is at least about 85%, homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
104. The polypeptide of claim 101, wherein said tail is at least about 90% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
105. The polypeptide of claim 101, wherein said tail is at least about 95% homologous to a polypeptide having the sequence GECAWLGVQKRWISGPFLS (SEQ ID NO: 185) in HUMAl ACM_PEA_2_P59 (SEQ ID NO:53).
106. The isolated oligonucleotide of claim 1, comprising an amplicon according to any one of the SEQ ID NOs: 56 or 59.
107. A primer pair, comprising a pair of isolated oligonucleotides capable of amplifying said amplicon of claim 106.
108. The primer pair of claim 107, comprising a pair of isolated oligonucleotides: SEQ NOs 54 and 55, or 57 and 58.
109. An antibody capable of specifically binding to an epitope of an amino acid sequence of claim 3.
110. The antibody of claim 109, wherein said amino acid sequence corresponds to a bridge, edge portion, tail, or head as in any of the previous claims.
111. The antibody of claim 109, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
112. A kit for detecting a Marker-detectable disease, comprising a kit detecting specific expression of a splice variant according to any of the above claims.
113. The kit of claim 112, wherein said kit comprises a NAT-based technology.
114. The kit of claim 113, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
115. The kit of claim 113, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence according to any of the above claims.
116. The kit of claim 112, wherein said kit comprises an antibody according to any of the above claims.
117. The kit of claim 116, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.
118. A method for detecting a Marker-detectable disease, comprising detecting specific expression of a splice variant according to any of the above claims.
119. The method of claim 118, wherein said marker-detectable disease is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer.
120. The method of claim 118, wherein said detecting specific expression is performed with a NAT-based technology.
121. The method of claim 118, wherein said detecting specific expression is performed with an immunoassay.
122. The method of claim 121, wherein said immunoassay comprises an antibody according to any of the above claims.
123. A biomarker capable of detecting Marker-detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
124. A method for screening for variant-detectable disease, comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
125. A method for diagnosing a marker-detectable disease, comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
126. A method for monitoring disease progression and/or treatment efficacy and/or relapse of Marker-detectable disease, comprising detecting cells affected by Marker- detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
127. A method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
128. The method of any one of the claims 124-127, wherein said marker-detectable disease is selected from the group consisting of chronic lung diseases, pulmonary embolism, stroke, lung cancer, colon cancer, ovarian cancer, and prostate cancer.
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