CA2403909A1

CA2403909A1 - Compositions and methods for the therapy and diagnosis of prostate cancer

Info

Publication number: CA2403909A1
Application number: CA002403909A
Authority: CA
Inventors: Jiangchun Xu; Davin C. Dillon; Jennifer L. Mitcham; Susan L. Harlocker; Yuqiu Jiang; Michael D. Kalos; Gary Richard Fanger; Marc W. Retter; John A. Stolk; Craig H. Day; Thomas S. Vedvick; Darrick Carter; Samuel X. Li; Aijun Wang; Yasir A. W. Skeiky; William T. Hepler; Robert A. Henderson
Original assignee: Individual
Current assignee: Corixa Corp
Priority date: 2000-03-27
Filing date: 2001-03-27
Publication date: 2001-10-04
Also published as: EP1311673A2; WO2001073032A3; AU2001249549A1; JP2004504808A; WO2001073032A2

Abstract

Compositions and methods for the therapy and diagnosis of cancer, particular ly prostate cancer, are disclosed. Illustrative compositions comprise one or mo re prostate-specific polypeptides, immunogenic portions thereof, polynucleotide s that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly prostate cancer.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

~~ TTENANT LES PAGES 212 A 288 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

COMPOSITIONS AND METHODS FOR THE THERAPY AND DIAGNOSIS OF
PROSTATE CANCER
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to therapy and diagnosis of cancer, such as prostate cancer. The invention is more specifically related to polypeptides, comprising at least a portion of a prostate-specific protein, and to polynucleotides encoding such polypeptides. Such polypeptides and polynucleotides are useful in pharmaceutical compositions, e.g., vaccines, and other compositions for the diagnosis and treatment of prostate cancer.
BACKGROUND OF THE INVENTION
Cancer is a significant health problem throughout the world. Although Cancer is a significant health problem throughout the world. Although advances have been made in detection and therapy of cancer, no vaccine or other universally successful method for prevention or treatment is currently available. Current therapies, which are generally based on a combination of chemotherapy or surgery and radiation, continue to prove inadequate in many patients.
Prostate cancer is the most common forth of cancer among males, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has the propensity to metastasize to bone, and the disease appears to progress inevitably from androgen dependent to androgen refractory status, leading to increased patient mortality. This prevalent disease is currently the second leading cause of cancer death among men in the U.S.
In spite of considerable research into therapies for the disease, prostate cancer remains difficult to treat. Commonly, treatment is based on surgery and/or radiation therapy, but these methods are ineffective in a significant percentage of cases.
Two previously identified prostate specific proteins - prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) - have limited therapeutic and diagnostic potential. For example, PSA levels do not always correlate well with the presence of prostate cancer, being positive in a percentage of non-prostate cancer cases, including benign prostatic hyperplasia (BPH). Furthermore, PSA measurements correlate with prostate volume, and do not indicate the level of metastasis.
In spite of considerable research into therapies for these and other cancers, prostate cancer remains difficult to diagnose and treat effectively.
Accordingly, there is a need in the art for improved methods for detecting and treating such cancers.
The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides polynucleotide compositions comprising a sequence selected from the group consisting of (a) sequences provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(b) complements of the sequences provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 93 8, 93 9 and 942;
(c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(d) sequences that hybridize to a sequence provided in SEQ ID NO:
1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942, under moderately stringent conditions;
(e) sequences having at least 75% identity to a sequence of SEQ ID
NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(f) sequences having at Ieast 90% identity to a sequence of SEQ ID
NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942; and (g) degenerate variants of a sequence provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942.
In one preferred embodiment, the polynucleotide compositions of the invention are expressed in at least about 20%, more preferably in at least about 30%, and most preferably in at least about 50% of prostate tissue samples tested, at a level that is at least about 2-fold, preferably at least about 5-fold, and most preferably at least about 10-fold higher than that for other normal tissues.
The present invention, in another aspect, provides polypeptide compositions comprising an amino acid sequence that is encoded by a polynucleotide sequence described above.
The present invention further provides polypeptide compositions comprising an amino acid sequence selected from the group consisting of sequences recited in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 866-877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943.
In certain preferred embodiments, the polypeptides and/or polynucleotides of the present invention are immunogenic, i. e., they are capable of eliciting an immune response, particularly a humoral and/or cellular immune response, as further described herein.
The present invention further provides fragments, variants and/or derivatives of the disclosed polypeptide and/or polynucleotide sequences, wherein the fragments, variants and/or derivatives preferably have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% of the level of immunogenic activity of a polypeptide sequence set forth in SEQ ID
NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858 or 860-862, or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID
NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942.
The present invention further provides polynucleotides that encode a polypeptide described above, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
Within other aspects, the present invention provides pharmaceutical compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
Within a related aspect of the present invention, pharmaceutical compositions, e.g., vaccine compositions, are provided for prophylactic or therapeutic applications. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the invention and an immunostimulant, such as an adjuvant, together with a physiologically acceptable carrier.

The present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of the present invention, or a fragment thereof; and (b) a physiologically acceptable carrier.

5 Within further aspects, the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient.
Illustrative antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.
Within related aspects, pharmaceutical compositions are provided that comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) an immunostimulant.
The present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins, typically in the form of pharmaceutical compositions, e.g., vaccine compositions, comprising a physiologically acceptable carrier and/or an immunostimulant. The fusions proteins may comprise multiple immunogenic polypeptides or portions/variants thereof, as described herein, and may further comprise one or more polypeptide segments for facilitating and/or enhancing the expression, purification and/or immunogenicity of the polypeptide(s).
Within further aspects, the present invention provides methods for stimulating an immune response in a patient, preferably a T cell response in a human patient, comprising administering a pharmaceutical composition described herein. The patient may be afflicted with prostate cancer, in which case the methods provide treatment for the disease, or a patient considered to be at risk for such a disease may be treated prophylactically.
Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition as recited above. The patient may be afflicted with prostate cancer, in which case the methods provide treatment for the disease, or a patient considered to be at risk for such a disease may be treated prophylactically.
The present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a polypeptide of the present invention, wherein the step of contacting is performed under conditions and for a time suff cient to permit the removal of cells expressing the polypeptide from the sample.
Within related aspects, methods are provided for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated as described above.
Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a polypeptide of the present invention, comprising contacting T cells with one or more of: (i) a polypeptide as described above;
(ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Isolated T cell populations comprising T cells prepared as described above are also provided.
Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
The present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of (a) incubating CD4+
and/or CD8+ T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of polypeptide disclosed herein;
(ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen-presenting cell that expressed such a polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, thereby inhibiting the development of a cancer in the patient.
Proliferated cells may, but need not, be cloned prior to administration to the patient.
Within further aspects, the present invention provides methods for determining the presence or absence of a cancer, preferably a prostate cancer, in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within preferred embodiments, the binding agent is an antibody, more preferably a monoclonal antibody.
The present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient. Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b), and therefrom monitoring the progression of the cancer in the patient.
The present invention further provides, within other aspects, methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide of the present invention; (b) detecting in the sample a level of a polynucleotide, preferably mRNA, that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within certain embodiments, the amount of mRNA is detected via polymerase chain reaction using, fox example, at least one oligonucleotide primer that hybridizes to' a polynucleotide of the present invention, or a complement of such a polynucleotide. Within other embodiments, the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to an inventive polynucleotide, or a complement of such a polynucleotide.
In related aspects, methods are provided for monitoring the progression of a cancer in a patient, comprising the steps of (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide of the present invention; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b), and therefrom monitoring the progression of the cancer in the patient.
Within further aspects, the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings.
All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS
Figure 1 illustrates the ability of T cells to kill fibroblasts expressing the representative prostate-specific polypeptide P502S, as compared to control fibroblasts.
The percentage lysis is shown as a series of effectoraarget ratios, as indicated.
Figures 2A and 2B illustrate the ability of T cells to recognize cells expressing the representative prostate-specific polypeptide P502S. In each case, the number of y-interferon spots is shown for different numbers of responders. In Figure 2A, data is presented for fibroblasts pulsed with the P2S-12 peptide, as compared to fibroblasts pulsed with a control E75 peptide. In Figure 2B, data is presented for fibroblasts expressing P502S, as compared to fibroblasts expressing HER-2/fzeu.
Figure 3 represents a peptide competition binding assay showing that the P1S#10 peptide, derived from PSOlS, binds HLA-A2. Peptide P1S#10 inhibits HLA-A2 restricted presentation of fluM58 peptide to CTL clone D150M58 in TNF
release bioassay. D150M58 CTL is specific for the HLA-A2 binding influenza matrix peptide fluMS 8.

Figure 4 illustrates the ability of T cell lines generated from P1S#10 immunized mice to specifically lyse P1S#10-pulsed Jurkat A2Kb targets and transduced Jurkat A2Kb targets, as compared to EGFP-transduced Jurkat A2Kb.
The percent lysis is shown as a series of effector to target ratios, as indicated.
Figure 5 illustrates the ability of a T cell clone to recognize and specifically lyse Jurkat A2Kb cells expressing the representative prostate-specific polypeptide PSO1S, thereby demonstrating that the P1S#10 peptide may be a naturally processed epitope of the P501S polypeptide.
Figures 6A and 6B are graphs illustrating the specificity of a CD8+ cell line (3A-1) for a representative prostate-specific antigen (PSO1S). Figure 6A
shows the results of a SICr release assay. The percent specific lysis is shown as a series of effectoraarget ratios, as indicated. Figure 6B shows the production of interferon-gamma by 3A-1 cells stimulated with autologous B-LCL transduced with PSO1S, at varying effectoraarget rations as indicated.
IS Figure 7 is a Western blot showing the expression of PSOlS in baculovirus.
Figure 8 illustrates the results of epitope mapping studies on PSO1S.
Figure 9 is a schematic representation of the P501 S protein showing the location of transmembrane domains and predicted intracellular and extracellulax domains.
Figure 10 is a genomic map showing the location of the prostate genes P775P, P704P, B305D, P712P and P774P within the Cat Eye Syndrome region of chromosome 22q11.2 Figure 11 shows the results of an ELISA assay to determine the specificity of rabbit polyclonal antisera raised against P501 S.
Figures 12A(1), 12A(2), 12A(3), and B are the full-length cDNA (SEQ
m N0:777) and predicted amino acid (SEQ ID N0:778) sequences, respectively, for the clone P788P.
SEQ ID NO: 1 is the determined cDNA sequence for F1-13 SEQ ID NO: 2 is the determined 3' cDNA sequence for F1-12 SEQ ID NO: 3 is the determined 5' cDNA sequence for F1-12 SEQ ID NO: 4 is the determined 3' cDNA sequence for F1-16 SEQ ID NO: 5 is the determined 3' cDNA sequence for Hl-1 SEQ ID NO: 6 is the determined 3' cDNA sequence for H1-9 5 SEQ ID NO: 7 is the determined 3' cDNA sequence for H1-4 SEQ ID NO: 8 is the determined 3' cDNA sequence fox J1-17 SEQ ID NO: 9 is the determined 5' cDNA sequence for J1-17 SEQ ID NO: 10 is the determined 3' cDNA sequence for L1-12 SEQ ID NO: 11 is the determined 5' cDNA sequence for L1-12 10 SEQ ID NO: 12 is the deternlined 3' cDNA sequence for N1-1862 SEQ ID NO: 13 is the determinedcDNA sequence for 5' N1-1862 SEQ ID NO: 14 is the determinedcDNA sequence for 3' J1-13 SEQ ID NO: 15 is the determinedcDNA sequence for 5' J1-13 SEQ ID NO: 16 is the determinedcDNA sequence for 3' J1-19 SEQ ID NO: 17 is the determined 5' cDNA sequence for J1-19 SEQ ID NO: 18 is the determined 3' cDNA sequence for Jl-25 SEQ ID NO: 19 is the determined 5' cDNA sequence for J1-25 SEQ ID NO: 20 is the determined 5' cDNA sequence for J1-24 SEQ ID NO: 21 is the determined 3' cDNA sequence for J1-24 SEQ ID NO: 22 is the determined 5' cDNA sequence for KI-58 SEQ ID NO: 23 is the determined 3' cDNA sequence for Kl-58 SEQ ID NO: 24 is the determined 5' cDNA sequence for K1-63 SEQ ID NO: 25 is the determined 3' cDNA sequence for K1-63 SEQ ID NO: 26 is the determined 5' cDNA sequence for L1-4 SEQ ID NO: 27 is the determined 3' cDNA sequence for Ll-4 SEQ ID NO: 28 is the determined 5' cDNA sequence for Ll-14 SEQ ID NO: 29 is the determined 3' cDNA sequence for L1-14 SEQ ID NO: 30 is the determined 3' cDNA sequence for Jl-12 SEQ ID NO: 31 is the determined 3' cDNA sequence for Jl-16 SEQ ID NO: 32 is the determined 3' cDNA sequence for J1-21 SEQ ID NO: 33 is the determined 3' cDNA sequence for K1-48 SEQ ID NO: 34 is the determined 3' cDNA sequence for Kl-SS
SEQ ID NO: 3S is the determined 3' cDNA sequence for L1-2 SEQ ID NO: 36 is the determined 3' cDNA sequence for L1-6 SEQ ID NO: 37 is the determined 3' cDNA sequence for N1-1858 SEQ ID NO: 38 is the determined 3' cDNA sequence for Nl-1860 SEQ ID NO: 39 is the determined 3' cDNA sequence for Nl-1861 SEQ ID NO: 40 is the determined 3' cDNA sequence for N1-1864 SEQ ID NO: 41 is the determined cDNA sequence for PS
SEQ ID NO: 42 is the determined cDNA sequence for P8 SEQ ID NO: 43 is the determined cDNA sequence for P9 l SEQ ID NO: 44 is the determined cDNA sequence for P 18 SEQ ID NO: 4S is the determined cDNA sequence for P20 SEQ ID NO: 46 is the determined cDNA sequence for P29 1 S SEQ ID NO: 47 is the determined cDNA sequence fox P30 SEQ ID NO: 48 is the determined cDNA sequence for P34 SEQ ID NO: 49 is the determined cDNA sequence for P36 SEQ ID NO: SO is the determined cDNA sequence for P38 SEQ ID NO: Sl is the determined cDNA sequence for P39 SEQ ID NO: S2 is the determined cDNA sequence for P42 SEQ ID NO: S3 is the determined cDNA sequence for P47 SEQ ID NO: S4 is the determined cDNA sequence for P49 SEQ ID NO: SS is the determined cDNA sequence for PSO
SEQ ID NO: S6 is the determined cDNA sequence for PS3 ZS SEQ ID NO: S7 is the determined cDNA sequence for PSS
SEQ ID NO: S8 is the determined cDNA sequence for P60 SEQ ID NO: S9 is the determined cDNA sequence for P64 SEQ ID NO: 60 is the determined cDNA sequence for P65 SEQ ID NO: 61 is the determined cDNA sequence for P73 SEQ ID NO: 62 is the determined cDNA sequence for P7S

SEQ ID NO: 63 is the determined cDNA sequence for P76 SEQ ID NO: 64 is the determined cDNA sequence for P79 SEQ ID NO: 65 is the determined cDNA sequence for P84 SEQ ID NO: 66 is the determined cDNA sequence for F68 SEQ ID NO: 67 is the determined cDNA sequence for P80 (also referred to as P704P) SEQ ID NO: 68 is the determined cDNA sequence for P82 SEQ ID NO: 69 is the determined cDNA sequence for U1-3064 SEQ ID NO: 70 is the determined cDNA sequence for U1-3065 SEQ ID NO: 71 is the determined cDNA sequence for V 1-3692 SEQ ID NO: 72 is the determined cDNA sequence for 1A-3905 SEQ ID NO: 73 is the determined cDNA sequence for V 1-3686 SEQ ID NO: 74 is the determined cDNA sequence for R1-2330 SEQ ID NO: 75 is the determined cDNA sequence for 1B-3976 SEQ ID NO: 76 is the determined cDNA sequence for Vl-3679 SEQ ID NO: 77 is the determined cDNA sequence for 1 G-4736 SEQ ID NO: 78 is the determined cDNA sequence for 1G-4738 SEQ ID NO: 79 is the determined cDNA sequence for 1 G-4741 SEQ ID NO: 80 is the determined cDNA sequence for 1 G-4744 SEQ ID NO: 81 is the determined cDNA sequence for 1 G-4734 SEQ ID NO: 82 is the determined cDNA sequence for 1 H-4774 SEQ ID NO: 83 is the determined cDNA sequence for 1 H-4781 SEQ ID NO: 84 is the determined cDNA sequence for 1H-4785 SEQ ID NO: 85 is the determined cDNA sequence for 1H-4787 SEQ ID NO: 86 is the determined cDNA sequence for 1H-4796 SEQ ID NO: 87 is the determined cDNA sequence for 1I-4807 SEQ ID NO: 88 is the determined cDNA sequence for 1I-4810 SEQ ID NO: 89 is the determined cDNA sequence for l I-4811 SEQ ID NO: 90 is the determined cDNA sequence for 1J-4876 SEQ ID NO: 91 is the determined cDNA sequence for lI~-4884 SEQ ID NO: 92 is the determined cDNA sequence for 1K-4896 SEQ ID NO: 93 is the determined cDNA sequence for 1G-4761 SEQ ID NO: 94 is the determined cDNA sequence for 1 G-4762 SEQ ID NO: 9S is the determined cDNA sequence for 1H-4766 S SEQ ID NO: 96 is tile determined cDNA sequence for 1H-4770 SEQ ID NO: 97 is the determined cDNA sequence for 1 H-4771 SEQ ID NO: 98 is the determined cDNA sequence for 1H-4772 SEQ ID NO: 99 is the determined cDNA sequence for 1D-4297 SEQ ID NO: 100 is the determined cDNA sequence for 1D-4309 SEQ ID NO: 101 is the determined cDNA sequence for 1D.1-4278 SEQ ID NO: 102 is the determined cDNA sequence for 1D-4288 SEQ ID NO: 103 is the determined cDNA sequence fox 1D-4283 SEQ ID NO: 104 is the determined cDNA sequence for 1D-4304 SEQ ID NO: l OS is the determined cDNA sequence for I D-4296 1 S SEQ ID NO: 106 is the determined cDNA sequence for 1 D-4280 SEQ ID NO: 107 is the determined full length cDNA sequence for Fl-12 (also referred to as PS04S) SEQ ID NO: 108 is the predicted amino acid sequence for Fl-12 SEQ ID NO: 109 is the determined full length cDNA sequence for Jl-17 SEQ ID NO: 110 is the determined full length cDNA sequence for Ll-12 (also referred to as PSOl S) SEQ ID NO: 111 is the determined full length cDNA sequence for Nl-1862 (also referred to as PS03S) SEQ ID NO: 112 is the predicted amino acid sequence for Jl-17 2S SEQ ID NO: 113 is the predicted amino acid sequence for LI-12 (also referred to as PSOl S) SEQ ID NO: 114 is the predicted amino acid sequence for N1-1862 (also referred to as PS03S) SEQ ID NO: 11 S is the determined cDNA sequence for P89 SEQ ID NO: 116 is the determined cDNA sequence for P90 SEQ ID NO: 117 is the determined cDNA sequence for P92 SEQ ID NO: 118 is the determined cDNA sequence for P95 SEQ ID NO: 119 is the determined cDNA sequence for P98 SEQ ID NO: 120 is the determined cDNA sequence for P102 SEQ ID NO: 121 is the determined cDNA sequence for P 110 SEQ ID NO: 122 is the determined cDNA sequence for P 111 SEQ ID NO: 123 is the determined cDNA sequence for P114 SEQ ID NO: 124 is the determined cDNA sequence for P115 SEQ ID NO: 125 is the determined cDNA sequence for Pl 16 SEQ ID NO: 126 is the determined cDNA sequence for P124 SEQ ID NO: 127 is the determined cDNA sequence for P126 SEQ ID NO: 128 is the determined cDNA sequence for P130 SEQ ID NO: 129 is the determined cDNA sequence for P133 SEQ ID NO: 130 is the determined cDNA sequence for P138 SEQ ID NO: 131 is the determined cDNA sequence for P143 SEQ ID NO: 132 is the determined cDNA sequence for P 151 SEQ ID NO: 133 is the determined cDNA sequence for P156 SEQ ID NO: 134 is the determined cDNA sequence for P157 SEQ ID NO: 135 is the determined cDNA sequence for P166 SEQ ID NO: 136 is the determined cDNA sequence for P176 SEQ ID NO: 137 is the determined cDNA sequence for P178 SEQ ID NO: 138 is the determined cDNA sequence for P179 SEQ ID NO: 139 is the determined cDNA sequence for P185 SEQ ID NO: 140 is the determined cDNA sequence for P192 SEQ ID NO: 141 is the determined cDNA sequence for P201 SEQ ID NO: 142 is the determined cDNA sequence for P204 SEQ ID NO: 143 is the determined cDNA sequence for P208 SEQ ID NO: 144 is the determined cDNA sequence for P211 SEQ ID NO: 145 is the determined cDNA sequence for P213 SEQ ID NO: 146 is the determined cDNA sequence for P219 SEQ ID NO: 147 is the determined cDNA sequence for P237 SEQ ID NO: 148 is the determined cDNA sequence for P239 SEQ ID NO: 149 is the determined cDNA sequence for P248 SEQ ID NO: 150 is the determined cDNA sequence for P251 5 SEQ ID NO: 151 is the determined cDNA sequence for P255 SEQ ID NO: 152 is the determined cDNA sequence for P256 SEQ ID NO: 153 is the determined cDNA sequence for P259 SEQ ID NO: 154 is the determined cDNA sequence for P260 SEQ ID NO: 155 is the determined cDNA sequence for P263 10 SEQ ID NO: 156 is the determined cDNA sequence for P264 SEQ ID NO: 157 is the determined cDNA sequence for P266 SEQ ID NO: 158 is the determined cDNA sequence for P270 SEQ ID NO: 159 is the determined cDNA sequence for P272 SEQ ID NO: 160 is the determined cDNA sequence for P278 15 SEQ ID NO: 161 is the determined cDNA sequence for P105 SEQ ID NO: 162 is the determined cDNA sequence for P 107 SEQ ID NO: 163 is the determined cDNA sequence for P137 SEQ ID NO: 164 is the determined cDNA sequence for P 194 SEQ ID NO: 165 is the determined cDNA sequence for P195 SEQ ID NO: 166 is the determined cDNA sequence for P196 SEQ ID NO: 167 is the determined cDNA sequence for P220 SEQ ID NO: 168 is the determined cDNA sequence for P234 SEQ ID NO: 169 is the determined cDNA sequence for P235 SEQ ID NO: 170 is the determined cDNA sequence for P243 SEQ ID NO: 171 is the determined cDNA sequence for P703P-DE1 SEQ ID NO: 172 is the predicted amino acid sequence for P703P-DEl SEQ ID NO: 173 is the determined cDNA sequence for P703P-DE2 SEQ ID NO: 174 is the determined cDNA sequence for P703P-DE6 SEQ ID NO: 175 is the determined cDNA sequence for P703P-DE13 SEQ ID NO: 176 is the predicted amino acid sequence for P703P-DE13 SEQ ID NO: 177 is the determined cDNA sequence for P703P-DE14 SEQ ID NO: 178 is the predicted amino acid sequence for P703P-DE14 SEQ ID NO: 179 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 180 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 181 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 182 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 183 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 184 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 185 is the determined extended cDNA sequence for 1H-SEQ ID NO: 186 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 187 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 188 is the determined extended cDNA sequence for 1I-SEQ ID NO: 189 is the determined 3' cDNA sequence for l I-4810 SEQ ID NO: 190 is the determined 3' cDNA sequence for l I-4811 SEQ ID NO: 191 is the determined extended cDNA sequence for 1J-SEQ ID NO: 192 is the determined extended cDNA sequence for 1 K-SEQ ID NO: 193 is the determined extended cDNA sequence for 1K-SEQ ID NO: 194 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 195 is the determined extended cDNA sequence for 1 G-SEQ ID NO: 196 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 197 is the determined 3' cDNA sequence for 1H-4770 SEQ ID NO: 198 is the determined 3' cDNA sequence for 1 H-4771 SEQ ID NO: 199 is the determined extended cDNA sequence for 1 H-SEQ ID NO: 200 is the determined extended cDNA sequence for 1D-SEQ ID NO: 201 is the determined extended cDNA sequence for 1D.1-SEQ ID NO: 202 is the determined extended cDNA sequence for 1D-SEQ ID NO: 203 is the determined extended cDNA sequence for 1 D-SEQ ID NO: 204 is the determined extended cDNA sequence for 1 D-SEQ ID NO: 205 is the determined extended cDNA sequence for 1D-SEQ ID NO: 206 is the determined extended cDNA sequence for 1 D-SEQ ID NO: 207 is the determined cDNA sequence for 10-d8fwd SEQ ID NO: 208 is the determined cDNA sequence for 10-HlOcon SEQ ID NO: 209 is the determined cDNA sequence for 11-CBrev SEQ ID NO: 210 is the determined cDNA sequence for 7.g6fwd SEQ ID NO: 211 is the determined cDNA sequence for 7.g6rev SEQ ID NO: 212 is the determined cDNA sequence for 8-b5fwd SEQ ID NO: 213 is the determined cDNA sequence for 8-b5rev SEQ ID NO: 214 is the determined cDNA sequence for 8-b6fwd SEQ ID NO: 215 is the determined cDNA sequence for 8-b6 rev SEQ ID NO: 216 is the determined cDNA sequence for 8-d4fwd SEQ ID NO: 217 is the determined cDNA sequence for 8-d9rev SEQ ID NO: 218 is the determined cDNA sequence for 8-g3fwd SEQ ID NO: 219 is the determined cDNA sequence for 8-g3rev SEQ ID NO: 220 is the determined cDNA sequence for 8-hl lrev SEQ ID NO: 221 is the determined cDNA sequence for g-fl2fwd SEQ ID NO: 222 is the determined cDNA sequence for g-f3rev SEQ ID NO: 223 is the determined cDNA sequence for P509S
SEQ ID NO: 224 is the determined cDNA sequence for PS l OS
SEQ ID NO: 225 is the determined cDNA sequence for P703DE5 SEQ ID NO: 226 is the determined cDNA sequence for 9-Al 1 SEQ ID NO: 227 is the determined cDNA sequence for 8-C6 SEQ ID NO: 228 is the determined cDNA sequence for 8-H7 SEQ ID NO: 229 is the determined cDNA sequence for JPTPN13 SEQ ID NO: 230 is the determined cDNA sequence for JPTPN14 SEQ ID NO: 231 is the determined cDNA sequence for JPTPN23 SEQ ID NO: 232 is the determined cDNA sequence for JPTPN24 SEQ ID NO: 233 is the determined cDNA sequence for JPTPN25 SEQ ID NO: 234 is the determined cDNA sequence for JPTPN30 SEQ ID NO: 235 is the determined cDNA sequence for JPTPN34 SEQ ID NO: 236 is the determined cDNA sequence for PTPN35 SEQ ID NO: 237 is the determined cDNA sequence for JPTPN36 SEQ ID NO: 238 is the determined cDNA sequence for JPTPN38 SEQ ID NO: 239 is the determined cDNA sequence for 3PTPN39 SEQ ID NO: 240 is the determined cDNA sequence for JPTPN40 SEQ ID NO: 241 is the determined cDNA sequence for JPTPN41 SEQ ID NO: 242 is the determined cDNA sequence for JPTPN42 SEQ ID NO: 243 is the determined cDNA sequence for JPTPN45 SEQ ID NO: 244 is the determined cDNA sequence for JPTPN46 SEQ ID NO: 245 is the determined cDNA sequence for JPTPN51 SEQ ID NO: 246 is the determined cDNA sequence for JPTPN56 SEQ ID NO: 247 is the determined cDNA sequence for PTPN64 SEQ ID NO: 248 is the determined cDNA sequence for JPTPN65 SEQ ID NO: 249 is the determined cDNA sequence for JPTPN67 SEQ ID NO: 250 is the determined cDNA sequence for JPTPN76 SEQ ID NO: 251 is the determined cDNA sequence for JPTPN84 SEQ ID NO: 252 is the determined cDNA sequence for JPTPN85 SEQ ID NO: 253 is the determined cDNA sequence for JPTPN86 SEQ ID NO: 254 is the determined cDNA sequence for JPTPN87 SEQ ID NO: 255 is the determined cDNA sequence for JPTPN88 SEQ ID NO: 256 is the determined cDNA sequence for JP1F1 SEQ ID NO: 257 is the determined cDNA sequence for JP1F2 SEQ ID NO: 258 is the determined cDNA sequence for JP1C2 SEQ ID NO: 259 is the determined cDNA sequence fox JP1B1 SEQ ID NO: 260 is the determined cDNA sequence for JP1B2 SEQ ID NO: 261 is the determined cDNA sequence for JP1D3 SEQ ID NO: 262 is the determined cDNA sequence fox JPlA4 SEQ ID NO: 263 is the determined cDNA sequence for JP 1 FS
SEQ ID NO: 264 is the determined cDNA sequence for JP 1 E6 SEQ ID NO: 265 is the determined cDNA sequence for JP1D6 SEQ ID NO: 266 is the determined cDNA sequence for JP1B5 SEQ ID NO: 267 is the determined cDNA sequence for JP1A6 SEQ ID NO: 268 is the determined cDNA sequence for JP1E8 SEQ ID NO: 269 is the determined cDNA sequence for JP1D7 SEQ ID NO: 270 is the determined cDNA sequence for JP 1 D9 SEQ ID NO: 271 is the determined cDNA sequence for JP 1 C 10 SEQ ID NO: 272 is the determined cDNA sequence for JP1A9 SEQ ID NO: 273 is the determined cDNA sequence for JP 1 F 12 SEQ ID NO: 274 is the determined cDNA sequence for JP 1 E 12 SEQ ID NO: 275 is the determined cDNA sequence for JP1D11 SEQ ID NO: 276 is the determined cDNA sequence for JP1C11 SEQ ID NO: 277 is the determined cDNA sequence for JP1C12 SEQ ID NO: 278 is the determined cDNA sequence for JP1B12 SEQ ID NO: 279 is the determined cDNA sequence for JPlAl2 SEQ ID NO: 280 is the determined cDNA sequence for JP8G2 SEQ ID NO: 281 is the determined cDNA sequence for JP8H1 10 SEQ ID NO: 282 is the determined cDNA sequence for JP8H2 SEQ ID NO: 283 is the determined cDNA sequence for JP8A3 SEQ ID NO: 284 is the determined cDNA sequence for JP8A4 SEQ ID NO: 285 is the determined cDNA sequence for JP8C3 SEQ ID NO: 286 is the determined cDNA sequence for JP8G4 1 S SEQ ID NO: 287 is the determined cDNA sequence for JP8B6 SEQ ID NO: 288 is the determined cDNA sequence for JP8D6 SEQ ID NO: 289 is the determined cDNA sequence for JP8F5 SEQ ID NO: 290 is the determined cDNA sequence for JP8A8 SEQ ID NO: 291 is the determined cDNA sequence for JP8C7 20 SEQ ID NO: 292 is the determined cDNA sequence for JP8D7 SEQ ID NO: 293 is the determined cDNA sequence for P8D8 SEQ ID NO: 294 is the determined cDNA sequence for JP8E7 SEQ ID NO: 295 is the determined cDNA sequence for JP8F8 SEQ ID NO: 296 is the determined cDNA sequence for JP8G8 SEQ ID NO: 297 is the determined cDNA sequence for JP8B10 SEQ ID NO: 298 is the determined cDNA sequence for JPBC 10 SEQ ID NO: 299 is the determined cDNA sequence for JP8E9 SEQ ID NO: 300 is the determined cDNA sequence for JP8E10 SEQ ID NO: 301 is the determined cDNA sequence for JP8F9 SEQ ID NO: 302 is the deterniined cDNA sequence for JP8H9 SEQ ID NO: 303 is the determined cDNA sequence for JP8C12 SEQ ID NO: 304 is the determined cDNA sequence for JP8E11 SEQ ID NO: 305 is the determined cDNA sequence for JP8E12 SEQ ID NO: 306 is the amino acid sequence for the peptide PS2#12 SEQ ID NO: 307 is the determined cDNA sequence for P711P
SEQ ID NO: 308 is the determined cDNA sequence for P712P
SEQ ID NO: 309 is the determined cDNA sequence for CLONE23 SEQ ID NO: 310 is the determined cDNA sequence for P774P
SEQ ID NO: 311 is the determined cDNA sequence for P775P
SEQ ID NO: 312 is the determined cDNA sequence for P715P
SEQ ID NO: 313 is the determined cDNA sequence for P710P
SEQ ID NO: 314 is the determined cDNA sequence for P767P
SEQ ID NO: 315 is the determined cDNA sequence for P768P
SEQ ID NO: 316-325 are the determined cDNA sequences of previously isolated genes SEQ ID NO: 326 is the determined cDNA sequence for P703PDE5 SEQ ID NO: 327 is the predicted amino acid sequence for P703PDE5 SEQ ID NO: 328 is the determined cDNA sequence for P703P6.26 SEQ ID NO: 329 is the predicted amino acid sequence for P703P6.26 SEQ ID NO: 330 is the determined cDNA sequence for P703PX-23 SEQ ID NO: 331 is the predicted amino acid sequence for P703PX-23 SEQ ID NO: 332 is the determined full length cDNA sequence for SEQ ID NO: 333 is the determined extended cDNA sequence for P707P
(also referred to as 11-C9) SEQ ID NO: 334 is the determined cDNA sequence for P714P
SEQ ID NO: 335 is the determined cDNA sequence for P705P (also referred to as 9-F3) SEQ ID NO: 336 is the predicted amino acid sequence for P705P
SEQ ID NO: 337 is the amino acid sequence of the peptide Pl S#10 SEQ ID NO: 338 is the amino acid sequence of the peptide p5 SEQ ID NO: 339 is the predicted amino acid sequence of P509S
SEQ ID NO: 340 is the determined cDNA sequence for P778P
SEQ ID NO: 341 is the determined cDNA sequence for P786P
SEQ ID NO: 342 is the determined cDNA sequence for P789P
SEQ ID NO: 343 is the determined cDNA sequence for a clone showing homology to Homo Sapiens MM46 mRNA
SEQ ID NO: 344 is the determined cDNA sequence for a clone showing homology to Homo Sapiens TNF-alpha stimulated ABC protein (ABC50) mRNA
SEQ ID NO: 345 is the determined cDNA sequence for a clone showing homology to Homo Sapiens mRNA for E-cadherin SEQ ID NO: 346 is the determined cDNA sequence for a clone showing homology to Human nuclear-encoded mitochondrial serine hydroxymethyltransferase (SHMT) SEQ ID NO: 347 is the determined cDNA sequence for a clone showing homology to Homo Sapiens natural resistance-associated macrophage protein2 (NRAMP2) SEQ ID NO: 348 is the determined cDNA sequence for a clone showing homology to Homo Sapiens phosphoglucomutase-related protein (PGMRP) SEQ ID NO: 349 is the determined cDNA sequence for a clone showing homology to Human mRNA for proteosome subunit p40 SEQ ID NO: 350 is the determined cDNA sequence for P777P
SEQ ID NO: 351 is the determined cDNA sequence for P779P
SEQ ID NO: 352 is the determined cDNA sequence for P790P
SEQ ID NO: 353 is the determined cDNA sequence for P784P
SEQ ID NO: 354 is the determined cDNA sequence for P776P
SEQ ID NO: 355 is the determined cDNA sequence for P780P
SEQ ID NO: 356 is the determined cDNA sequence for P544S
SEQ ID NO: 357 is the determined cDNA sequence for P745S
SEQ ID NO: 358 is the determined cDNA sequence for P782P

SEQ ID NO: 359 is the determined cDNA sequence for P783P
SEQ ID NO: 360 is the determined cDNA sequence for unknown 17984 SEQ ID NO: 361 is the determined cDNA sequence for P787P
SEQ ID NO: 362 is the determined cDNA sequence for P788P
SEQ ID NO: 363 is the determined cDNA sequence for unknown 17994 SEQ ID NO: 364 is the determined cDNA sequence for P781P
SEQ ID NO: 365 is the determined cDNA sequence for P785P
SEQ ID NO: 366-375 are the determined cDNA sequences for splice variants of B305D.
SEQ ID NO: 376 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 366.
SEQ ID NO: 377 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 372.
SEQ ID NO: 378 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 373.
SEQ ID NO: 379 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 374.
SEQ ID NO: 380 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 375.
SEQ ID NO: 381 is the determined cDNA sequence for B716P.
SEQ ID NO: 382 is the determined full-length cDNA sequence for P711 P.
SEQ ID NO: 383 is the predicted amino acid sequence for P711P.
SEQ ID NO: 384 is the cDNA sequence for P1000C.
SEQ ID NO: 385 is the cDNA sequence for CGI-82.
SEQ ID N0:386 is the cDNA sequence for 23320.
SEQ ID N0:387 is the cDNA sequence fox CGI-69.
SEQ ID N0:388 is the cDNA sequence for L-iditol-2-dehydrogenase.
SEQ ID N0:389 is the cDNA sequence for 23379.
SEQ ID N0:390 is the cDNA sequence fox 23381.

SEQ ID N0:391 is the cDNA sequence for KIAA0122.
SEQ ID N0:392 is the cDNA sequence for 23399.
SEQ ID N0:393 is the cDNA sequence for a previously identified gene.
SEQ ID N0:394 is the cDNA sequence for HCLBP.
SEQ ID N0:395 is the cDNA sequence for transglutaminase.
SEQ ID N0:396 is the cDNA sequence for a previously identified gene.
SEQ ID N0:397 is the cDNA sequence for PAP.
SEQ ID N0:398 is the cDNA sequence for Ets transcription factor PDEF.
SEQ ID N0:399 is the cDNA sequence for hTGR.
SEQ ID N0:400 is the cDNA sequence for KIAA0295.
SEQ ID N0:40I is the cDNA sequence for 22545.
SEQ ID N0:402 is the cDNA sequence for 22547.
SEQ ID N0:403 is the cDNA sequence for 22548.
SEQ ID N0:404 is the cDNA sequence for 22550.
SEQ ID N0:405 is the cDNA sequence for 22551.
SEQ ID N0:406 is the cDNA sequence for 22552.
SEQ ID N0:407 is the cDNA sequence for 22553 (also known as P 1020C).
SEQ ID N0:408 is the cDNA sequence for 22558.
SEQ ID N0:409 is the cDNA sequence for 22562.
SEQ ID N0:410 is the cDNA sequence for 22565.
SEQ ID N0:411 is the cDNA sequence for 22567.
SEQ ID N0:412 is the cDNA sequence for 22568.
SEQ ID N0:413 is the cDNA sequence for 22570.
SEQ ID N0:414 is the cDNA sequence for 22571.
SEQ ID N0:415 is the cDNA sequence for 22572.
SEQ ID N0:416 is the cDNA sequence for 22573.
SEQ ID N0:417 is the cDNA~sequence for 22573.
SEQ ID N0:418 is the cDNA sequence for 22575.

SEQ ID N0:4I9 is the cDNA sequence for 22580.
SEQ ID N0:420 is the cDNA sequence for 22581.
SEQ ID N0:421 is the cDNA sequence for 22582.
SEQ ID N0:422 is the cDNA sequence for 22583.
5 SEQ ID N0:423 is the cDNA sequence for 22584.
SEQ ID N0:424 is the cDNA sequence for 22585.
SEQ ID N0:425 is the cDNA sequence for 22586.
SEQ ID N0:426 is the cDNA sequence for 22587.
SEQ ID N0:427 is the cDNA sequence for 22588.
10 SEQ ID N0:428 is the cDNA sequence for 22589.
SEQ ID N0:429 is the cDNA sequence for 22590.
SEQ ID N0:430 is the cDNA sequence for 22591.
SEQ ID N0:431 is the cDNA sequence for 22592.
SEQ ID N0:432 is the cDNA sequence for 22593.
15 SEQ ID N0:433 is the cDNA sequence for 22594.
SEQ ID N0:434 is the cDNA sequence for 22595.
SEQ ID N0:435 is the cDNA sequence fox 22596.
SEQ ID N0:436 is the cDNA sequence for 22847.
SEQ ID N0:437 is the cDNA sequence for 22848.
20 SEQ ID NO:438 is the cDNA sequence fox 22849.
SEQ ID N0:439 is the cDNA sequence for 22851.
SEQ ID N0:440 is the cDNA sequence for 22852.
SEQ ID NO:441 is the cDNA sequence for 22853.
SEQ ID N0:442 is the cDNA sequence for 22854.

25 SEQ ID N0:443 is the cDNA sequence for 22855.
SEQ ID N0:444 is the cDNA sequence for 22856.
SEQ ID N0:445 is the cDNA sequence for 22857.
SEQ ID N0:446 is the cDNA sequence for 23601.
SEQ ID N0:447 is the cDNA sequence for 23602.
SEQ ID N0:448 is the cDNA sequence for 23605.

SEQ ID N0:449 is the cDNA sequence for 23606.
SEQ ID N0:4S0 is the cDNA sequence for 23612.
SEQ ID N0:4S1 is the cDNA sequence for 23614.
SEQ ID N0:4S2 is the cDNA sequence for 23618.
S SEQ ID N0:4S3 is the cDNA sequence for 23622.
SEQ ID N0:4S4 is the cDNA sequence for folate hydrolase.
SEQ ID N0:4SS is the cDNA sequence for LIM protein.
SEQ ID N0:4S6 is the cDNA sequence for a known gene.
SEQ ID N0:4S7 is the cDNA sequence for a known gene.
SEQ ID N0:4S8 is the cDNA sequence for a previously identified gene.
SEQ ID N0:4S9 is the cDNA sequence for 23045.
SEQ ID N0:460 is the cDNA sequence for 23032.
SEQ ID N0:461 is the cDNA sequence for clone 23054.
SEQ ID N0:462-467 are cDNA sequences for known genes.
1 S SEQ ID N0:468-471 are cDNA sequences for P710P.
SEQ ID N0:472 is a cDNA sequence for P 1001 C.
SEQ ID NO: 473 is the determined cDNA sequence for a first splice variant of P77SP (referred to as 27SOS).
SEQ ID NO: 474 is the determined cDNA sequence for a second splice variant of P77SP (referred to as 19947).
SEQ ID NO: 47S is the determined cDNA sequence for a third splice variant of P77SP (referred to as 19941).
SEQ ID NO: 476 is the determined cDNA sequence for a fourth splice variant of P77SP (referred to as 19937).
2S SEQ ID NO: 477 is a first predicted amino acid sequence encoded by the sequence of SEQ ID NO: 474.
SEQ ID NO: 478 is a second predicted amino acid sequence encoded by the sequence of SEQ ID NO: 474.
SEQ ID NO: 479 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: 475.

SEQ ID NO: 480 is a first predicted amino acid sequence encoded by the sequence of SEQ ID NO: 473.
SEQ ID NO: 481 is a second predicted amino acid sequence encoded by the sequence of SEQ ID NO: 473.
SEQ ID NO: 482 is a third predicted amino acid sequence encoded by the sequence of SEQ ID NO: 473.
SEQ ID NO: 483 is a fourth predicted amino acid sequence encoded by the sequence of SEQ ID NO: 473.
SEQ ID NO: 484 is the first 30 amino acids of the M. tuberculosis 10, antigen Ral2.
SEQ ID NO: 485 is the PCR primer AW025.
SEQ ID NO: 486 is the PCR primer AW003.
SEQ ID NO: 487 is the PCR primer AW027.
SEQ ID NO: 488 is the PCR primer AW026.
SEQ ID NO: 489-501 are peptides employed in epitope mapping studies.
SEQ ID NO: 502 is the determined cDNA sequence of. the complementarity determining region for the anti-P503S monoclonal antibody 20D4.
SEQ ID NO: 503 is the determined cDNA sequence of the complementarity determining region for the anti-P503S monoclonal antibody JA1.
SEQ ID NO: 504 & 505 are peptides employed in epitope mapping studies.
SEQ ID NO: 506 is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 8H2.
SEQ ID NO: 507 is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 7H8.
SEQ ID NO: 508 . is the determined cDNA sequence of the complementarity determining region for the anti-P703P monoclonal antibody 2D4.
SEQ ID NO: 509-522 are peptides employed in epitope mapping studies.
SEQ ID NO: 523 is a mature form of P703P used to raise antibodies against P703P.

SEQ ID NO: 524 is the putative full-length cDNA sequence of P703P.
SEQ ID NO: 525 is the predicted amino acid sequence encoded by SEQ
ID NO: 524.
SEQ ID NO: 526 is the full-length cDNA sequence for P790P.
SEQ ID NO: 527 is the predicted amino acid sequence for P790P.
SEQ ID NO: 528 & 529 are PCR primers.
SEQ ID NO: 530 is the cDNA sequence of a splice variant of SEQ ID
NO: 366.
SEQ ID NO: 531 is the cDNA sequence of the open reading frame of SEQ ID NO: 530.
SEQ ID NO: 532 is the predicted amino acid encoded by the sequence of SEQ ID NO: 531.
SEQ ID NO: 533 is the DNA sequence of a putative ORF of P775P.
SEQ ID NO: 534 is the predicted amino acid sequence encoded by SEQ
ID NO: 533.
SEQ ID NO: 535 is a first full-length cDNA sequence for PS l OS.
SEQ ID NO: 536 is a second full-length cDNA sequence for PS l OS.
SEQ ID NO: 537 is the predicted amino acid sequence encoded by SEQ
ID NO: 535.
SEQ ID NO: 538 is the predicted amino acid sequence encoded by SEQ
ID NO: 536.
SEQ ID NO: 539 is the peptide P501 S-370.
SEQ ID NO: 540 is the peptide P501 S-376.
SEQ ID NO: 541-551 are epitopes of PSOlS.
SEQ ID NO: 552 is an extended cDNA sequence for P712P.
SEQ ID NO: 553-568 are the amino acid sequences encoded by predicted open reading frames within SEQ ID NO: 552.
SEQ ID NO: 569 is an extended cDNA sequence for P776P.
SEQ ID NO: 570 is the determined cDNA sequence for a splice variant of P776P referred to as contig 6.

SEQ ID NO: 571 is the determined cDNA sequence for a splice variant of P776P referred tows contig 7.
SEQ ID NO: 572 is the determined cDNA sequence for a splice variant of P776P referred to as contig 14.
SEQ ID NO: 573 is the amino acid sequence encoded by a first predicted ORF of SEQ ID NO: 570.
SEQ ID NO: 574 is the amino acid sequence encoded by a second predicted ORF of SEQ ID NO: 570.
SEQ ID NO: 575 is the amino acid sequence encoded by a predicted ORF of SEQ ID NO: 571.
SEQ ID NO: 576-586 are amino acid sequences encoded by predicted ORFs of SEQ ID NO: 569.
SEQ ID NO: 587 is a DNA consensus sequence of the sequences of P767P and P777P.
SEQ ID NO: 588-590 are amino acid sequences encoded by predicted ORFs of SEQ ID NO: 587.
SEQ ID NO: 591 is an extended cDNA sequence for P1020C.
SEQ ID NO: 592 is the predicted amino acid sequence encoded by the sequence of SEQ ID NO: P 1020C.
SEQ ID NO: 593 is a splice variant of P775P referred to as 50748.
SEQ ID NO: 594 is a splice variant of P775P referred to as 50717.SEQ
ID NO: 595 is a splice variant of P775P referred to as 45985.
SEQ ID NO: 596 is a splice variant of P775P referred to as 38769.
SEQ ID NO: 597 is a splice variant of P775P referred to as 37922.
SEQ ID NO: 598 is a splice variant of P510S referred to as 49274.
SEQ ID NO: 599 is a splice variant of P510S referred to as 39487.
SEQ ID NO: 600 is a splice variant of P504S referred to as 5167.16.
SEQ ID NO: 601 is a splice variant of P504S referred to as 5167.1.
SEQ ID NO: 602 is a splice variant of P504S referred to as 5163.46.
SEQ ID NO: 603 is a splice variant of P504S referred to as 5163.42.

SEQ ID NO: 604 is a splice variant of P504S referred to as 5163.34.
SEQ ID NO: 605 is a splice variant of P504S referred to as 5163.17.
SEQ ID NO: 606 is a splice variant of P501 S referred to as 10640.
SEQ ID NO: 607-615 are the sequences of PCR primers.
5 SEQ ID NO: 616 is the determined cDNA sequence of a fusion of P703P
and PSA.
SEQ ID NO: 617 is the amino acid sequence of the fusion of P703P and PSA.
SEQ ID NO: 618-689 axe determined cDNA sequences of prostate-10 specific clones.
SEQ ID NO: 690 is the cDNA sequence of the gene DD3.
SEQ ID NO: 691-697 are determined cDNA sequences of prostate-specific clones.
SEQ ID NO: 698 is an extended cDNA sequence for P714P.
15 SEQ ID NO: 699-701 are the cDNA sequences for splice variants of P704P.
SEQ ID NO: 702 is the cDNA sequence of a spliced variant of P553S
referred to as P553S-14.
SEQ ID NO: 703 is the cDNA sequence of a spliced variant of P553S
20 referred to as P553S-12.
SEQ ID NO: 704 is the cDNA sequence of a spliced variant of P553S
referred to as P553S-10.
SEQ ID NO: 705 is the cDNA sequence of a spliced variant of P553S
referred to as P553S-6.
25 o SEQ ID NO: 706 is the amino acid sequence encoded by SEQ ID NO:
705.
SEQ ID NO: 707 is the amino acid sequence encoded by SEQ ID NO:
702 SEQ ID NO: 708 is a second amino acid sequence encoded by SEQ ID NO: 702.
SEQ ID NO: 709-772 are determined cDNA sequences of prostate-30 specific clones.

SEQ ID NO: 773 is a first full-length cDNA sequence for prostate-specific transglutaminase gene (also referred to herein as PS58S).
SEQ ID NO: 774 is a second full-length cDNA sequence for prostate-specific transglutarninase gene.
S SEQ ID NO: 77S is the amino acid sequence encoded by the sequence of SEQ ID NO: 773.
SEQ ID NO: 776 is the amino acid sequence encoded by the sequence of SEQ ID NO: 774.
SEQ ID NO: 777 is the full-length cDNA sequence for P788P.
SEQ ID NO: 778 is the amino acid sequence encoded by SEQ ID NO:
777.
SEQ ID NO: 779 is the determined cDNA sequence for a polymorphic variant of P788P.
SEQ ID NO: 780 is the amino acid sequence encoded by SEQ ID NO:
1 S 779.
SEQ ID NO: 781 is the amino acid sequence of peptide 4 from P703P.
SEQ ID NO: 7$2 is the cDNA sequence that encodes peptide 4 from P703P.
SEQ ID NO: 783-798 are the cDNA sequence encoding epitopes of P703P.
SEQ ID NO: 799-814 are the amino acid sequences of epitopes of P703P.
SEQ ID NO: 81 S and 816 are PCR primers.
SEQ ID NO: 817 is the cDNA sequence encoding an N-terminal portion 2S of P788P expressed in E. coli.
SEQ ID NO: 818 is the amino acid sequence of the N-terminal portion of P788P expressed in E coli.
SEQ ID NO: 819 is the amino acid sequence of the M. tubey~culosis antigen Ral2.
SEQ ID NO: 820 and 821 are PCR primers.

SEQ ID NO: 822 is the cDNA sequence for the Ral2-PS l OS-C
construct.
SEQ ID NO: 823 is the cDNA sequence for the P510S-C construct.
SEQ ID NO: 824 is the cDNA sequence for the PS l OS-E3 construct.
SEQ ID NO: 825 is the amino acid sequence for the Ral2-PS l OS-C
construct.
SEQ ID NO: 826 is the amino acid sequence for the PS l OS-C construct.
SEQ ID NO: 827 is the amino acid sequence for the PS l OS-E3 construct.
SEQ ID NO: 828-833 are PCR primers.
SEQ ID NO: 834 is the cDNA sequence of the construct Ral2-P775P-ORF3.
SEQ ID NO: 835 is the amino acid sequence of the construct Ral2-P775P-ORF3.
SEQ ID NO: 836 and 837 are PCR primers.
SEQ ID NO: 838 is the determined amino acid sequence for a P703P His tag fusion protein.
SEQ ID NO: 839 is the determined cDNA sequence for a P703P His tag fusion protein.
SEQ ID NO: 840 and 841 are PCR primers.
SEQ ID NO: 842 is the determined amino acid sequence for a P705P His tag fusion protein.
SEQ ID NO: 843 is the determined cDNA sequence for a P705P His tag fusion protein.
SEQ ID NO: 844 and 845 axe PCR primers.
SEQ ID NO: 846 is the determined amino acid sequence for a P711P His tag fusion protein.
SEQ ID NO: 847 is the determined cDNA sequence for a P711P His tag fusion protein.
SEQ ID NO: 848 is the amino acid sequence of the M. tubey~culosis antigen Ral2.

SEQ ID NO: 849 and 850 are PCR primers.
SEQ ID NO: 851 is the determined cDNA sequence for the construct Ral2-P501 S-E2.
SEQ ID NO: 852 is the determined amino acid sequence for the construct Ral2-P501 S-E2.
SEQ ID NO: 853 is the amino acid sequence for an epitope of P501 S.
SEQ ID NO: 854 is the DNA sequence encoding SEQ ID NO: 853.
SEQ ID NO: 855 is the amino acid sequence for an epitope of P501 S.
SEQ ID NO: 856 is the DNA sequence encoding SEQ ID NO: 855.
SEQ ID NO: 857 is a peptide employed in epitope mapping studies.
SEQ ID NO: 858 is the amino acid sequence for an epitope of P501 S.
SEQ ID NO: 859 is the DNA sequence encoding SEQ ID NO: 858.
SEQ ID NO: 860-862 are the amino acid sequences for CD4 epitopes of P501 S.
1 S SEQ ID NO: 863-865 axe the DNA sequences encoding the sequences of SEQ ID NO: 860-862.
SEQ ID NO: 866-877 are the amino acid sequences for putative CTL
epitopes of P703P.
SEQ ID NO: 878 is the full-length cDNA sequence for P789P.
SEQ ID NO: 879 is the amino acid sequence encoded by SEQ ID NO:
878.
SEQ ID NO: 880 is the determined full-length cDNA sequence fox the splice variant of P776P referred to as contig 6.
SEQ ID NO: 881-882 are determined full-length cDNA sequences for the splice variant of P776P referred to as contig 7.
SEQ ID NO: 883-887 are amino acid sequences encoded by SEQ ID NO:
880.
SEQ ID NO: 888-893 are amino acid sequences encoded by the splice variant of P776P referred to as contig 7.

SEQ ID NO: 894 is the full-length cDNA sequence for human transmembrane protease serine 2.
SEQ ID NO: 895 is the amino acid sequence encoded by SEQ ID NO:
894.
SEQ ID NO: 896 is the cDNA sequence encoding the first 209 amino acids of human transmembrane protease serine 2.
SEQ ID NO: 897 is the first 209 amino acids of human transmembrane protease serine 2.
SEQ ID NO: 898 is the amino acid sequence of peptide 296-322 of PSOlS.
SEQ ID NO: 899-902 are PCR primers.
SEQ ID NO: 903 is the determined cDNA sequence of the Vb chain of a T cell receptor for the P501 S-specif c T cell clone 4E5.
SEQ ID NO: 904 is the determined cDNA sequence of the Va chain of a T cell receptor for the P501 S-specific T cell clone 4E5.
SEQ ID NO: 905 is the amino acid sequence encoded by SEQ ID NO
903.
SEQ ID NO: 906 is the amino acid sequence encoded by SEQ ID NO
904.
SEQ ID NO: 907 is the full-length open reading frame for P768P
including stop codon.
SEQ ID NO: 908 is the full-length open reading frame for P768P without stop codon.
SEQ ID NO: 909 is the amino acid sequence encoded by SEQ ID NO:
908.
SEQ ID NO: 910-915 are the amino acid sequences for predicted domains of P768P.
SEQ ID NO: 916 is the full-length cDNA sequence of P835P.
SEQ ID NO: 917 is the cDNA sequence of the previously identified clone FLJ13581.

SEQ ID NO: 918 is the cDNA sequence of the open reading frame for P835P with stop codon.
SEQ ID NO: 919 is the cDNA sequence of the open reading frame for P835P without stop codon.
5 SEQ ID NO: 920 is the full-length amino acid sequence for P835P.
SEQ ID NO: 921-928 are the amino acid sequences of extracellular and intracellular domains of P835P.
SEQ ID NO: 929 is the full-length cDNA sequence for P1000C.
SEQ ID NO: 930 is the cDNA sequence of the open reading frame for 10 P1000C, including stop codon.
SEQ ID NO: 931 is the cDNA sequence of the open reading frame for P1000C, without stop codon.
SEQ ID NO: 932 is the full-length amino acid sequence for P1000C.
SEQ ID NO: 933 is amino acids 1-100 of SEQ ID NO: 932.
15 SEQ ID NO: 934 is amino acids 100-492 of SEQ ID NO: 932.
SEQ ID NO: 935-937 are PCR primers.
SEQ TD NO: 938 is the cDNA sequence of the expressed full-length P767P coding region.
SEQ ID NO: 939 is the cDNA sequence of an expressed truncated P767P
20 coding region.
SEQ ID NO: 940 is the amino acid sequence encoded by SEQ ID NO:
939.
SEQ TD NO: 941 is the amino acid sequence encoded by SEQ ID NO:
938.
25 SEQ ID NO: 942 is the DNA sequence of a CD4 epitope of P703P.
SEQ ID NO: 943 is the amino acid sequence of a CD4 epitope of P703P.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed generally to compositions and their use in the therapy and diagnosis of cancer, particularly prostate cancer. As described further below, illustrative compositions of the present invention include, but are not restricted to, polypeptides, particularly immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (e.g., T cells).
The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration.
Such techniques are explained fully in the literature. See, e.g., Sambrook, et al.
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning:
A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D.
Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B.
Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986);
Perbal, A Practical Guide to Molecular Cloning (1984).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
Polypeptide Compositions As used herein, the term "polypeptide"' " is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.
Particularly illustrative polypeptides of the present invention comprise those encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs:
1-11 l, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942, or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions, to a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942. In specific embodiments, the polypeptides of the invention comprise amino acid sequences as set forth in any one of SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 866-877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943.
The polypeptides of the present invention are sometimes herein referred to as prostate-specific proteins or prostate-specific polypeptides, as an indication that their identification has been based at least in part upon their increased levels of expression in prostate tissue samples. Thus, a "prostate-specific polypeptide"
or "prostate-specific protein," refers generally to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide, that is expressed in a substantial proportion of prostate tissue samples, for example preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50% or more of prostate tissue samples tested, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in other normal tissues, as determined using a representative assay provided herein. A
prostate-specific polypeptide sequence of the invention, based upon its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.
In certain preferred embodiments, the polypeptides of the invention are I0 immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with prostate cancer. Screening fox immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide.
Unbound sera may then be removed and bound antibodies detected using, for example, lasl-labeled Protein A.
As would be recognized by the skilled artisan, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention. An "immunogenic portion," as used herein, is a fragment of an immunogenic polypeptide of the invention that itself is immunologically reactive (i. e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide.
Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific" if they specifically bind to an antigen (i.e., they react with the protein in an ELISA
or other immunoassay, and do not react detestably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well-known techniques.
In one preferred embodiment, an immunogenic portion of a polypeptide of the present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Preferably, the level of immunogenic activity of the ixnmunogenic portion is at least about 50%, preferably at least about 70%
and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide. In some instances, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.
In certain other embodiments, illustrative immunogenic portions may include peptides in which an N-terminal leader sequence and/or transmembrane domain has been deleted. Other illustrative immunogenic portions will contain a small N-and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.
In another embodiment, a polypeptide composition of the invention may also comprise one or more polypeptides that are immunologically reactive with T cells and/or antibodies generated against a polypeptide of the invention, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof In another embodiment of the invention, polypeptides are provided that comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.

The present invention, in another aspect, provides polypeptide fragments comprising at least about S, 10, 1S, 20, 2S, S0, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide composition set forth herein, such as those set forth in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, S 380, 383, 477-483, 496, 504, SOS, 519, 520, 522, S2S, 527, 532, 534, S37-SSl, SS3-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 8SS, 858, 860-862, 866-877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID NO: 1-111, 11S-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 10 335, 340-375, 381, 382 and 384-476, 524, S26, 530, 531, 533, S3S, 536, SS2, S69-572, 587, 591, S93-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942.
In another aspect, the present invention provides variants of the polypeptide compositions described herein. Polypeptide variants generally 1 S encompassed by the present invention will typically exhibit at least about 70%, 7S%, 80%, 8S%, 90%, 91%, 92%, 93%, 94%, 9S%, 96%, 97%, 98%; or 99% or more identity (determined as described below), along its length, to a polypeptide sequence set forth herein.
In one preferred embodiment, the polypeptide fragments and variants 20 provided by the present invention are immunologically reactive with an antibody andlor T-cell that reacts with a full-length polypeptide specifically set forth herein.
In another preferred embodiment, the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about SO%, preferably at least about 70%, and most preferably at least about 90% or 2S more of that exhibited by a full-length polypeptide sequence specifically set forth herein.
A polypeptide "variant," as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally 30 occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity as described herein using any of a number of techniques well known in the art.
For example, certain illustrative variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transrnembrane domain, have been removed. Other illustrative variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.
In many instances, a variant will contain conservative substitutions. A
"conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a vaxiant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, immunogenic variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table 1.
For , example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

TABLE I
Amino Acids Codons Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic Asp D GAC GAU
acid Glutamic Glu E GAA GAG
acid PhenylalaninePhe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

IsoleucineIle I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

MethionineMet M AUG

AsparagineAsn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gln Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Sex S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

TryptophanTrp W UGG

Tyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are:
isoleucine +4.5 ~ valine +4.2 ; leucine +3.8 ; phen lalanine +2.8 ; c steine/c stine ( )~ ( ) ( ) Y ( ) Y Y
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7);
serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i. e. still obtain a biological functionally equivalent protein.
In making such changes, the substitution of amino acids whose hydropathic indices axe within ~2 is preferred, those within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.
S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophiliciiy of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0 ~ 1 ); glutamate (+3.0 ~ 1 ); serine (+0.3); asparagine (+0.2);
glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5 ~ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8);
tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ~2 is preferred, those within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate;
serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-methyl-, thio- and other modif ed forms of adenine, cytidine, guanine, thymine and uridine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine;
and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

When comparing polypeptide sequences, two sequences are said to be "identical" if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison 5 window to identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
10 Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. ( 1978) A
model of evolutionary change in proteins - Matrices for detecting distant relationships.
15 In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Ev~zymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, 20 E.D. (1971) Comb. Theo. 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Pr°inciples and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be 25 conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST
2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or, below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one preferred approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i. e., gaps) of 20 percent or less, usually 5 to I S percent, or I O to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.
Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA
sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors:
(1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Py°oc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No.
4,935,233 and U.S.
Patent No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86-91, 1997).
In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ral2 fragment. Ral2 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. Patent Application 60/158,585, the disclosure of which is incozporated herein by reference in its entirety. Briefly, Ral2 refers to a polynucleotide region that is a subsequence of a Mycobacteriurn tuberculosis MTB32A nucleic acid.
MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. Patent Application 60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as a soluble polypeptides throughout the purification process. Moreover, Ral2 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ral2 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ral2 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ral2 polypeptide. Ral2 polynucleotides may comprise a native sequence (i. e., an endogenous sequence that encodes a Ral2 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ral2 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ral2 polypeptide. Variants preferably exhibit at least about 70%
identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ral2 polypeptide or a portion thereof.
Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-I10 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer).
The lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
Other fusion partners include the non-structural protein from influenzae virus, NS 1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneurnoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986).
LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been 5 exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA
fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at 10 residue 178. A particularly preferred repeat portion incorporates residues 188-305.
Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wheiein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Patent No. 5,633,234. An immunogenic polypeptide of the invention, 15 when fused with this targeting signal, will associate more efficiently with MHC class II
molecules and thereby provide enhanced in vivo stimulation of CD4+ T-cells specific for the polypeptide.
Polypeptides of the invention are prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further 20 described below. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a 25 growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.
Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
In general, polypeptide compositions (including fusion polypeptides) of 30 the invention are isolated. An "isolated" polypeptide is one that is removed from its original environment. For example, a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, e.g., are at least about 90%
pure, more preferably at Ieast about 95% pure and most preferably at least about 99%
pure.
Polynucleotide Compositions The present invention, in other aspects, provides polynucleotide compositions. The terms "DNA" and "polynucleotide" are used essentially interchangeably herein to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. "Isolated," as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA
molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions.
Of course, this refers to the DNA molecule as originally isolated, and does not exclude I S genes or coding regions later added to the segment by the hand of man.
As will be understood by those skilled in the art, the polynucleotide compositions of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
As will be also recognized by the skilled artisan, polynucleotides of the .
invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which~do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i. e., an endogenous sequence that encodes a polypeptide/protein of the invention or a portion thereof) or may comprise a sequence that encodes a variant or derivative, preferably an immunogenic variant or derivative, of such a sequence.
Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that comprise some or all of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-11 l, 115-171, 173-175, 177, 179-305, 307-3I5, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942, complements of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942, and degenerate variants of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above.
In other related embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID NOs: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942, for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein). The term "variants" should also be understood to encompasses homologous genes of xenogenic origin.
In additional embodiments, the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to, or complementary to, one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, ~ 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);
hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, O.SX and 0.2X SSC containing 0.1% SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.
In certain preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
The polynucleotides of the present invention, or fragments thereof, I 5 regardless of the length of the coding sequence itself, may be combined with other DNA
sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate Lengths) are contemplated to be useful in many implementations of this invention.
When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, preferably 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using 5 the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A
model of evolutionary change in proteins - Matrices for detecting distant relationships.
In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical 10 Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-15 425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxohomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
20 Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Softwaxe Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), 25 or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity axe the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST
30 2.0 can be used, for example with the parameters described herein, to determine percent 56 .
sequence identity for the polynucleotides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always <0).
Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments;
or the end of either sequence is reached. The BLAST algorithm parameters W, T
and X
IO determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl.
Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i. e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i. e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the, genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of immunogenic variants and/or derivatives of the polypeptides described herein.
By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.
Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA
molecule. In such embodiments, a primer comprising typically about 14 to about nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the ant. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I
I~lenow fragment, in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Ruby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.
In another approach for the production of polypeptide variants of the present invention, recursive sequence recombination, as described in U.S.
Patent No.
5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.
In other embodiments of the present invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15 contiguous nucleotides that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Polynucleotide molecules having sequence regions consisting of 5 contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in 10 various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger 15 contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 1 S-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
Molecules having contiguous complementary sequences over stretches greater than 15 bases in 20 length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.
25 Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.
Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM
technology of U. S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
The nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M
salt, at temperatures ranging from about 20°C to about 55°C.
Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature.
Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
According to another embodiment of the present invention, polynucleotide compositions comprising antisense oligonucleotides are provided.
Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U. S. Patent 5,739,119 and U. S. Patent 5,759,829).
Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDGl), ICAM-1, E-selectin, STIR-l, striatal GABAA receptor and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U. S.
Patent 5,801,154; U.S. Patent 5,789,573; U. S. Patent 5,718,709 and U.S. Patent 5,610,288).
Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U. S. Patent 5,747,470; U. S.
Patent 5,591,317 and U. S. Patent 5,783,683).
Therefore, in certain embodiments, the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof. In one embodiment, the antisense oligonucleotides comprise DNA
or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein.
Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and xelative stability. Antisense compositions may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
Highly preferred target regions of the mRNA, are those which are at or near the AUG
translation initiation codon, and those sequences which are substantially complementary to 5' regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software and/or the BLASTN 2Ø5 algorithm software (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
The use of an antisense delivery method employing a short peptide vector, termed MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul 15;25(14):2730-6). It has been demonstrated that several molecules of the MPG peptide coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%).
Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.
According to another embodiment of the invention, the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of the tumor polypeptides and proteins of the present invention in tumor cells. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec;27(3 Pt 2):487-96;
Michel and Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature.
1992 May 14;357(6374):173-6). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in traps (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct site, acts enzyrnatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7305-9). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis 8 virus, group I intron or RNaseP RNA (in association with an RNA
5 guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65.
Examples of hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP
0360257), Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25;18(2):299-304 and U. S. Patent 5,631,359. An example of the 10 hepatitis 8 virus motif is described by Perrotta and Been, Biochemistry.
1992 Dec 1;31(47):11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al., Cell. 1983 Dec;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. 1990 May 18;61 (4):685-96;
Saville and Collins, Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8826-30; Collins and Olive, 15 Biochemistry. 1993 Mar 23;32(11):2795-9); and an example of the Group I
intron is described in (U. S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an 20 RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.
Ribozymes may be designed as described in Int. Pat. Appl. Publ. No.
WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as 25 described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA
targets in other species can be utilized when necessary.
Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that 30 pxevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl.
Publ. No. WO

92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO
91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U. S. Patent 5,334,711; and Int. Pat.
Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA
synthesis times and reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted. to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stmt. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ.
No. WO
94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.
Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA
expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells Ribozymes expressed from such promoters have been shown to function in mammalian cells.
Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA
vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).
In another embodiment of the invention, peptide nucleic acids (PNAs) compositions are provided. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 Jun;lS(6):224-9). As such, in certain embodiments, one may prepare PNA
sequences that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell to which such PNA compositions have been administered.
PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec 6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan;4(1):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.
PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., Bioorg Med Chem. 1995 Apr;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.
Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine.
Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem.

Apr;3(4):437-45; Petersen et al., J Pept Sci. 1995 May-Jun;l(3):175-83; Orum et al., Biotechniques. 1995 Sep;l9(3):472-80; Footer et al., Biochemistry. 1996 Aug 20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug 11;23(15):3003-8;
Pardridge et al., Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1901-5; Gambacorti-Passerine et al., Blood. 1996 Aug 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U S A.

Nov 11;94(23):12320-5; Seeger et al., Biotechniques. 1997 Sep;23(3):512-7).
U.S.
Patent No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. 1993 Dec 15;65(24):3545-9) and Jensen et al.
(Biochemistry. 1997 Apr 22;36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcoreTM technology.
Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.
Polynucleotide Identification, Characterization and Expression Polynucleotide compositions of the present invention may be identified, prepared and/or manipulated using any of a variety of well established techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other like references).
For example, a polynucleotide may be identified, as described in more detail below, by screening a microaxray of cDNAs for tumor-associated expression (i. e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using the microarray technology of Affymetrix, Inc. (Santa Clara, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., P~oc. Natl.
Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci.
USA
94:2150-2155, 1997). Alternatively, polynucleotides may be amplified from cDNA
prepared from cells expressing the proteins described herein, such as tumor cells.
Many template dependent processes are available to amplify a target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCRTM) which is described in detail in U.S.
Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCRTM, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present 5 in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse 10 ~ transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
Any of a number of other template dependent processes, many of which are variations of the PCR TM amplification technique, are readily known and available in 15 the art. Illustratively, some such methods include the ligase chain reaction (referred to as LCR), described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and LT.S. Patent No. 4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.
PCT/LTS87/00880; Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in Great Britain Pat.
20 Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/LTS89/01025.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822 describes a nucleic acid amplification process involving cyclically synthesizing single-stranded 25 RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). PCT Intl. Pat. Appl.
Publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA
("ssDNA") followed by transcription of many RNA copies of the sequence. Other amplification methods such as "RACE" (Frohman, 1990), and "one-sided PCR"
(Ohara, 30 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a tumor cDNA
library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification.
Preferably, a library is size-selected to include larger molecules. Random primed libraries may' also be preferred for identifying 5' and upstream regions of genes.
Genomic libraries are preferred fox obtaining introns and extending S' sequences.
For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32P) using well known techniques. A
bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis, cDNA
clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
Alternatively, amplification techniques, such as those described above, can be useful for obtaining a full length coding sequence from a partial cDNA
sequence.
One such amplification technique is inverse PCR (see Triglia et al., Nucl.
Acids Res.
16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences axe typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO
96/38591. Another such technique is known as "rapid amplification of cDNA
ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR
(Lagerstrom et al., PCR Methods Applic. 1:l 11-19, 1991) and walking PCR (Parker et al., Nucl. Acids.
Res. 19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
In certain instances, if is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA
sequences may also be obtained by analysis of genomic fragments.
In other embodiments of the invention, polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, cadons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half life which is longer than that of a transcript generated from the naturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
For example, I)NA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H.
et al.
(1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof.
For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (I995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, CA).
A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. ( 1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
N.Y.
A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors;
insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.
Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.

For example, when cloning in bacterial systems, inducible promoters such as the hybrid IacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses 5 are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV
may be advantageously used with an appropriate selectable marker.
In bacterial systems, any of a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, 10 when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E.
coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with 15 sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G.
and S.
M. Schuster (1989) .I. Biol. Chena. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are 20 soluble and can easily be purified from lysed Bells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST
moiety at will.
25 In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzyr~zol. 153:516-544.
In cases where plant expression vectors are used, the expression of 30 sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results P~obl. Cell Diffey~. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
An insect system may also be used to express a polypeptide of interest.
For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S.
frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard, E. K. et al. (1994) Pr~oc. Natl. Acad. Sci. 91 :3224-3227).
In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficient translation of w~uences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and WI3S, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for I-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk^- or aprt^- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70);
npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 1SO:I-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Marry, supra).
Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci.
8S:8047-51). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. SS:121-131).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter.
Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells that contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection andlor quantification of nucleic acid or protein.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACE). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed.
These and other assays are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D.
E. et al. (1983; J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR
amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector fox the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA
probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of 5 interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity 10 purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen.
San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine 15 residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Pot. Exp. Pu~if. 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J.
20 et al. (1993; DNA Cell Biol. 12:441-453).
In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. X5:2149-2154).
Protein synthesis may be performed using manual techniques or by automation. Automated 25 synthesis may be achieved, for example, using Applied Biosystems 431A
Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that exhibit immunological binding to a tumor polypeptide disclosed herein, or to a portion, variant or derivative thereof. An antibody, or antigen-binding fragment thereof, is said to "specifically bind," "immunogically bind," and/or is "immunologically reactive" to a polypeptide of the invention if it reacts at a detectable level (within, for example, an ELISA assay) with the polypeptide, and does not react detectably with unrelated polypeptides under similar conditions.
Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
Thus, both the "on rate constant" (Ko") and the "off rate constant" (Ko~) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
The ratio of Ko~ /Ko" enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Kd. See, generally, Davies et al.
(1990) Annual Rev. Biochem. 59:439-473.
An "antigen-binding site," or "binding portion" of an antibody refers to the part of the immunoglobulin molecule that participates in antigen binding.
The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" which are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus the term "FR" refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an~ antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs."
Binding agents may be further capable of differentiating between patients with and without a cancer, such as prostate cancer, using the representative assays provided herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients.
Alternatively, or in addition, the antibody will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. Preferably, a statistically significant number of samples with and without the disease will be assayed.
Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.
Any agent that satisfies the above requirements rnay be a binding agent.
For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur.
J.
Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT
(hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, geI filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, fox example, an affinity chromatography step.
A number of therapeutically useful molecules are known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the "F(ab)"
fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the "F(ab')2 " fragment which comprises both antigen-binding sites. An "Fv"
fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule.
mbar et al. (1972) Proc. Nat. Acad.-Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et a1. (1980) Biochem 19:4091-4096.
A single chain Fv ("sFv") polypeptide is a covalently linked VH::VL
heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat.
Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated--but chemically separated--light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et aL;
and U.S. Pat. No. 4,946,778, to Ladner et al.
Each of the above-described molecules includes a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain FR
5 set which provide support to the CDRS and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDRl,"
"CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, 10 comprising the CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDRl, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the 15 heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.
As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V
region.
Some FR residues may contact bound antigen; however, FRs are primarily responsible 20 for folding the V region into the antigen-binding site, particularly the FR
residues directly adjacent to the CDRS. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-25 binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical"
structures--regardless of the precise CDR amino acid sequence. Further, certain FR
residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al.
(1989) Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J Ixnmunol.
I38:4534-4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No.
519,596, published Dec. 23, 1992). These "humanized" molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
As used herein, the terms "veneered FRs" and "recombinantly veneered FRs" refer to the selective replacement of FR residues from, e.g., a rodent heavy or Iight chain V region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen-binding site which retains substantially all of the native FR
polypeptide folding structure. Veneering techniques are based on the understanding that the ligand binding characteristics of an antigen-binding site are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473.
Thus, antigen binding specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other, and their interaction with the rest of the V region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.

The process of veneering makes use of the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S.
Government Printing Office, 1987), updates to the Kabat database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities of V
region amino acids can be deduced from the known three-dimensional structure for human and marine antibody fragments. There axe two general steps in veneering a marine antigen-binding site. Initially, the FRs of the variable domains of an antibody molecule of interest are compared with corresponding FR sequences of human variable domains obtained from the above-identified sources. The most homologous human V
regions axe then compared residue by residue to corresponding marine amino acids. The residues in the marine FR which differ from the human counterpart are replaced by the residues present in the human moiety using recombinant techniques well known in the art. Residue switching is only carried out with moieties which axe at least partially I S exposed (solvent accessible), and care is exercised in the replacement of amino acid residues which may have a significant effect on the tertiary structure of V
region domains, such as proline, glycine and charged amino acids.
In this manner, the resultant "veneered" marine antigen-binding sites are thus designed to retain the marine CDR residues, the residues substantially adjacent to the CDRs, the residues identified as buried or mostly buried (solvent inaccessible), the residues believed to participate in non-covalent (e.g., electrostatic and hydrophobic) contacts between heavy and light chain domains, and the residues from conserved structural regions of the FRs which are believed to influence the "canonical"
tertiary structures of the CDR loops. These design criteria axe then used to prepare recombinant nucleotide sequences which combine the CDRs of both the heavy and light chain of a marine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies which exhibit the antigen specificity of the rnurine antibody molecule.
In another embodiment of the invention, monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 9°Y, ~23I, i2sh 13th rs6Re, ~88Re, 2~jAt, and alaBi. preferred drugs include methotrexate, and pyrimidine and purine analogs.
Preferred differentiation inducers include phorbol esters and butyric acid.
Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A
direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A
number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spider), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody.
Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A
carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
For example, U.S. Patent No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.
T Cell Compositions The present invention, in another aspect, provides T cells specific for a tumor polypeptide disclosed herein, or for a variant or derivative thereof.
Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the IsolexTM System, available from Nexell Therapeutics, Inc.
(Irvine, 5 CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO
89/06280; WO
91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
T cells may be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide.
10 Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide of interest.
Preferably, a tumor polypeptide or polynucleotide of the invention is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
T cells are considered to be specific for a polypeptide of the present 15 invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T
cell specificity may be evaluated using any of a variety of standard techniques.
For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, 20 indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer' Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
For example, T cell proliferation can be detected by measuring an increased rate of DNA
synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and 25 measuring the amount of tritiated thymidine incorporated into DNA). Contact with a tumor polypeptide (100 ng/ml - 100 ~,g/ml, preferably 200 ng/ml - 25 ~g/ml) for 3 - 7 days will typically result in at least a two fold increase in proliferation of the T cells.
Contact as described above for 2-3 hours should result in activation of the T
cells, as measured using standard cytokine assays in which a two fold increase in the level of 30 cytokine release (e.g., TNF or IFN-y) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. l, Wiley Interscience (Greene 1998)). T
cells that have been activated in response to a tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4+ and/or CD8+. Tumor polypeptide-specific T
cells may be expanded using standard techniques. Within preferred embodiments, the T
cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in response to a tumor polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells ifZ vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a tumor polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a tumor polypeptide. Alternatively, one or more T cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
Pharmaceutical Compositions In additional embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody compositions disclosed herein in pharmaceutically-acceptable carriers for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions.
Therefore, in another aspect of the present invention, pharmaceutical compositions are provided comprising one or more of the polynucleotide, polypeptide, antibody, and/or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise irmnunogenic polynucleotide and/or polypeptide compositions of the invention for use in prophylactic and theraputic vaccine applications. Vaccine preparation is generally described in, for example, M.F.
Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach),"
Plenum Press (NY, 1995). Generally, such compositions will comprise one or more polynucleotide and/or polypeptide compositions of the present invention in combination with one or more immunostimulants.
It will be apparent that any of the pharmaceutical compositions described herein can contain pharmaceutically acceptable salts.. of the polynucleotides and polypeptides of the invention. Such salts can be prepared, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
In another embodiment, illustrative immunogenic compositions, e.g., vaccine compositions, of the present invention comprise DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered within any of a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques axe well known in the art, such as those described by Rolland, Crit. Rev.
They°ap. Dy°ug Cay-rier~ Systems 15:143-198, 1998, and references cited therein.
Appropriate polynucleotide expression systems will, of course, contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and terminating signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as Bacillus-Calmette-Gue~~in) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems.
In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S.
Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D.
(1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;
Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.
(1993) J.
Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729;
Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;
Berkner, K. L.
(1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).
Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;
International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al.
(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129;

Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
Additional viral vectors useful for delivering the polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK(-) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant 5 Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO
91/12882; WO
89/03429; and WO 92/03545.
Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in 10 U.S. Patent Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Patent Nos. 5,505,947 and 5,643,576.
Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et aI. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et 15 al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.
Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al., Pf°oc. Natl. Acad. Sci.
USA 86:317-321, 1989; Flexner et al., A~~. N Y. Acad. Sci. 569:86-103, 1989;
Flexner 20 et al., Yaccifze 8:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487;
WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242;
WO 91/02805; Berkner, Biotechhiques 6:616-627, 1988; Rosenfeld et al., Scie~zce 252:431-434, 1991; Dolls et al., P~oc. Natl. Acad. Sci. USA 91:215-219, 1994;
Lass-Eisler et al., P~oc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., 25 Cif°culation 88:2838-2848, 1993; and Guzman et al., Cir. Res.
73:1202-1207, 1993.
In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in a specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the 30 polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in syncluonization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.
In another embodiment of the invention, a polynucleotide is administered/delivered as "naked" DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
The uptake of naked DNA may be increased by coating the°DNA onto biodegradable beads, which are efficiently transported into the cells.
In still another embodiment, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described.
In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI), some examples of which are described in U.S. Patent Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No.

799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.
In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in U.S. Patent Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163;
5,520,639 and 5,993,412.
According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC
compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Myeobacterium tuberculosis derived proteins.
Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate;
salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine;
acylated sugars; canonically or anionically derivatized polysaccharides;
polyphosphazenes;
biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
Within certain embodiments of the invention, the adjuvant composition is preferably one that induces an immune response predominantly of the Thl type. High levels of Thl-type cytokines (e.g., IFN-y, TNFa,, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl-and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl-type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Aun. Rev. Immunol. 7:145-173, 1989.
Certain preferred adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL'2 adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US
Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, (3-escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such. as CarbopolR to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPLm adjuvant,e as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL~ adjuvant and tocopherol in an oil-in-water emulsion is described in WO
95/17210.
Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF
(Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS
series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn~; Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other axninoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general formula (I): HO(CH2CH20)n A-R, wherein, n is 1-50, A is a bond or -C(O)-, R is C1_so alkyl or Phenyl C1_so alkyl.
One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is Cj_SO, preferably C4-C2o alkyl and most preferably CI2 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO

99/52549. The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.
According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Anu. Rev. Med. 50:507-529, 1999).
In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitr°o), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T
cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not connnonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Natm°e Med. 4:594-600, 1998).
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compounds) that induce differentiation, maturation and proliferation of dendritic cells.
Dendritic cells are conveniently categorized as "immature" and "mature"
cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will typically vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.
Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release.
In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S.
Patent No.
5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S.
Patent Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;

5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems.
such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S.
Patent No.
5,928,647, which are capable of inducing a class I-restricted cytotoxic T
lymphocyte responses in a host.
The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, IO polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
Alternatively, compositions of the present invention may be formulated as a lyophilizate.
The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature 1997 Mar 27;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S.
Patent 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
Typically, these formulations will contain at least about 0.1 % of the active compound or more, although the percentage of the active ingredients) may, of course, be varied and may conveniently be between about 1 or 2% and about 60%
or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compounds) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

lOS
For oral administration, the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can 'be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent 5,756,353 and U.
S. Patent 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 Mar 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drag delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 Ju1;16(7):307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 Aug;35(8):801-9;
Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434;
U.S.
Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically incorporated herein by reference in its entirety).
Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem.
1990 Sep 25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-9). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.
I S In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm.
1998 Dec;24(12):1113-28). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20;
zur Muhlen et al., Eur J Pharm Biopharm. 1998 Mar;45(2):149-55; Zambaux et al. J
Controlled Release. 1998 Jan 2;50(1-3):31-40; and U. S. Patent 5,145,684.

Cancer Therapeutic Methods In further aspects of the present invention, the pharmaceutical compositions described herein may be used for the treatment of cancer, particularly for the immunotherapy of prostate cancer. Within such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer.
Accordingly, the above pharmaceutical compositions may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. As discussed above, administration of the pharmaceutical compositions may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.
Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).
Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T
lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy.
The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a suff cient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo.
Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., If~z~raunological Reviews 157:177, 1997).
Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.
Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
Preferably, between l and 10 doses may be administered over a 52 week period.
Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i. e., untreated) level.
Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 ~g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
In general, an appropriate dosage and treatment regimen provides the active compounds) in an amount sufficient to provide therapeutic andlor prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
Cancer Detection and Diagnostic Compositions, Methods and Kits In general, a cancer may be detected in a patient based on the presence of one or more prostate tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as prostate cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer. In general, a prostate tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample.
See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) 1 S comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length prostate tumor proteins and polypeptide portions thereof to which the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S.
Patent No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific Literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 fig, and preferably about 100 ng to about 1 ~.g, is sufficient to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. Fox example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody.
Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with prostate cancer.
Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally Buff cient.
Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. The second antibody, which contains a reporter group, may then be added to the solid support.
Preferred reporter groups include those groups recited above.
The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme).
Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of a cancer, such as prostate cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology.~ A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, 'the cut-off value nay be determined from a plot of pairs of true positive rates (i. e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result.
The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate 'cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.

In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 fig, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such tumor protein specific antibodies may correlate with the presence of a cancer.

A cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample.
Within certain methods, a biological sample comprising CD4+ and/or CD8+ T cells isolated from a patient is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected.
Suitable biological samples include, but are not limited to, isolated T cells.
For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T
cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C
with polypeptide (e.g., 5 - 25 ~,g/ml). It may be desirable to incubate another aliquot of a T
cell sample in the absence of tumor polypeptide to serve as a control. For CD4~ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8+ T
cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that 'is at least two fold greater and/or a level of cytolytic activity that is at least 20%
greater than in disease-free patients indicates the presence of a cancer in the patient.
As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the tumor protein. The amplified cDNA
is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.
To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a tumor protein of the invention that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above.
Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA
molecule having a sequence as disclosed herein. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Synap. Quaf2t. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA
molecules.
PCR amplification using at least one specific primer generates a cDNA
molecule, which may be separated and visualized using, for example, gel electrophoresis.
Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions~ of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.
In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.
Certain i~ vivo diagnostic assays may be performed directly on a tumor.
One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.
As noted above, to improve sensitivity, multiple tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay.
Further, multiple primers or probes may be used concurrently. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.
The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein.
Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding a tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a tumor protein. Such an oligonucleotide may be used, fox example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a tumor protein.
The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

S ISOLATION AND CHARACTERIZATION OF PROSTATE-SPECIFIC POLYPEPTIDES
This Example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.
A human prostate tumor cDNA expression library was constructed from prostate tumor poly A+ RNA using a Superscript Plasmid System for cDNA
Synthesis and Plasmid Cloning kit (BRL Life Technologies, Gaithersburg, MD 20897) following the manufacturer's protocol. Specifically, prostate tumor tissues were homogenized with polytron (Kinematica, Switzerland) and total RNA was extracted using Trizol reagent (BRL Life Technologies) as directed by the manufacturer. The poly A+
RNA
was then purified using a Qiagen oligotex spin column mRNA purification kit (Qiagen, Santa Clarita, CA 91355) according to the manufacturer's protocol. First-strand cDNA
was synthesized using the NotI/Oligo-dTl8 primer. Double-stranded cDNA was synthesized, ligated with EcoRI/BAXI adaptors (Invitrogen, San Diego, CA) and digested with NotI. Following size fractionation with Chroma Spin-1000 columns (Clontech, Palo Alto, CA), the cDNA was ligated into the EcoRI/NotI site of pCDNA3.1 (Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRL
Life Technologies) by electroporation.
Using the same procedure, a normal human pancreas cDNA expression library was prepared from a pool of six tissue specimens (Clontech). The cDNA
libraries were characterized by determining the number of independent colonies, the percentage of clones that carried insert, the average insert size and by sequence analysis.
The prostate tumor library contained 1.64 x 10~ independent colonies, with 70%
of clones having an insert and the average insert size being 1745 base pairs. The normal pancreas cDNA library contained 3.3 x 106 independent colonies, with 69% of clones having inserts and the average insert size being 1120 base pairs. For both libraries, sequence analysis showed that the majority of clones had a full length cDNA
sequence and were synthesized from mRNA, with minimal rRNA and mitochondria) DNA
contamination.
cDNA library subtraction was performed using the above prostate tumor and normal pancreas cDNA libraries, as described by Hara et al. (Blood, 84:189-199, 1994) with some modifications. Specifically, a prostate tumor-specific subtracted cDNA library was generated as follows. Normal pancreas cDNA library (70 ~,g) was digested with EcoRI, NotI, and SfuI, followed by a filling-in reaction with DNA
polymerase Klenow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA was dissolved in 100 ~1 of H20, heat-denatured and mixed with 100 ~,1 (100 ~.g) of Photoprobe biotin (Vector Laboratories, Burlingame, CA).
As recommended by the manufacturer, the resulting mixture was irradiated with a sunlamp on ice for 20 minutes. Additional Photoprobe biotin (50 ~l) was added and the biotinylation reaction was repeated. After extraction with butanol five times, the DNA was ethanol-precipitated and dissolved in 23 ~,1 H20 to form the driver DNA.
To form the tracer DNA, 10 ~g prostate tumor cDNA library was digested with BamHI and XhoI, phenol chloroform extracted and passed through Chroma spin-400 columns (Clontech). Following ethanol precipitation, the tracer DNA
was dissolved in 5 p.1 H20. Tracer DNA was mixed with 15 p,1 driver DNA and 20 p1 of 2 x hybridization buffer (1.5 M NaCI/10 mM EDTA/50 mM HEPES pH 7.5/0.2%
sodium dodecyl sulfate), overlaid with mineral oil, and heat-denatured completely. The sample was immediately transferred into a 68 °C water bath and incubated for 20 hours (long hybridization [LH]). The reaction mixture was then subjected to a streptavidin treatment followed by phenol/chloroform extraction. This process was repeated three more times. Subtracted DNA was precipitated, dissolved in 12 p1 H20, mixed with 8 ~1 driver DNA and 20 ~,l of 2 x hybridization buffer, and subjected to a hybridization at 68 °C for 2 hours (short hybridization [SH]). After removal of biotinylated double-stranded DNA, subtracted cDNA was ligated into BamHI/XhoI site of chloramphenicol resistant pBCSK+ (Stratagene, La Jolla, CA 92037) and transformed into ElectroMax E.

coli DHlOB cells by electroporation to generate a prostate tumor specific subtracted cDNA library (referred to as "prostate subtraction 1").
To analyze the subtracted cDNA library, plasmid DNA was prepared from 100 independent clones, randomly picked from the subtracted prostate tumor specific library and grouped based on insert size. Representative cDNA clones were further characterized by DNA sequencing with a Perkin Elmer/Applied Biosystems Division Automated Sequencer Model 373A (Foster City, CA). Six cDNA clones, hereinafter referred to as F1-13, Fl-12, F1-16, Hl-l, Hl-9 and Hl-4, were shown to be abundant in the subtracted prostate-specific cDNA library. The determined 3' and 5' cDNA sequences for Fl-12 are provided in SEQ ID NO: 2 and 3, respectively, with determined 3' cDNA sequences for F1-13, Fl-16, H1-1, H1-9 and Hl-4 being provided in SEQ ID NO: l and 4-7, respectively.
The cDNA sequences for the isolated clones were compared to known sequences in the gene bank using the EMBL and GenBank databases (release 96).
Four of the prostate tumor cDNA clones, Fl-13, F1-16, Hl-1, and Hl-4, were determined to encode the following previously identified proteins: prostate specific antigen (PSA), human glandular kallikrein, human tumor expression enhanced gene, and mitochondria cytochrome C oxidase subunit II. H1-9 was found to be identical to a previously identified human autonomously replicating sequence. No significant homologies to the cDNA sequence for F1-12 were found.
Subsequent studies led to the isolation of a full-length cDNA sequence for F1-12 (also referred to as P504S). This sequence is provided in SEQ ID NO:
107, with the corresponding predicted amino acid sequence being provided in SEQ ID
NO:
108. cDNA splice variants of P504S are provided in SEQ ID NO: 600-605.
To clone less abundant prostate tumor specific genes, cDNA library subtraction was performed by subtracting the prostate tumor cDNA library described above with the normal pancreas cDNA library and with the three most abundant genes in the previously subtracted prostate tumor specific cDNA library: human glandular kallikrein, prostate specific antigen (PSA), and mitochondria cytochrome C
oxidase subunit II. Specifically, 1 ~g each of human glandular kallikrein, PSA and mitochondria cytochrome C oxidase subunit II cDNAs in pCDNA3.l were added to the driver DNA and subtraction was performed as described above to provide a second subtracted cDNA library hereinafter referred to as the "subtracted prostate tumor specific cDNA library with spike".
Twenty-two cDNA clones were isolated from the subtracted prostate tumor specific cDNA library with spike. The determined 3' and 5' cDNA
sequences for the clones referred to as J1-17, L1-12, N1-1862, J1-13, Jl-19, J1-25, J1-24, Kl-58, K1-63, L1-4 and L1-14 are provided in SEQ ID NOS: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24-25, 26-27 and 28-29, respectively. The determined 3' cDNA
sequences for the clones referred to as Jl-12, J1-16, J1-21, K1-48, Kl-55, L1-2, Ll-6, N1-1858, Nl-1860, N1-1861, N1-1864 are provided in SEQ ID NOS: 30-40, respectively. Comparison of these sequences with those in the gene bank as described above, revealed no significant homologies to three of the five most abundant DNA
species, (J1-17, Ll-12 and N1-1862; SEQ ID NOS: 8-9, 10-11 and 12-13, respectively).
IS Of the remaining two most abundant species, one (J1-12; SEQ ID N0:30) was found to be identical to the previously identified human pulmonary surfactant-associated protein, and the other (K1-48; SEQ ID N0:33) was determined to have some homology to R.
no~vegicus mRNA for 2-arylpropionyl-CoA epimerase. Of the 17 less abundant cDNA
clones isolated from the subtracted prostate tumor specific cDNA library with spike, four (J1-16, K1-55, Ll-6 and N1-1864; SEQ ID NOS:31, 34, 36 and 40, respectively) were found to be identical to previously identified sequences, two (J1-21 and N1-1860;
SEQ ID NOS: 32 and 38, respectively) were found to show some homology to non-human sequences, and two (L1-2 and Nl-1861; SEQ ID NOS: 35 and 39, respectively) were found to show some homology to known human sequences. No significant homologies were found to the polypeptides J1-13, J1-19, Jl-24, J1-25, Kl-58, K1-63, L1-4, L1-14 (SEQ ID NOS: 14-15, 16-17, 20-21, 18-19, 22-23, 24-25, 26-27, 2829, respectively).
Subsequent studies led to the isolation of full length cDNA sequences for Jl-17, Ll-12 and Nl-1862 (SEQ ID NOS: 109-111, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NOS: 112-114. L1-12 is also referred to as P501 S. A cDNA splice variant of P501 S is provided in SEQ ID
NO: 606.
In a further experiment, four additional clones were identified by subtracting a prostate tumor cDNA library with normal prostate cDNA prepared from a pool of three normal prostate poly A+ RNA (referred to as "prostate subtraction 2").
The determined cDNA sequences for these clones, hereinafter referred to as U1-3064, U1-3065, V1-3692 and 1A-3905, are provided in SEQ ID NO: 69-72, respectively.
Comparison of the determined sequences with those in the gene bank revealed no significant homologies to Ul-3065.
A second subtraction with spike (referred to as "prostate subtraction spike 2") was performed by subtracting a prostate tumor specific cDNA library with spike with normal pancreas cDNA library and further spiked with PSA, Jl-17, pulmonary surfactant-associated protein, mitochondria) DNA, cytochrome c oxidase subunit II, N1-1862, autonomously replicating sequence, L1-12 and tumor expression enhanced gene. Four additional clones, hereinafter referred to as Vl-3686, R1-2330, 1B-3976 and V1-3679, were isolated. The determined cDNA sequences for these clones are provided in SEQ ID N0:73-76, respectively. Comparison of these sequences with those in the gene bank revealed no significant homologies to V1-3686 and RI-2330.
Further analysis of the three prostate subtractions described above (prostate subtraction 2, subtracted prostate tumor specific cDNA library with spike, and prostate subtraction spike 2) resulted in the identif cation of sixteen additional clones, referred to as 1 G-4736, 1 G-473 8, 1 G-4741, 1 G-4744, 1 G-4734, 1 H-4774, ~
1 H-4781, 1H-4785, 1H-4787, 1H-4796, 1I-4810, 1I-4811, 1J-4876, 1K-4884 and 1K-4896. The determined cDNA sequences for these clones are provided in SEQ ID NOS: 77-92, respectively. Comparison of these sequences with those in the gene bank as described above, revealed no significant homologies to 1 G-4741, 1 G-4734, l I-4807, 1J-4876 and 1K-4896 (SEQ ID NOS: 79, 81, 87, 90 and 92, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences for 1 G-4736, 1 G-4738, 1 G-4741, 1 G-4744, 1 H-.4774, 1 H-4781, 1 H-4785, 1 H-4787, 1 H-4796, l I-4807, 1J-4876, 1K-4884 and 1K-4896, provided in SEQ ID NOS: 179-188 and 191-193, respectively, and to the determination of additional partial cDNA sequences for 1I-4810 and 1I-481 l, provided in SEQ ID NOS: 189 and 190, respectively.
Additional studies with prostate subtraction spike 2 resulted in the S isolation of three more clones. Their sequences were determined as described above and compared to the most recent GenBank. All three clones were found to have homology to known genes, which are Cysteine-rich protein, KIAA0242, and (SEQ ID NO: 317, 319, and 320, respectively). Further analysis of these clones by Synteni microarray (Synteni, Palo Alto, CA) demonstrated that all three clones were over-expressed in most prostate tumors and prostate BPH, as well as in the majority of normal prostate tissues tested, but low expression in all other normal tissues.
An additional subtraction was performed by subtracting a normal prostate cDNA library with normal pancreas cDNA (referred to as "prostate subtraction 3"). This led to the identification of six additional clones referred to as 1G-4761, 16-4762, 1H-4766, 1H-4770, 1H-4771 and 1H-4772 (SEQ ID NOS: 93-98). Comparison of these sequences with those in the gene bank revealed no significant homologies to 1G-4761 and 1H-4771 (SEQ ID NOS: 93 and 97, respectively). Further analysis of the isolated clones led to the determination of extended cDNA sequences for 1 G-4761, 1 6-4762, 1H-4766 and 1H-4772 provided in SEQ ID NOS: 194-196 and 199, respectively, and to the determination of additional partial cDNA sequences for 1 H-4770 and 4771, provided in SEQ ID NOS: 197 and 198, respectively.
Subtraction of a prostate tumor cDNA library, prepared from a pool of polyA+ RNA from three prostate cancer patients, with a normal pancreas cDNA
library (prostate subtraction 4) led to the identification of eight clones, referred to as 1 D-4297, 1D-4309, 1D.1-4278, 1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280 (SEQ ID
NOS: 99-I07). These sequences were compared to those in the gene bank, as described above. No significant homologies were found to 1D-4283 and 1D-4304 (SEQ ID
NOS:
103 and 104, respectively). Further analysis of the isolated clones Ied to the determination of extended cDNA sequences for 1D-4309, 1D.1-4278, 1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280, provided in SEQ ID NOS: 200-206, respectively.
cDNA clones isolated in prostate subtraction 1 and prostate subtraction 2, described above, were colony PCR amplified and their mRNA expression levels in prostate tumor, normal prostate and in various other normal tissues were determined using microarray technology (Synteni, Palo Alto, CA). Briefly, the PCR
amplification products were dotted onto slides in an array format, with each product occupying a unique location in the array. mRNA was extracted from the tissue sample to be tested, reverse transcribed, and fluorescent-labeled cDNA probes were generated. The microarrays were probed with the labeled cDNA probes, the slides scanned and fluorescence intensity was measured. This intensity correlates with the hybridization intensity. Two clones (referred to as P509S and PS l OS) were found to be over-expressed in prostate tumor and normal prostate and expressed at low levels in all other normal tissues tested (liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testes, salivary gland, large intestine, kidney, ovary, Iung, spinal cord, skeletal muscle and colon). The determined cDNA sequences for P509S and PS l OS are provided in SEQ ID NO: 223 and 224, respectively. Comparison of these sequences with those in the gene bank as described above, revealed some homology to previously identified ESTs.
Additional, studies Ied to the isolation of the full-length cDNA sequence for P509S. This sequence is provided in SEQ ID NO: 332, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 339. Two variant full-length cDNA sequences for PS10S are provided iri SEQ ID NO: 535 and 536, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 537 and 538, respectively. Additional splice variants of PS lOS axe provided in SEQ ID
NO: 598 and 599.
The determined cDNA sequences for additional prostate-specific clones isolated during characterization of prostate specific cDNA libraries are provided in SEQ
ID NO: 618-689, 691-697 and 709-772. Comparison of these sequences with those in the public databases revealed no significant homologies to any of these sequences.

DETERMINATION OF TISSUE SPECIFICITY OF PROSTATE-SPECIFIC POLYPEPTIDES
Using gene specific primers, mRNA expression levels for the representative prostate-specific polypeptides F1-16, H1-l, J1-17 (also referred to as P502S), L1-12 (also referred to as PSO1S), F1-12 (also referred to as P504S) and Nl-1862 (also referred to as P503S) were examined in a variety of normal and tumor tissues using RT-PCR.
Briefly, total RNA was extracted from a variety of normal and tumor tissues using Trizol reagent as described above. First strand synthesis was carried out using 1-2 ~.g of total RNA with Superscript II reverse transcriptase (BRL Life Technologies) at 42 °C for one hour. The cDNA was then amplified by PCR
with gene-specific primers. To ensure the semi-quantitative nature of the RT-PCR, (3-actin was used as an internal control for each of the tissues examined. First, serial dilutions of the first strand cDNAs were prepared and RT-PCR assays were performed using (3-actin specific primers. A dilution was then chosen that enabled the linear range amplification of the (3-actin template and which was sensitive enough to reflect the differences in the initial copy numbers. Using these conditions, the (3-actin levels were determined for each reverse transcription reaction from each tissue. DNA contamination was minimized by DNase treatment and by assuring a negative PCR result when using first strand cDNA that was prepared without adding reverse transcriptase.
mRNA Expression levels were examined in four different types of tumor tissue (prostate tumor from 2 patients, breast tumor from 3 patients, colon tumor, lung tumor), and sixteen different normal tissues, including prostate, colon, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin, stomach, testes, bone marrow and brain.
F1-16 was found to be expressed at high levels in prostate tumor tissue, colon tumor and normal prostate, and at lower levels in normal liver, skin and testes, with expression being undetectable in the other tissues examined. Hl-1 was found to be expressed at high levels in prostate tumor, lung tumor, breast tumor, normal prostate, normal colon and normal brain, at much lower levels in normal lung, pancreas, skeletal muscle, skin, small intestine, bone marrow, and was not detected in the other tissues tested. J1-17 (P502S) and Ll-12 (P501 S) appear to be specifically over-expressed in prostate, with both genes being expressed at high levels in prostate tumor and normal prostate but at low to undetectable levels in all the other tissues examined. N1-1862 (P503S) was found to be over-expressed in 60% of prostate tumors and detectable in normal colon and kidney. The RT-PCR results thus indicate that F1-16, Hl-1, J1-17 (P502S), 1862 (P503S) and L1-12 (PSO1S) are either prostate specific or are expressed at significantly elevated levels in prostate.
Further RT-PCR studies showed that Fl-12 (P504S) is over-expressed in 60% of prostate tumors, detectable in normal kidney but not detectable in all other tissues tested. Similarly, Rl-2330 was shown to be over-expressed in 40% of prostate tumors, detectable in normal kidney and liver, but not detectable in all other tissues tested. U1-3064 was found to be over-expressed in 60% of prostate tumors, and also I 5 expressed in breast and colon tumors, but was not detectable in normal tissues.
RT-PCR characterization of Rl-2330, Ul-3064 and 1D-4279 showed that these three antigens are over-expressed in prostate and/or prostate tumors.
Northern analysis with four prostate tumors, two normal prostate samples, two BPH prostates, and normal colon, kidney, liver, lung, pancrease, skeletal muscle, brain, stomach, testes, small intestine and bone marrow, showed that (P501 S) is over-expressed in prostate tumors and normal prostate, while being undetectable in other normal tissues tested. J1-17 (P502S) was detected in two prostate tumors and not in the other tissues tested. N1-1862 (P503S) was found to be over-expressed in three prostate tumors and to be expressed in normal prostate, colon and kidney, but not in other tissues tested. F1-12 (P504S) was found to be highly expressed in two prostate tumors and to be undetectable in all other tissues tested.
The microarray technology described above was used to determine the expression levels of representative antigens described herein in prostate tumor, breast tumor and the following normal tissues: prostate, liver, pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder, testes, salivary gland, large intestine, kidney, ovary, lung, spinal cord, skeletal muscle and colon. L1-12 (PSOlS) was found to be over-expressed in normal prostate and prostate tumor, with some expression being detected in normal skeletal muscle. Both Jl-12 and F1-12 (P504S) were found to be over-expressed in prostate tumor, with expression being lower or undetectable in all other tissues tested. N1-1862 (P503S) was found to be expressed at high levels in prostate tumor and normal prostate, and at low levels in normal large intestine and normal colon, with expression being undetectable in all other tissues tested.

was found to be over-expressed in prostate tumor and normal prostate, and to be expressed at lower levels in all other tissues tested. 1D-4279 was found to be over-expressed in prostate tumor and normal prostate, expressed at lower levels in normal spinal cord, and to be undetectable in all other tissues tested.
Further microarray analysis to specifically address the extent to which P501 S (SEQ ID NO: 110) was expressed in breast tumor revealed moderate over-expression not only in breast tumor, but also in metastatic breast tumor (2/31), with negligible to Iow expression in normal tissues. This data suggests that P501 S
may be over-expressed in various breast tumors as well as in prostate tumors.
The expression levels of 32 ESTs (expressed sequence tags) described by Vasmatzis et al. (Proc. Natl. Acad. Sci. USA 95:300-304, 1998) in a variety of tumor and normal tissues were examined by microarray technology as described above.
Two of these clones (referred to as P1000C and P1001C) were found to be over-expressed in prostate tumor and normal prostate, and expressed at low to undetectable levels in aII
other tissues tested (normal aorta, thymus, resting and activated PBMC, epithelial cells, spinal cord, adrenal gland, fetal tissues, skin, salivary gland, large intestine, bone marrow, liver, lung, dendritic cells, stomach, lymph nodes, brain, heart, small intestine, skeletal muscle, colon and kidney. The determined cDNA sequences for P1000C
and P1001C are provided in SEQ ID NO: 384 and 472, respectively. The sequence of P 1001 C was found to show some homology to the previously isolated Human mRNA
for JM27 protein. Subsequent comparison of the sequence of SEQ ID NO: 384 with sequences in the public databases, Ied to the identification of a full-length cDNA
sequence of P1000C (SEQ ID NO: 929), which encodes a 492 amino acid sequence.

Analysis of the amino acid sequence using the PSORT II program led to the identification of a putative transmembrane domain from amino acids 84-100. The cDNA sequence of the open reading frame of P 1000C, including the stop codon, is provided in SEQ ID NO: 930, with the open reading frame without the stop codon being provided in SEQ ID NO: 931. The full-length amino acid sequence of P1000C is provided in SEQ ID NO: 932. SEQ ID NO: 933 and 934 represent amino acids 1-100 and 100-492 of P1000C, respectively.
The expression of the polypeptide encoded by the full length cDNA
sequence for F1-12 (also referred to as P504S; SEQ ID NO: 108) was investigated by immunohistochemical analysis. Rabbit-anti-P504S polyclonal antibodies were generated against the full length P504S protein by standard techniques.
Subsequent isolation and characterization of the polyclonal antibodies were also performed by techniques well known in the art. Immunohistochemical analysis showed that the P504S polypeptide was expressed in I00% of prostate carcinoma samples tested (n=5).
The rabbit-anti-P504S polyclonal antibody did not appear to label benign prostate cells with the same cytoplasmic granular staining, but rather with light nuclear staining. Analysis of normal tissues revealed that the encoded polypeptide was found to be expressed in some, but not all normal human tissues. Positive cytoplasmic staining with rabbit-anti-P504S polyclonal antibody was found in normal human kidney, liver, brain, colon and lung-associated macrophages, whereas heart and bone marrow were negative.
This data indicates that the P504S polypeptide is present in prostate cancer tissues, and that there are qualitative and quantitative differences in the staining between benign prostatic hyperplasia tissues and prostate cancer tissues, suggesting that this polypeptide may be detected selectively in prostate tumors and therefore be useful in the diagnosis of prostate cancer.

ISOLATION AND CHARACTERIZATION OF PROSTATE-SPECIFIC
POLYPEPTIDES BY PCR-BASED SUBTRACTION
A cDNA subtraction library, containing cDNA from normal prostate subtracted with ten other normal tissue cDNAs (brain, heart, kidney, liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) and then submitted to a first round of PCR amplification, was purchased from Clontech. This library was subjected to a second round of PCR amplification, following the manufacturer's protocol. The resulting cDNA fragments were subcloned into the vector pT7 Blue T-vector (Novagen, Madison, WI) and transformed into XL-1 Blue MRF' E. coli (Stratagene). DNA was isolated from independent clones and sequenced using a Perkin Elmer/Applied Biosystems Division Automated Sequencer Model 373A.
Fifty-nine positive clones were sequenced. Comparison of the DNA
sequences of these clones with those in the gene bank, as described above, revealed no significant homologies to 25 of these clones, hereinafter referred to as P5, P8, P9, P18, P20, P30, P34, P36, P38, P39, P42, P49, P50, P53, P55, P60, P64, P65, P73, P75, P76, P79 and P84. The determined cDNA sequences for these clones are provided in SEQ
ID NO: 41-45, 47-52 and 54-65, respectively. P29, P47, P68, P80 and P82 (SEQ
ID
NO: 46, 53 and 66-68, respectively) were found to show some degree of homology to previously identified DNA sequences. To the best of the inventors' knowledge, none of these sequences have been previously shown to be present in prostate.
Further studies employing the sequence of SEQ ID NO: 67 as a probe in standard full-length cloning methods, resulted in the isolation of three cDNA
sequences which appear to be splice variants of P80 (also known as P704P). These sequences are provided in SEQ ID NO: 699-701.
Further studies using the PCR-based methodology described above resulted in the isolation of more than 180 additional clones, of which 23 clones were found to show no significant homologies to known sequences. The determined cDNA
sequences for these clones are provided in SEQ ID NO: 115-123, 127, 131, 137, 145, 147-151, 153, 156-158 and 160. Twenty-three clones (SEQ ID NO: 124-126, 128-130, 132-136, 138-144, 146, 152, 154, 155 and 159) were found to show some homology to previously identified ESTs. An additional ten clones (SEQ ID NO: 161-170) were found to have some degree of homology to known genes. Larger cDNA clones containing the P20 sequence represent splice variants of a gene referred to as P703P.
The determined DNA sequence for the variants referred to as DE1, DE13 and DE14 are provided in SEQ ID NOS: 171, 175 and 177, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 172, 176 and 178, respectively. The determined cDNA sequence for an extended spliced form of P703 is provided in SEQ ID NO: 225. The DNA sequences for the splice variants referred to as DE2 and DE6 are provided in SEQ ID NOS: 173 and 174, respectively.
mRNA Expression levels for representative clones in tumor tissues (prostate (n=5), breast (n=2), colon and lung) normal tissues (prostate (n=5), colon, kidney, liver, lung (n=2), ovary (n=2), skeletal muscle, skin, stomach, small intestine and brain), and activated and non-activated PBMC was determined by RT-PCR as described above. Expression was examined in one sample of each tissue type unless otherwise indicated.
P9 was found to be highly expressed in normal prostate and prostate tumor compared to all normal tissues tested except for normal colon which showed comparable expression. P20, a portion of the P703P gene, was found to be highly expressed in normal prostate and prostate tumor, compared to all twelve normal tissues tested. A modest increase in expression of P20 in breast tumor (n=2), colon tumor and lung tumor was seen compared to all normal tissues except lung (1 of 2).
Increased expression of P18 was found in normal prostate, prostate tumor and breast tumor compared to other normal tissues except lung and stomach. A modest increase in expression of PS was observed in normal prostate compared to most other normal tissues. However, some elevated expression was seen in normal lung and PBMC.
Elevated expression of PS was also observed in prostate tumors (2 of 5), breast tumor and one lung tumor sample. For P30, similar expression levels were seen in normal prostate and prostate tumor, compared to six of twelve other normal tissues tested.

Increased expression was seen in breast tumors, one lung tumor sample and one colon tumor sample, and also in normal PBMC. P29 was found to be over-expressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to the majority of normal tissues. However, substantial expression of P29 was observed in normal colon and normal lung (2 of 2). P80 was found to be over-expressed in prostate tumor (5 of 5) and normal prostate (5 of 5) compared to all other normal tissues tested, with increased expression also being seen in colon tumor.
Further studies resulted in the isolation of twelve additional clones, hereinafter referred to as 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3, 8-hl l, 9-fl2 and 9-f3. The determined DNA sequences for 10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-hl l, 9-f12 and 9-f3 are provided in SEQ ID NO: 207, 208, 209, 216, 217, 220, 221 and 222, respectively. The determined forward and reverse DNA sequences for 7-g6, 8-b5, 8-b6 and 8-g3 are provided in SEQ ID NO: 210 and 211; 212 and 213;

and 215; and 218 and 219, respectively. Comparison of these sequences with those in the gene bank revealed no significant homologies to the sequence of 9-f3. The clones 10-d8, 11-c8 and 8-hll were found to show some homology to previously isolated ESTs, while 10-h10, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3 and 9-f12 were found to show some homology to previously identified genes. Further characterization of 7-G6 and showed identity to the known genes PAP and PSA, respectively.
mRNA expression levels for these clones were determined using the micro-array technology described above. The clones 7-G6, 8-G3, 8-B5, 8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10-A2, 10-A4, 11-C9 and 11-F2 were found to be over-expressed in prostate tumor and normal prostate, with expression in other tissues tested being low or undetectable. Increased expression of 8-F 11 was seen in prostate tumor and normal prostate, bladder, skeletal muscle and colon. Increased expression of 10-H10 was seen in prostate tumor and normal prostate, bladder, lung, colon, brain and large intestine. Increased expression of 9-B 1 was seen in prostate tumor, breast tumor, and normal prostate, salivary gland, large intestine and skin, with increased expression of 11-C8 being seen in prostate tumor, and normal prostate and large intestine.

An additional cDNA fragment derived from the PCR-based normal prostate subtraction, described above, was found to be prostate specific by both micro array technology and RT-PCR. The determined cDNA sequence of this clone (referred to as 9-A11) is provided in SEQ ID NO: 226. Comparison of this sequence with those in the public databases revealed 99% identity to the known gene HOXB13.
Further studies led to the isolation of the clones 8-C6 and 8-H7. The determined cDNA sequences for these clones are provided in SEQ ID NO: 227 and 228, respectively. These sequences were found to show some homology to previously isolated ESTs.
PCR and hybridization-based methodologies were employed to obtain longer cDNA sequences for clone P20 (also referred to as P703P), yielding three additional cDNA fragments that progressively extend the 5' end of the gene.
These fragments, referred to as P703PDE5, P703P6.26, and P703PX-23 (SEQ ID NO: 326, 328 and 330, with the predicted corresponding amino acid sequences being provided in SEQ ID NO: 327, 329 and 331, respectively) contain additional 5' sequence.
P703PDE5 was recovered by screening of a cDNA library (#141-26) with a portion of P703P as a probe. P703P6.26 was recovered from a mixture of three prostate tumor cDNAs and P703PX 23 was recovered from cDNA library (#438-48). Together, the additional sequences include all of the putative mature serine protease along with part of the putative signal sequence. The full-length cDNA sequence for P703P is provided in SEQ ID NO: 524, with the corresponding amino acid sequence being provided in SEQ
ID NO: 525.
Using computer algorithms, the following regions of P703P were predicted to represent potential HLA A2-binding CTL epitopes: amino acids 164-of SEQ ID NO: 525 (SEQ ID NO: 866); amino acids 160-168 of SEQ ID NO: 525 (SEQ ID NO: 867); amino acids 239-247 of SEQ ID NO: 525 (SEQ ID NO: 868);
amino acids 118-126 of SEQ ID NO: 525 (SEQ ID NO: 869); amino acids 112-120 of SEQ ID NO: 525 (SEQ ID NO: 870); amino acids 155-164 of SEQ ID NO: 525 (SEQ
ID NO: 871); amino acids 117-126 of SEQ ID NO: 525 (SEQ ID NO: 872); amino acids 164-173 of SEQ ID NO: 525 (SEQ ID NO: 873); amino acids 154-163 of SEQ ID NO:

525 (SEQ ID NO: 874); amino acids 163-172 of SEQ ID NO: 525 (SEQ ID NO: 875);
amino acids 58-66 of SEQ ID NO: 525 (SEQ ID NO: 876); and amino acids 59-67 of SEQ ID NO: 525 (SEQ ID NO: 877).
P703P was found to show some homology to previously identified proteases, such as thrombin. The thrombin receptor has been shown to be preferentially expressed in highly metastatic breast carcinoma cells and breast carcinoma biopsy samples. Introduction of thrombin receptor antisense cDNA has been shown to inhibit the invasion of metastatic breast carcinoma cells in culture. Antibodies against thrombin receptor inhibit thrombin receptor activation and thrombin-induced platelet activation. Furthermore, peptides that resemble the receptor's tethered ligand domain inhibit platelet aggregation by thrombin. P703P may play a role in prostate cancer through a protease-activated receptor on the cancer cell or on stromal cells.
The potential trypsin-like protease activity of P703P may either activate a protease-activated receptor on the cancer cell membrane to promote tumorgenesis or activate a protease-IS activated receptor on the adjacent cells (such as stromal cells) to secrete growth factors and/or proteases (such as matrix metalloproteinases) that could promote tumor angiogenesis, invasion and metastasis. P703P may thus promote tumor progression and/or metastasis through the activation of protease-activated receptor.
Polypeptides and antibodies that block the P703P-receptor interaction may therefore be usefully employed in the treatment of prostate cancer.
To determine whether P703P expression increases with increased severity of Gleason grade, an indicator of tumor stage, quantitative PCR
analysis was performed on prostate tumor samples with a range of Gleason scores from 5 to >
8. The mean level of P70~3P expression increased with increasing Gleason score, indicating that P703P expression may correlate with increased disease severity.
Further studies using a PCR-based subtraction library of a prostate tumor pool subtracted against a pool of normal tissues (referred to as JP: PCR
subtraction) resulted in the isolation of thirteen additional clones, seven of which did not share any significant homology to known GenBank sequences. The determined cDNA sequences for these seven clones (P711P, P712P, novel 23, P774P, P775P, P710P and P768P) are provided in SEQ ID NO: 307-311, 313 and 315, respectively. The remaining six clones (SEQ ID NO: 316 and 321-32S) were shown to share some homology to known genes.
By microarray analysis, all thirteen clones showed three or more fold over-expression in prostate tissues, including prostate tumors, BPH and normal prostate as compared to S normal non-prostate tissues. Clones P711P, P712P, novel 23 and P768P showed over-expression in most prostate tumors and BPH tissues tested (n=29), and in the majority of normal prostate tissues (n=4), but background to low expression levels in all normal tissues. Clones P774P, P77SP and P710P showed comparatively lower expression and expression in fewer prostate tumors and BPH samples, with negative to low expression in normal prostate.
Further studies Ied to the isolation of an extended cDNA sequence for P712P (SEQ ID NO: SS2). The amino acid sequences encoded by 16 predicted open reading frames present within the sequence of SEQ ID NO: SS2 are provided in SEQ ID
NO: SS3-568.
1S The full-length cDNA for P711P was obtained by employing the partial sequence of SEQ ID NO: 307 to screen a prostate cDNA library. Specifically, a directionally cloned prostate cDNA library was prepared using standard techniques.
One million colonies of this library were plated onto LB/Amp plates. Nylon membrane filters were used to lift these colonies, and the cDNAs which were picked up by these filters were denatured and cross-linked to the filters by UV light. The P711P
cDNA
fragment of SEQ ID NO: 307 was radio-labeled and used to hybridize with these filters.
Positive clones were selected, and cDNAs were prepared and sequenced using an automatic Perkin Elmer/Applied Biosystems sequences. The determined full-length sequence of P711P is provided in SEQ ID NO: 382, with the corresponding predicted 2S amino acid sequence being provided in SEQ ID NO: 383.
Using PCR and hybridization-based methodologies, additional cDNA
sequence information was derived for two clones described above, 11-C9 and 9-F3, herein after referred to as P707P and P714P, respectively (SEQ ID NO: 333 and 334).
After comparison with the most recent GenBank, P707P was found to be a splice variant of the known gene HoxB 13. In contrast, no signif cant homologies to were found. Further studies employing the sequence of SEQ ID NO: 334 as a probe in standard full-length cloning methods, resulted in an extended cDNA sequence for P714P. This sequence is provided in SEQ ID NO: 698. This sequence was found to show some homology to the gene that encodes human ribosomal L23A protein.
Clones 8-B3, P89, P98, P130 and P201 (as disclosed in U.S. Patent Application No. 09/020,956, filed February 9, 1998) were found to be contained within one contiguous sequence, referred to as P705P (SEQ ID NO: 335, with the predicted amino acid sequence provided in SEQ ID NO: 336), which was determined to be a splice variant of the known gene NKX 3.1.
Further studies on P775P resulted in the isolation of four additional sequences (SEQ ID NO: 473-476) which are all splice variants of the P775P
gene. The sequence of SEQ ID NO: 474 was found to contain two open reading frames (ORFs).
The predicted amino acid sequences encoded by these ORFs are provided in SEQ
ID
NO: 477 and 478. The cDNA sequence of SEQ ID NO: 475 was found to contain an ORF which encodes the amino acid sequence of SEQ ID NO: 479. The cDNA
sequence of SEQ ID NO: 473 was found to contain four ORFs. The predicted amino acid sequences encoded by these ORFs are provided in SEQ ID NO: 480-483.
Additional splice variants of P775P are provided in SEQ ID NO: 593-597.
Subsequent studies led to the identification of a genomic region on chromosome 22q11.2, known as the Cat Eye Syndrome region, that contains the five prostate genes P704P, P712P, P774P, P775P and B305D. The relative location of each of these five genes within the genomic region is shown in Fig. 10. This region may therefore be associated with malignant tumors, and other potential tumor genes may be contained within this region. These studies also led to the identification of a potential open reading frame (ORF) for P775P (provided in SEQ ID NO: 533), which encodes the amino acid sequence of SEQ ID NO: 534.
Comparison of the clone of SEQ ID NO: 325 (referred to as P558S) with sequences in the GenBank and GeneSeq DNA databases showed that P558S is identical to the prostate-specific transglutaminase gene, which is known to have two forms. The full-length sequences for the two forms are provided in SEQ ID NO: 773 and 774, with the corresponding amino acid sequences being provided in SEQ ID NO: 775 and 776, respectively. The cDNA sequence of SEQ ID NO: 774 has a 15 pair base insert, resulting in a 5 amino acid insert in the corresponding amino acid sequence (SEQ ID
NO: 776). This insert is not present in the sequence of SEQ ID NO: 773.
. Further studies on P768P (SEQ ID NO: 315) led to the identification of the putative full-length open reading frame (ORF). The cDNA sequence of the ORF
with stop codon is provided in SEQ ID NO: 907. The cDNA sequence of the ORF
without stop codon is provided in SEQ ID NO: 908, with the corresponding amino acid sequence being provided in SEQ ID NO: 909. This sequence was found to show 86%
identity to a rat calcium transporter protein, indicating that P768P may represent a human calcium transporter protein. The locations of transmembrane domains within P768P were predicted using the PSORT II computer algorithm. Six transmembrane domains were predicted at amino acid positions 118-134, 172-188, 211-227, 230-246, 282-298 and 348-364. The amino acid sequences of SEQ ID NO: 910-915 represent amino acids 1-134, 135-188, 189-227, 228-246, 247-298 and 299-511 of P768P, respectively.

SYNTHESIS OF POLYPEPTIDES
Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiolahioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides may be precipitated in cold methyl-t-butyl-ether. The peptide pellets may then be dissolved in water containing 0.1 %
trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A
gradient of 0%-60% acetonitrile (containing 0.1 % TFA) in water (containing 0.1 % TFA) may be used to elute the peptides. Following lyophilization of the pure fractions, the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

FURTHER ISOLATION AND CHARACTERIZATION OF
PROSTATE-SPECIFIC POLYPEPTIDES BY PCR-BASED SUBTRACTION
A cDNA library generated from prostate primary tumor mRNA as described above was subtracted with cDNA from normal prostate. The subtraction was performed using a PCR-based protocol (Clontech), which was modified to generate larger fragments. Within this protocol, tester and driver double stranded cDNA
were separately digested with five restriction enzymes that recognize six-nucleotide restriction sites (MIuI, MscI, PvuII, SaII and StuI). This digestion resulted in an average cDNA size of 600 bp, rather than the average size of 300 by that results from digestion with RsaI according to the Clontech protocol. This modification did not affect the subtraction efficiency. Two tester populations were then created with different adapters, and the driver library remained without adapters.
The tester and driver libraries were then hybridized using excess driver cDNA. In the first hybridization step, driver was separately hybridized with each of the two tester cDNA populations. This resulted in populations of (a) unhybridized tester cDNAs, (b) tester cDNAs hybridized to other tester cDNAs, (c) tester cDNAs hybridized to driver cDNAs and (d) unhybridized driver cDNAs. The two separate hybridization reactions were then combined, and rehybridized in the presence of additional denatured driver cDNA. Following this second hybridization, in addition to populations (a) through (d), a fifth population (e) was generated in which tester cDNA
with one adapter hybridized to tester cDNA with the second adapter.
Accordingly, the second hybridization step resulted in enrichment of differentially expressed sequences which could be used as templates for PCR amplification with adaptor-specific primers.

The ends were then filled in, and PCR amplification was performed using adaptor-specific primers. Only population (e), which contained tester cDNA that did not hybridize to driver cDNA, was amplified exponentially. A second PCR
amplification step was then performed, to reduce background and further enrich differentially expressed sequences.
This PCR-based subtraction technique normalizes differentially expressed cDNAs so that rare transcripts that are overexpressed in prostate tumor tissue may be recoverable. Such transcripts would be difficult to recover by traditional subtraction methods.
In addition to genes known to be overexpressed in prostate tumor, seventy-seven further clones were identified. Sequences of these partial cDNAs are provided in SEQ ID NO: 29 to 305. Most of these clones had no significant homology to database sequences. Exceptions were JPTPN23 (SEQ ID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQ ID NO: 234; similarity to rat mRNA
for proteasome subunit), JPTPN45 (SEQ ID NO: 243; similarity to rat norvegicus cytosolic NADP-dependent isocitrate dehydrogenase), JPTPN46 (SEQ ID NO: 244; similarity to human subclone H8 4 d4 DNA sequence), JP1D6 (SEQ ID NO: 265; similarity to G.
gallus dynein light chain-A), JP8D6 (SEQ ID NO: 288; similarity to human BAC
clone RG016J04), JP8F5 (SEQ ID NO: 289; similarity to human subclone H8 3 b5 DNA
sequence), and JP8E9 (SEQ ID NO: 299; similarity to human Alu sequence).
Additional studies using the PCR-based subtraction library consisting of a prostate tumor pool subtracted against a normal prostate pool (referred to as PT-PN
PCR subtraction) yielded three additional clones. Comparison of the cDNA
sequences of these clones with the most recent release of GenBank revealed no significant homologies to the two clones referred to as P715P and P767P (SEQ ID NO: 312 and 314). The remaining clone was found to show some homology to the known gene KIAA0056 (SEQ ID NO: 318). Using microarray analysis to measure mRNA
expression levels in various tissues, all three clones were found to be over-expressed in prostate tumors and BPH tissues. Specifically, clone P715P was over-expressed in most prostate tumors and BPH tissues by a factor of three or greater, with elevated expression seen in the majority of normal prostate samples and in fetal tissue, but negative to low expression in all other normal tissues. Clone P767P was over-expressed in several prostate tumors and BPH tissues, with moderate expression levels in half of the normal prostate samples, and background to low expression in all other normal tissues tested.
Further analysis, by microarray as described above, of the PT-PN PCR
subtraction library and of a DNA subtraction library containing cDNA from prostate tumor subtracted with a pool of normal tissue cDNAs, Ied to the isolation of additional clones (SEQ ID NO: 340-365 and 381) which were determined to be over-expressed in prostate tumor. The clones of SEQ ID NO: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and 364 were also found to be expressed in normal prostate.
Expression of all 26 clones in a variety of normal tissues was found to be low or undetectable, with the exception of P544S (SEQ ID NO: 356) which was found to be expressed in small intestine. Of the 26 clones, 11 (SEQ ID NO: 340-349 and 362) were found to show some homology to previously identified sequences. No significant homologies were found to the clones of SEQ ID NO: 350, 351, 353-361, and 363-365.
Comparison of the sequence of SEQ ID NO: 362 with sequences in the GenBank and GeneSeq DNA databases showed that this clone (referred to as P788P) is identical to GeneSeq Accession No. X27262, which encodes a protein found in the GeneSeq protein Accession No. Y00931. The fuel length cDNA sequence of P788P
is shown in Figure 12A (SEQ ID NO: 777), with the corresponding predicted amino acid being shown in Figure 12B (SEQ ID NO: 778). Subsequently, a full-length cDNA
sequence for P788P that contains polymorphisms not found in the sequence of SEQ ID
NO: 779, was cloned multiple times by PCR amplification from cDNA prepared from several RNA templates from three individuals. This determined cDNA sequence of this polymorphic variant of P788P is provided in SEQ ID NO: 779, with the corresponding amino acid sequence being provided in SEQ ID NO: 780. The sequence of SEQ ID
NO: 780 differs from that of SEQ ID NO: 778 by six amino acid residues. The protein has 7 potential transmembrane domains at the C-terminal portion and is predicted to be a plasma membrane protein with an extracellular N-terminal region.

Further studies on the clone of SEQ ID NO: 352 (referred to as P790P) led to the isolation of the full-length cDNA sequence of SEQ ID NO: 526. The corresponding predicted amino acid is provided in SEQ ID NO: 527. Data from two quantitative PCR experiments indicated that P790P is over-expressed in 11/15 tested prostate tumor samples and is expressed at low levels in spinal cord, with no expression being seen in all other normal samples tested. Data from further PCR
experiments and microarray experiments showed over-expression in normal prostate and prostate tumor with little or no expression in other tissues tested. P790P was subsequently found to show significant homology to a previously identified G-protein coupled prostate tissue receptor.
Additional studies on the clone of SEQ ID NO: 354 (referred to as P776P) led to the isolation of an extended cDNA sequence, provided in SEQ ID
NO:
569. The determined cDNA sequences of three additional splice variants of P776P are provided in SEQ ID NO: 570-572. The amino acid sequences encoded by two predicted open reading frames (ORFs) contained within SEQ ID NO: 570, one predicted ORF
contained within SEQ ID NO: 571, and 11 predicted ORFs contained within SEQ ID
NO: 569, are provided in SEQ ID NO: 573-586, respectively. Further studies led to the isolation of the full-length sequence for the clone of SEQ ID NO: S70 (provided in SEQ
ID NO: 880). Full-length cloning efforts on the clone of SEQ ID NO: 571 led to the isolation of two sequences (provided in SEQ ID NO: 881 and 882), representing a single clone, that are identical with the exception of a polymorphic insertion/deletion at position 1293. Specifically, the clone of SEQ ID NO: 882 (referred to as clone Fl) has a C at position 1293. The clone of SEQ ID NO: 881 (referred to as clone F2) has a single base pair deletion at position 1293. The predicted amino acid sequences encoded by 5 open reading frames located within SEQ ID NO: 880 are provided in SEQ ID
NO:
883-887, with the predicted amino acid sequences encoded by the clone of SEQ
ID NO:
881 and 882 being provided in SEQ ID NO: 888-893.
Comparison of the cDNA sequences for the clones P767P (SEQ ID NO:
c 314) and P777P (SEQ ID NO: 350) with sequences in the GenBank human EST
database showed that the two clones matched many EST sequences in common, suggesting that P767P and P777P may represent the same gene. A DNA consensus sequence derived from a DNA sequence alignment of P767P, P777P and multiple EST
clones is provided in SEQ ID NO: 587. The amino acid sequences encoded by three putative ORFs located within SEQ ID NO: 587 are provided in SEQ ID NO: 588-590.
The clone of SEQ ID NO: 342 (referred to as P789P) was found to show homology to a previously identified gene. The full length cDNA sequence for and the corresponding amino acid sequence are provided in SEQ ID NO: 878 and 879, respectively.

PEPTIDE PRIMING OF MICE AND PROPAGATION OF CTL LINES
6.1. This Example illustrates the preparation of a CTL cell line specific for cells expressing the P502S gene.
Mice expressing the transgene for human HLA A2I~b (provided by Dr L.
Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which is derived from the P502S gene (also referred to herein as J1-17, SEQ ID NO: 8), as described by Theobald et al., Ps°oc. Natl.
Acad. Sci. USA 92:11993-11997, 1995 with the following modifications. Mice were immunized with 100~,g of P2S#12 and 120~.g of an I-Ab binding peptide derived from hepatitis B Virus protein emulsified in incomplete Freund's adjuvant. Three weeks later these mice were sacrificed and using a nylon mesh single cell suspensions prepared.
Cells were then resuspended at 6 x 106 cells/ml in complete media (RPMI-1640;
Gibco BRL, Gaithersburg, MD) containing 10% FCS, 2mM Glutamine (Gibco BRL), sodium pyruvate (Gibco BRL), non-essential amino acids (Gibco BRL), 2 x 10-5 M 2-mercaptoethanol, SOU/ml penicillin and streptomycin, and cultured in the presence of irradiated (3000 rads) P2S#12-pulsed (Smg/ml P2S#12 and l Omg/ml (32-microglobulin) LPS blasts (A2 transgenic spleens cells cultured in the presence of 7~g/ml dextran sulfate and 25~g/ml LPS for 3 days). Six days later, cells (5 x 105/m1) were restimulated with 2.5 x 106/m1 peptide pulsed irradiated (20,000 rads) EL4A2Kb cells (Sherman et a1, Science 258:815-818, 1992) and 3 x 106/m1 A2 transgenic spleen feeder cells. Cells were cultured in the presence of 20U/ml IL-2. Cells continued to be .
restimulated on a weekly basis as described, in preparation for cloning the line.
P2S#12 line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (I x 104 cells/ well) as stimulators and A2 transgenic spleen cells as feeders ( 5 x 105 cells/ well) grown in the presence of 30U/ml IL-2.
On day 14, cells were restimulated as before. On day 21, clones that were growing were isolated and maintained in culture. Several of these clones demonstrated significantly higher reactivity (lysis) against human fibroblasts (HLA A2Kb expressing) transduced with P502S than against control fibroblasts. An example is presented in Figure 1.
This data indicates that P2S #12 represents a naturally processed epitope of the P502S protein that is expressed in the context of the human HLA A2Kb molecule.
6.2. This Example illustrates the preparation of murine CTL lines and CTL clones specific for cells expressing the P501 S gene.
This series of experiments were performed similarly to that described above. Mice were immunized with the P1S#10 peptide (SEQ ID NO: 337), which is derived from the P501 S gene (also referred to herein as Ll-12, SEQ ID NO: 1 IO). The P1S#10 peptide was derived by analysis of the predicted polypeptide sequence for P501 S for potential HLA-A2 binding sequences as defined by published HLA-A2 binding motifs (Parker, KC, et al, J. Imrnunol., 152:I63, 1994). P1S#10 peptide was synthesized as described in Example 4, and empirically tested for HLA-A2 binding using a T cell based competition assay. Predicted A2 binding peptides were tested for their ability to compete HLA-A2 specific peptide presentation to an HLA-A2 restricted CTL clone (D150M58), which is specific for the HLA-A2 binding influenza matrix peptide fluM58. D150M58 CTL secretes TNF in response to self presentation of peptide f1uM58. In the competition assay, test peptides at I00-200 ~g/mI were added to cultures of D150M58 CTL in order to bind HLA-A2 on the CTL. After thirty minutes, CTL cultured with test peptides, or control peptides, were tested for their antigen dose response to the fluM58 peptide in a standard TNF bioassay. As shown in Figure 3, peptide P1S#10 competes HLA-A2 restricted presentation of f1uM58, demonstrating that peptide P 1 S# 10 binds HLA-A2.
Mice expressing the transgene for human HLA A2Kb were immunized as described by Theobald et al. (Proc. Natl. Acad. Sci. USA 92:11993-11997, 1995) with the following modifications. Mice were immunized with 62.Spg of P1S #10 and 120~g of an I-Ab binding peptide derived from Hepatitis B Virus protein emulsified in incomplete Freund's adjuvant. Three weeks Iater these mice were sacrificed and single cell suspensions prepared using a nylon mesh. Cells were then resuspended at 6 x 106 cells/ml in complete media (as described above) and cultured in the presence of irradiated (3000 rads) P1S#10-pulsed (2~g/ml P1S#10 and lOmg/ml (32-microglobulin) LPS blasts (A2 transgenic spleens cells cultured in the presence of 7~,g/ml dextran sulfate and 25~,g/ml LPS for 3 days). Six days later cells (5 x 105/m1) were restimulated with 2.5 x 106/m1 peptide-pulsed irradiated (20,000 rads) EL4A2Kb cells, as described above, and 3 x 106/m1 A2 transgenic spleen feeder cells. Cells were cultured in the presence of 20 U/ml IL-2. Cells were restimulated on a weekly basis in preparation for cloning. After three rounds of in vitro stimulations, one line was generated that recognized P 1 S# 10-pulsed Jurkat A2Kb targets and P501 S-transduced Jurkat targets as shown in Figure 4.
A P1S#10-specific CTL line was cloned by limiting dilution analysis with peptide pulsed EL4 A2Kb tumor cells (1 x 104 cells/ well) as stimulators and A2 transgenic spleen cells as feeders (5 x 105 cells/ well) grown in the presence of 30U/ml IL-2. On day 14, cells were restimulated as before. On day 21, viable clones were isolated and maintained in culture. As shown in Figure 5, five of these clones demonstrated specific cytolytic reactivity against P501 S-transduced Jurkat A2Kb targets. This data indicates that P1S#10 represents a naturally processed epitope of the P501 S protein that is expressed in the context of the human HLA-A2.1 molecule.

PRIMING OF CTL IN 1~IY0 USING NAKED DNA IMMUNIZATION
WITH A PROSTATE ANTIGEN
The prostate-specific antigen L1-12, as described above, is also referred to as P501 S. HLA A2Kb Tg mice (provided by Dr L. Sherman, The Scripps Research Institute, La Jolla, CA) were immunized with 100 pg P501 S in the vector either intramuscularly or intradermally. The mice were immunized three times, with a two week interval between immunizations. Two weeks after the last immunization, immune spleen cells were cultured with Jurkat A2Kb-P501 S transduced stimulator cells. CTL lines were stimulated weekly. After two weeks of in vitro stimulation, CTL
activity was assessed against P501 S transduced targets. Two out of 8 mice developed strong anti-P501 S CTL responses. These results demonstrate that P501 S
contains at least one naturally processed HLA-A2-restricted CTL epitope.

ABILITY OF HUMAN T CELLS TO RECOGNIZE PROSTATE-SPECIFIC POLYPEPTIDES
This Example illustrates the ability of T cells specific for a prostate tumor polypeptide to recognize human tumor.
Human CD8+ T cells were primed in vitro to the P2S-12 peptide (SEQ
ID NO: 306) derived from P502S (also referred to as J1-17) using dendritic cells according to the protocol of Van Tsai et al. (Critical Reviews in Im~iunology 18:65-75, 1998). The resulting CD8+ T cell microcultures were tested for their ability to recognize the P2S-12 peptide presented by autologous fibroblasts or fibroblasts which were transduced to express the P502S gene in a y-interferon ELISPOT assay (see Lalvani et al., J. Exp. Med. 186:859-865, 1997). Briefly, titrating numbers of T cells were assayed in duplicate on 104 fibroblasts in the presence of 3 ~,g/ml human (32-microglobulin and 1 ~,g/ml P2S-12 peptide or control E75 peptide. In addition, T cells were simultaneously assayed on autologous fibroblasts transduced with the P502S gene or as a control, fibroblasts transduced with HER-2/neu. Prior to the assay, the fibroblasts were treated with 10 ng/ml y-interferon for 48 hours to upregulate class I
MHC expression. One of the microcultures (#5) demonstrated strong recognition of both peptide pulsed fibroblasts as well as transduced fibroblasts in a y-interferon ELISPOT assay. Figure 2A demonstrates that there was a strong increase in the number of y-interferon spots with increasing numbers of T cells on fibroblasts pulsed with the P2S-12 peptide (solid bars) but not with the control E75 peptide (open bars).
This shows the ability of these T cells to specifically recognize the P2S-12 peptide. As shown in Figure 2B, this microculture also demonstrated an increase in the number of y-interferon spots with increasing numbers of T cells on fibroblasts transduced to express the P502S gene but not the HER-2/neu gene. These results provide additional confirmatory evidence that the P2S-12 peptide is a naturally processed epitope of the P502S protein. Furthermore, this also demonstrates that there exists in the human T cell repertoire, high affinity T cells which are capable of recognizing this epitope. These T
cells should also be capable of recognizing human tumors which express the gene.

ELICITATION OF PROSTATE ANTIGEN-SPECIFIC CTL RESPONSES
IN HUMAN BLOOD
This Example illustrates the ability of a prostate-specific antigen to elicit a CTL response in blood of normal humans.
Autologous dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal donors by growth for five days in RPMI
medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4.
Following culture, DC were infected overnight with recombinant P501 S-expressing vaccinia virus at an M.O.I. of 5 and matured for 8 hours by the addition of 2 micrograms/ml CD40 ligand. Virus was inactivated by UV irradiation, CD8+ cells were isolated by positive selection using magnetic beads, and priming cultures were initiated in 24-well plates. Following five stimulation cycles using autologous fibroblasts retrovirally transduced to express P501 S and CD80, CD8+ lines were identified that specifically produced interferon-gamma when stimulated with autologous P501 S-transduced fibroblasts. The P501 S-specific activity of cell line 3A-1 could be maintained following additional stimulation cycles on autologous B-LCL
transduced with P501 S. Line 3A-1 was shoran to specifically recognize autologous B-LCL
transduced to express P501 S, but not EGFP-transduced autologous B-LCL, as measured by cytotoxicity assays (5' Cr release) and interferon-gamma production (Interferon-gamma Elispot; see above and Lalvani et al., J. Exp. Med. 186:859-865, 1997).
The results of these assays are presented in Figures 6A and 6B.

IDENTIFICATION OF A NATURALLY PROCESSED CTL EPITOPE.CONTAINED WITHIN THE

The 9-mer peptide p5 (SEQ ID NO: 338) was derived from the P703P
antigen (also referred to as P20). The p5 peptide is immunogenic in human HLA-donors and is a naturally processed epitope. Antigen specif c human CD8+ T
cells can be primed following repeated in vitf°o stimulations with monocytes pulsed with p5 peptide. These CTL specifically recognize p5-pulsed and P703P-transduced target cells in both ELISPOT (as described above) and chromium release assays.
Additionally, immunization of HLA-A2Kb transgenic mice with p5 leads to the generation of CTL
lines which recognize a variety of HLA-A2Kb or HLA-A2 transduced target cells expressing P703P.
Initial studies demonstrating that p5 is a naturally processed epitope were done using HLA-A2Kb transgenic mice. HLA-A2Kb transgenic mice were immunized subcutaneously in the footpad with 100 ~g of p5 peptide together with 140 ~g of hepatitis B virus core peptide (a Th peptide) in Freund's incomplete adjuvant.
Three weeks post immunization, spleen cells from immunized mice were stimulated in vitro with peptide-pulsed LPS blasts. CTL activity was assessed by chromium release assay five days after primary if2 vitro stimulation. Retrovirally transduced cells expressing the control antigen P703P and HLA-A2Kb were used as targets. CTL lines that specifically recognized both p5-pulsed targets as well as P703P-expressing targets were identified.
Human in vitro priming experiments demonstrated that the p5 peptide is immunogenic in humans. Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal human donors by culturing for five days in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL-4. Following culture, the DC were pulsed with 1 ug/ml p5 peptide and cultured with CD8+ T cell enriched PBMC. CTL lines were restimulated on a weekly basis with p5-pulsed monocytes. Five to six weeks after initiation of the CTL
cultures, CTL recognition of p5-pulsed target cells was demonstrated. CTL were additionally shown to recognize human cells transduced to express P703P, demonstrating that p5 is a naturally processed epitope.
Studies identifying a further peptide epitope (referred to as peptide 4) derived from the prostate tumor-specific antigen P703P that is capable of being recognized by CD4 T cells on the surface of cells in the context of HLA class II
molecules were carried out as follows. The amino acid sequence for peptide 4 is provided in SEQ ID NO: 78I, with the corresponding cDNA sequence being provided in SEQ ID NQ: 782.
Twenty 15-mer peptides overlapping by 10 amino acids and derived from the carboxy-terminal fragment of P703P were generated using standard procedures. Dendritic cells (DC) were derived from PBMC of a normal female donor using GM-CSF and IL-4 by standard protocols. CD4 T cells were generated from the same donor as the DC using MACS beads and negative selection. DC were pulsed overnight with pools of the 15-mer peptides, with each peptide at a final concentration of 0.25 microgram/ml. Pulsed DC were washed and plated at 1 x 104 cells/well of 96-well V-bottom plates and purified CD4 T cells were added at 1 x 105/well.
Cultures were supplemented with 60 ng/ml IL-6 and 10 ng/ml IL-12 and incubated at 37 °C.
Cultures were restimulated as above on a weekly basis using DC generated and pulsed as above as antigen presenting cells, supplemented with 5 ng/ml IL-7 and 10 ulml IL-2.
Following 4 ire vitro stimulation cycles, 96 lines (each line corresponding to one well) were tested for specific proliferation and cytokine production in response to the stimulating pools with an irrelevant pool of peptides derived from mammaglobin being used as a control.
One line (referred to as 1-F9) was identified from pool #1 that demonstrated specific proliferation (measured by 3H proliferation assays) and cytokine production (measured by interferon-gamma ELISA assays) in response to pool #1 of P703P peptides. This line was further tested for specific recognition of the peptide pool, specific recognition of individual peptides in the pool, and in HLA
mismatch analyses to identify the relevant restricting allele. Line 1-F9 was found to specifically proliferate and produce interferon-gamma in response to peptide pool #l, and also to peptide 4 (SEQ ID NO: 781). Peptide 4 corresponds to amino acids 126-140 of SEQ ID
NO: 327. Peptide titration experiments were conducted to assess the sensitivity of line 1-F9 for the specific peptide. The line was found to specifically respond to peptide 4 at concentrations as low as 0.25 ng/ml, indicating that the T cells are very sensitive and therefore likely to have high affinity for the epitope.
To determine the HLA restriction of the P703P response, a panel of antigen presenting cells (APC) was generated that was partially matched with the donor used to generate the T cells. The APC were pulsed with the peptide and used in proliferation and cytokine assays together with line 1-F9. APC matched with the donor at HLA-DRB0701 and HLA-DQB02 alleles were able to present the peptide to the T
cells, indicating that the P703P-specific response is restricted to one of these alleles.
Antibody blocking assays were utilized to determine if the restricting allele was HLA-DR0701 or HLA-DQ02. The anti-HLA-DR blocking antibody L243 or an irrelevant isotype matched IgG2a were added to T cells and APC cultures pulsed with the peptide RMPTVLQCVNVSVVS (SEQ ID NO: 781) at 250 ng/ml.
Standard interferon-gamma and proliferation assays were performed. Whereas the control antibody had no effect on the ability of the T cells to recognize peptide-pulsed APC, in both assays the anti-HLA-DR antibody completely blocked the ability of the T cells to specifically recognize peptide-pulsed APC.
To determine if the peptide epitope RMPTVLQCVNVSVVS (SEQ ID
NO: 781) was naturally processed, the ability of line 1-F9 to recognize APC
pulsed with recombinant P703P protein was examined. For these experiments a number of recombinant P703P sources were utilized; E. coli-derived P703P, Pichia-derived and baculovirus-derived P703P. Irrelevant protein controls used were E. coli-derived L3E a lung-specific antigen) and baculovirus-derived mammaglobin. In interferon-gamma ELISA assays, Line 1-F9 was able to efficiently recognize both E. coli forms of S P703P as well as Pichia-derived recombinant P703P, while baculovirus-derived was recognized Less efficiently. Subsequent Western blot analysis revealed that the E
coli and Pichia P703P protein preparations were intact while the baculovirus preparation was approximately 7S% degraded. Thus, peptide RMPTVLQCVNVSVVS
(SEQ ID NO: 781) from P703P is a naturally processed peptide epitope derived from P703P and presented to T cells in the context of HLA-DRB-0701 In further studies, twenty-four 1 S-mer peptides overlapping by 10 amino acids and derived from the N-terminal fragment of P703P (corresponding to amino acids 27-1 S4 of SEQ ID NO: S2S) were generated by standard procedures and their ability to be recognized by CD4 cells was determined essentially as described above.
1 S DC were pulsed overnight with pools of the peptides with each peptide at a final concentration of 10 microgram/ml. A large number of individual CD4 T cell lines (6S/480) demonstrated significant proliferation and cytokine release (IFN-gamma) in response to the P703P peptide pools but not to a control peptide pool. The CD4 T cell lines which demonstrated specific activity were restimulated on the appropriate pool of P703P peptides and reassayed on the individual peptides of each pool as well as a peptide dose titration of the pool of peptides in a IFN-gamma release assay and in a proliferation assay.
Sixteen immunogenic peptides were recognized by the T cells from the entire set of peptide antigens tested. The amino acid sequences of these peptides are 2S provided in SEQ ID NO: 799-814, with the corresponding cDNA sequences being provided in SEQ ID NO: 783-798, respectively. In some cases the peptide reactivity of the T cell line could be mapped to a single peptide, however some could be mapped to more than one peptide in each pool. Those CD4 T cell Lines that displayed a representative pattern of recognition from each peptide pool with a reasonable affinity for peptide were chosen for further analysis (I-lA, -6A; II-4C, -SE; III-6E, IV-4B, -3F, -9B, -lOF, V-SB, -4D, and -lOF). These CD4 T cells lines were restimulated on the appropriate individual peptide and reassayed on autologous DC pulsed with a truncated form of recombinant P703P protein made in E. coli (a.a. 96 - 254 of SEQ ID NO:
525), full-length P703P made in the baculovirus expression system, and a fusion between influenza virus NS1 and P703P made in E. coli. Of the T cell lines tested, line I-lA
recognized specifically the truncated form of P703P (E. coli) but no other recombinant form of P703P. This line also recognized the peptide used to elicit the T
cells. Line 2-4C recognized the truncated form of P703P (E. coli) and the full length form of P703P
made in baculovirus, as well as peptide. The remaining T cell lines tested were either peptide-specific only (II-SE, II-6F, IV-4B, IV-3F, IV-9B, IV-lOF, V-SB and V-4D) or were non-responsive to any antigen tested (V-lOF). These results demonstrate that the peptide sequence RPLLANDLMLIKLDE (SEQ ID NO: 814; corresponding to a.a. 110-124 of SEQ ID NO: 525) recognized by the T cell line I-lA, and the peptide sequences SVSESDTIRSISIAS (SEQ ID NO: 811; corresponding to a.a. I25-139 of SEQ ID NO:
525) and ISIASQCPTAGNSCL (SEQ ID NO: 810; corresponding to a.a. 135-149 of ' 15 SEQ ID NO: 525) recognized by the T cell line II-4C may be naturally processed epitopes of the P703P protein.
In further studies, forty 15-mer peptides overlapping by 10 amino acids and derived spanning amino acids 47 to 254 of P703P (SEQ ID NO: 525) were generated by standard procedures and their ability to be recognized by CD4 cells was determined essentially as described above. DC were prepared from PBMC of a donor having distinct HLA DR and DQ alleles from that used in previous experiments.
DC
were pulsed overnight with pools of the peptides with each peptide at a final concentration of 0.25 microgram/ml, and each pool containing 10 peptides.
Twelve lines were identified that demonstrated specific proliferation and cytokine production (measured in gamma-interferon ELISA assays) in response to the stimulating peptide pool. These lines were further tested for specific recognition of the peptide pool, specific recognition of individual peptides in the pool, and specific recognition of recombinant P703P protein. Lines 3ASH and 3A9H specifically proliferated and produced gamma-interferon in response to recombinant protein and one individual peptide as well as the peptide pool. Following re-stimulation on targets loaded with the specific peptide, only 3A9H responded specifically to targets exposed to lysates of fibroblasts infected adenovirus expressing full-length P703P. These results indicates that the line 3A9H can respond to antigenic peptide derived from protein synthesized in mammalian cells. The peptide to which the specific CD4 line responded correspond to amino acids 155-170 of P703P (SEQ ID NO: 943). The DNA sequence for this peptide is provided in SEQ ID NO: 942.

EXPRESSION OF A BREAST TUMOR-DERIVED ANTIGEN
IN PROSTATE
Isolation of the antigen B305D from breast tumor by differential display is described in US Patent Application No. 08/700,014, filed August 20, 1996.
Several different splice forms of this antigen were isolated. The determined cDNA
sequences for these splice forms are provided in SEQ ID NO: 366-375, with the predicted amino acid sequences corresponding to the sequences of SEQ ID NO: 292, 298 and 301-being provided in SEQ ID NO: 299-306, respectively. In further studies, a splice variant of the cDNA sequence of SEQ ID NO: 366 was isolated which was found to contain an additional guanine residue at position 884 (SEQ ID NO: 530), leading to a frameshift in the open reading frame. The determined DNA sequence of this ORF
is provided in SEQ ID NO: 531. This frameshift generates a protein sequence (provided in SEQ ID NO: 532) of 293 amino acids that contains the C-terminal domain common to the other isoforms of B305D but that differs in the N-terminal region.
The expression levels of B305D in a variety of tumor and normal tissues were examined by real time PCR and by Northern analysis. The results indicated that B305D is highly expressed in breast tumor, prostate tumor, normal prostate and normal testes, with expression being low or undetectable in all other tissues examined (colon tumor, lung tumor, ovary tumor, and normal bone marrow, colon, kidney, liver, lung, ovary, skin, small intestine, stomach). Using real,-time PCR on a panel of prostate tumors, expression of B305D in prostate tumors was shown to increase with increasing Gleason grade, demonstrating that expression of .B30SD increases as prostate cancer progresses.

S GENERATION OF HUMAN CTL IN Y ITRO USING WHOLE GENE PRIMING AND STIMULATION

Using irz vita°o whole-gene priming with PSO1S-vaccinia infected DC
(see, for example, Yee et al, The Journal of Immuhology, 1S7(9):4079-86, 1996), human CTL lines were derived that specifically recognize autologous fibroblasts transduced with PSOlS (also known as Ll-12), as determined by interferon-y ELISPOT
analysis as described above. Using a panel of HLA-mismatched B-LCL lines transduced with PSOlS, these CTL lines were shown to be likely restricted to HLAB
class I allele. Specifically, dendritic cells (DC) were differentiated from monocyte 1 S cultures derived from PBMC of normal human donors by growing for five days in RPMI medium containing 10% human serum, SO ng/ml human GM-CSF and 30 ng/ml human IL-4. Following culture, DC were infected overnight with recombinant vaccinia virus at a multiplicity of infection (M.O.I) of f ve, and matured overnight by the addition of 3 ~.glml CD40 ligand. Virus was inactivated by UV irradiation.
CD8+
T cells were isolated using a magnetic bead system, and priming cultures were initiated using standard culture techniques. Cultures were restimulated every 7-10 days using autologous primary fibroblasts retrovirally transduced with PSO1S and CD80.
Following four stimulation cycles, CD8+ T cell lines were identified that specifically produced interferon-y when stimulated with PSO1S and CD80-transduced autologous 2S fibroblasts. A panel of HLA-mismatched B-LCL lines transduced with PSOl S
were generated to define the restriction allele of the response. By measuring interferon-y in an ELISPOT assay, the PSOl S specific response was shown to be likely restricted by HLA B alleles. These results demonstrate that a CD8+ CTL response to PSOl S
can be elicited.

To identify the epitope(s) recognized, cDNA encoding P501 S was fragmented by various restriction digests, and sub-cloned into the retroviral expression vector pBIB-KS. Retroviral supernatants were generated by transfection of the helper packaging line Phoenix-Ampho. Supernatants were then used to transduce Jurkat/A2Kb cells for CTL screening. CTL were screened in IFN-gamma ELISPOT
assays against these A2Kb targets transduced with the "library" of PSOl S
fragments.
Initial positive fragments P50I S/H3 and P50I S/F2 were sequenced and found to encode amino acids 106-553 and amino acids 136-547, respectively, of SEQ ID NO: 113.
A
truncation of H3 was made to encode amino acid residues 106-351 of SEQ ID NO:
113, which was unable to stimulate the CTL, thus localizing the epitope to amino acid residues 351-547. Additional fragments encoding amino acids 1-472 (Fragment A) and amino acids 1-351 (Fragment B) were also constructed. Fragment A but not Fragment B stimulated the CTL thus localizing the epitope to amino acid residues 351-472.
Overlapping 20-mer and 18-mer peptides representing this region were tested by pulsing Jurkat/A2Kb cells versus CTL in an IFN-gamma assay. Only peptides P501 S-369(20) and PSOlS-369(18) stimulated the CTL. Nine-mer and 10-mer peptides representing this region were synthesized and similarly tested. Peptide P501 S-370 (SEQ ID
NO:
539) was the minimal 9-mer giving a strong response. Peptide P501 S-376 (SEQ
ID NO:
540) also gave a weak response, suggesting that it might represent a cross-reactive epitope.
In subsequent studies, the ability of primary human B cells transduced with P501 S to prime MHC class I-restricted, P501 S-specific, autologous CD8 T
cells was examined. Primary B cells were derived from PBMC of a homozygous HLA-A2 donor by culture in CD40 ligand and IL-4, transduced at high frequency with recombinant P501 S in the vector pBIB, and selected with blastocidin-S. For in vitro priming, purified CD8+ T cells were cultured with autologous CD40 ligand + IL-derived, P501 S-transduced B cells in a 96-well microculture format. These CTL
microcultures were re-stimulated with P501 S-transduced B cells and then assayed for specificity. Following this initial screen, microcultures with significant signal above background were cloned on autologous EBV-transformed B cells (BLCL), also transduced with P501 S. Using IFN-gamma ELISPOT for detection, several of these CD8 T cell clones were found to be specific for P501 S, as demonstrated by reactivity to BLCL/P501 S but not BLCL transduced with control antigen. It was further demonstrated that the anti-P501 S CD8 T cell specificity is HLA-A2-restricted.
First, antibody blocking experiments with anti-HLA-A,B,C monoclonal antibody (W6.32), anti-HLA-B,C monoclonal antibody (B1.23.2) and a control monoclonal antibody showed that only the anti-HLA-A,B,C antibody blocked recognition of P501 S-expressing autologous BLCL. Secondly, the anti-P501 S CTL also recognized an HLA
A2 matched, heterologous BLCL transduced with P501 S, but not the corresponding EGFP transduced control BLCL.
A naturally processed, CDB, class I-restricted peptide epitope of P501 S
was identified as follows. Dendritic Cells (DC) were isolated by Percol gradient followed by differential adherence, and cultured for 5 days in the presence of RPMI
medium containing 1% human serum, SOng/ml GM-CSF and 30ng/ml IL-4. Following culture, DC were infected for 24 hours with P501 S-expressing adenovirus at an MOI of 10 and matured for an additional 24 hours by the addition of 2ug/ml CD40 ligand. CD8 cells were enriched for by the subtraction of CD4+, CD14+ and CD16+
populations from PBMC with magnetic beads. Priming cultures containing 10,000 P501 S-expressing DC and 100,000 CD8+ T cells per well were set up in 96-well V-bottom plates with RPMI containing 10% human serum, Sng/ml IL-12 and lOnglml IL-6.
Cultures were stimulated every 7 days using autologous fibroblasts retrovirally transduced to express PSOlS and CD80, and were treated with IFN-gamma for 48-hours to upregulate MHC Class I expression. l0u/ml IL-2 was added at the time of stimulation and on days 2 and 5 following stimulation. Following 4 stimulation cycles, one P501 S-specific CD8+ T cell line (referred to as ZA2) was identified that produced IFN-gamma in response to IFN-gamma-treated PSO1S/CD80 expressing autologous fibroblasts, but not in response to IFN-gamma-treated P703P/CD80 expressing autologous fibroblasts in a y-IFN Elispot assay. Line 2A2 was cloned in 96-well plates with 0.5 cell/well or 2 cells/well in the presence of 75,000 PBMClwell, 10,000 B-LCL/well, 30ng/ml OKT3 and SOu/ml IL-2. Twelve clones were isolated that showed strong P501 S specificity in response to transduced fibroblasts.

Fluorescence activated cell sorting (FACS) analysis was performed on P501 S-specific clones using CD3-, CD4- and CD8-specific antibodies conjugated to PercP, FITC and PE respectively. Consistent with the use of CD8 enriched T
cells in the priming cultures, P5401S-specific clones were determined to be CD3+, CD8+
and CD4-.
To identify the relevant P501 S epitope recognized by P501 S specific CTL, pools of 18-20 mer or 30-mer peptides that spanned the majority of the amino acid sequence of P501 S were loaded onto autologous B-LCL and tested in y-IFN
Elispot assays for the ability to stimulate two P501 S-specific CTL clones, referred to as 4E5 and 4E7. One pool, composed of five 18-20 mer peptides that spanned amino acids 411-486 of P501 S (SEQ ID NO: 113), was found to be recognized by both P501 S-specific clones. To identify the specific 18-20 mer peptide recognized by the clones, each of the 18-20 mer peptides that comprised the positive pool were tested individually in y-IFN
Elispot assays for the ability to stimulate the two P501 S-specific CTL
clones, 4E5 and 4E7. Both 4E5 and 4E7 specifically recognized one 20-mer peptide (SEQ ID NO:
853;
cDNA sequence provided in SEQ ID NO: 854) that spanned amino acids 453-472 of P501 S. Since the minimal epitope recognized by CD8+ T cells is almost always either a 9 or 10-mer peptide sequence, 10-mer peptides that spanned the entire sequence of SEQ ID NO: 853 were synthesized that differed by 1 amino acid. Each of these 10-mer peptides was tested for the ability to stimulate two P501 S-specific clones, (referred to as 1D5 and 1E12). One 10-mer peptide (SEQ ID NO: 855; cDNA sequence provided in SEQ ID NO: 856) was identified that specifically stimulated the P501 S-specific clones.
This epitope spans amino acids 463-472 of P501 S. This sequence defines a minimal 10-mer epitope from P501 S that can be naturally processed and to which CTL
responses can be identified in normal PBMC. Thus, this epitope is a candidate for use as a vaccine moiety, and as a therapeutic and/or diagnostic reagent for prostate cancer.
To identify the class I restriction element for the P501 S-derived sequence of SEQ ID NO: 855, HLA blocking and mismatch analyses were performed. In y-IFN
Elispot assays, the specific response of clones 4A7 and 4E5 to P501 S-transduced autologous fibroblasts was blocked by pre-incubation with 25ug/ml W6/32 (pan-Class I
blocking antibody) and B 1.23.2 ~ (HLA-B/C blocking antibody). These results demonstrate that the SEQ ID NO: 855-specific response is restricted to an HLA-B or HLA-C allele.
For the HLA mismatch analysis, autologous B-LCL (HLA
Al,A2,B8,B51, Cwl, Cw7) and heterologous B-LCL (HLA
A2,A3,B18,BS1,Cw5,Cw14) that share the HLAB51 allele were pulsed for one hour with 20ug/ml of peptide of SEQ ID NO: 855, washed, and tested in y-IFN Elispot assays for the ability to stimulate clones 4A7 and 4E5. Antibody blocking assays with the B1.23.2 (HLA-B/C blocking antibody) were also performed. SEQ ID NO: 855-specific response was detected using both the autologous (D326) and heterologous (D107) B-LCL, and furthermore the responses were blocked by pre-incubation with 25ug/ml of B 1.23.2 HLA-B/C blocking antibody. Together these results demonstrate that the P501 S-specific response to the peptide of SEQ ID NO: 855 is restricted to the HLA-B51 class I allele. Molecular cloning and sequence analysis of the HLA-B51 allele from D3326 revealed that the HLA-B51 subtype of D326 is HLA-B51011.
Based on the 10-mer P501 S-derived epitope of SEQ ID NO: 855, two 9-mers with the sequences of SEQ ID NO: 857 and 858 were synthesized and tested in Elispot assays for the ability to stimulate two P501 S-specific CTL clones derived from line 2A2. The 10-mer peptide of SEQ ID'NO: 855, as well as the 9-mer peptide of SEQ
ID NO: 858, but not the 9-mer peptide of SEQ ID NO: 857, were capable of stimulating the P501 S-specific CTL to produce IFN-gamma. These results demonstrate that the peptide of SEQ ID NO: 858 is a 9-mer PSO1S-derived epitope recognized by PSO1S-specific CTL. The DNA sequence encoding the epitope of SEQ ID NO: 858 is provided in SEQ ID NO: 859.
To identify the class I restricting allele for the P501 S-derived peptide of SEQ ID NO: 855 and 858 specific response, each of the HLA B and C alleles were cloned from the donor used in the in vitro priming experiment. Sequence analysis indicated that the relevant alleles were HLA-B8, HLA-B51, HLA-Cw01 and HLA-Cw07. Each of these alleles were subcloned into an expression vector and co-transfected together with the PSO1S gene into VA-13 cells. Transfected VA-13 cells were then tested for the ability to specifically stimulate the P501 S-specific CTL in ELISPOT assays. VA-13 cells transfected with P501 S and HLA-B51 were capable of stimulating the P501 S-specific CTL to secrete gamma-IFN. VA-13 cells transfected with HLA-B51 alone or P501S + the other HLA-alleles were not capable of stimulating the P501 S-specific CTL. These results demonstrate that the restricting allele for the P501 S-specific response is the HLAB51 allele. Sequence analysis revealed that the subtype of the relevant restricting allele is HLA-B51011.
To determine if the P501 S-specific CTL could recognize prostate tumor cells that express P501 S, the P501 S-positive lines LnCAP and CRL2422 (both expressing "moderate" amounts of P501 S mRNA and protein), and PC-3 (expressing low amounts of P501 S mRNA and protein), plus the P501 S-negative cell line DU-were retrovirally transduced with the HLA-B51011 allele that was cloned from the donor used to generate the P501 S-specific CTL. HLA-B51011- or EGFP-transduced and selected tumor cells were treated with gamma-interferon and androgen (to upregulate stimulatory functions and P501 S, respectively) and used in gamma interferon Elispot assays with the P501 S-specific CTL clones 4E5 and 4E7.
Untreated cells were used as a control.
Both 4E5 and 4E7 efficiently and specifically recognized LnCAP and CRL2422 cells that were transduced with the HLA-B51011 allele, but not the same cell lines transduced with EGFP. Additionally, both CTL clones specifically recognized PC-3 cells transduced with HLA-B5101 l, but not the P501 S-negative tumor cell line DU-145. Treatment with gamma-interferon or androgen did not enhance the ability of CTL to recognize tumor cells. These results demonstrate that P501 S-specific CTL, generated by in vitro whole gene priming, specifically and efficiently recognize prostate tumor cell lines that express P501 S.
A naturally processed CD4 epitope of P501 S was identified as follows.
CD4 cells specific for P501 S were prepared as described above. A series of 16 overlapping peptides were synthesized that spanned approximately 50% of the amino terminal portion of the P501S gene (amino acids 1- 325 of SEQ ID NO:
113).
For priming, peptides were combined into pools of 4 peptides, pulsed at 4 ~.g/ml onto dendritic cells (DC) for 24 hours, with TNF-alpha. DC were then washed and mixed with negatively selected CD4+ T cells in 96 well U-bottom plates. Cultures were re-stimulated weekly on fresh DC loaded with peptide pools. Following a total of stimulation cycles, cells were rested for an additional week and tested for specificity to APC pulsed with peptide pools using y-IFN ELISA and proliferation assays. For these assays, adherent monocytes loaded with either the relevant peptide pool at 4ug/ml or an irrelevant peptide at ~,g/ml were used as APC. T cell lines that demonstrated either specific cytokine secretion or proliferation were then tested for recognition of individual peptides that were present in the pool. T cell lines could be identified from pools A and B that recognized individual peptides from these pools.
From pool A, lines AD9 and AE10 specifically recognized peptide 1 (SEQ ID NO: 862), and line AFS recognized peptide 39 (SEQ ID NO: 861). From pool B, line BC6 could be identified that recognized peptide 58 (SEQ ID NO: 860).
Each of these lines were stimulated on the specific peptide and tested for specific recognition of the peptide in a titration assay as well as cell lysates generated by infection of HEK 293 cells with adenovirus expressing either P501 S or an irrelevant antigen. For these assays, APC-adherent monocytes were pulsed with either 10, 1, or 0.1 ~g/ml individual peptides, and DC were pulsed overnight with a 1:5 dilution of adenovirally infected cell lysates. Lines AD9, AE10 and AFS retained significant recognition of the relevant P501 S-derived peptides even at 0.1 mg/ml. Furthermore, ,line AD9 demonstrated significant (8.1 fold stimulation index) specific activity for lysates from adenovirus-P501 S infected cells. These results demonstrate that high affinity CD4 T cell lines can be generated toward P501 S-derived epitopes, and that at least a subset of these T cells specific for the P501 S derived sequence of SEQ ID NO: 862 are specific for an epitope that is naturally processed by human cells. The DNA sequences encoding the amino acid sequences of SEQ ID NO: 860-862 are provided in SEQ ID NO: 863-865, respectively.
To further characterize the P501 S-specific activity of AD9, the line was cloned using anti-CD3. Three clones, referred to as 1A1, 1A9 and 1F5, were identified that were specific for the P501 S-1 peptide (SEQ ID NO: 862). To determine the HLA
restriction allele for the P501 S-specific response, each of these clones was tested in class II antibody blocking and HLA mismatch assays using proliferation and gamma-interferon assays. In antibody blocking assays and measuring gamma-interferon production using ELISA assays, the ability of all three clones to recognize peptide pulsed APC was specifically blocked by co-incubation with either a pan-class II
blocking antibody or a HLA-DR blocking antibody, but not with a HLA-DQ or an irrelevant antibody. Proliferation assays performed simultaneously with the same cells confirmed these results. These data indicate that the P501 S-specific response of the clones is restricted by an HLA-DR allele. Further studies demonstrated that the restricting allele for the PSO1S-specific response is HLA-DRB1501.

IDENTIFICATION OF PROSTATE-SPECIFIC ANTIGENS

This Example describes the isolation of certain prostate-specific polypeptides from a prostate tumor cDNA library.
A human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones that display at least a three fold over-expression in prostate tumor and/or normal prostate tissue, as compared to non-prostate normal tissues (not including testis). 372 clones were identified, and 319 were successfully sequenced. Table I presents a summary of these clones, which are shown in SEQ ID NOs:385-400. Of these sequences SEQ ID NOs:386, 389, 390 and 392 correspond to novel genes, and SEQ ID NOs: 393 and 396 correspond to previously identified sequences. The others (SEQ ID NOs:385, 387, 388, 391, 394, 395 and 400) correspond to known sequences, as shown in Table I.

Table I
Summary of Prostate Tumor Antigens Known Genes Previously IdentifiedNovel Genes Genes T-cell gamma chain P504S 23379 (SEQ ID
N0:389) Kallikrein P1000C 23399 (SEQ ID
N0:392) Vector P501 S 23320 (SEQ ID
N0:386) CGI-82 protein mRNA (23319;P503S 23381 (SEQ ID
SEQ N0:390) ID N0:385) Ald. 6 Dehyd. P784P

L-iditol-2 dehydrogenaseP502S
(23376; SEQ
ID N0:388) Ets transcription factorP706P
PDEF (22672;
SEQ ID N0:398) hTGR (22678; SEQ ID N0:399)19142.2, bangur.seq (22621; SEQ
ID N0:396) KIAA0295(22685; SEQ ID 5566.1 Wang (23404;
N0:400) SEQ ID
N0:393) Prostatic Acid Phosphatase(22655;P712P
SEQ ID N0:397) transglutaminase (22611;P778P
SEQ ID
N0:395) HDLBP (23508; SEQ ID
N0:394) CGI-69 Protein(23367;
SEQ ID
N0:3 87) KIAA0122(23383; SEQ ID
N0:391) TEEG

CGI-82 showed 4.06 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 43% of prostate tumors, 2S% normal prostate, not detected in other normal tissues tested. L-iditol-2 dehydrogenase showed 4.94 fold over-expression in prostate tissues as compared to S other normal tissues tested. It was over-expressed in 90% of prostate tumors, 100% of normal prostate, and not detected in other normal tissues tested. Ets transcription factor PDEF showed S.SS fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 47% prostate tumors, 2S% normal prostate and not detected in other normal tissues tested. hTGRl showed 9.11 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 63% of prostate tumors and is not detected in normal tissues tested including normal prostate. KIAA029S showed S.S9 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 47% of prostate tumors, low to undetectable in normal tissues tested including normal prostate tissues.
Prostatic acid 1 S phosphatase showed 9.14 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 67% of prostate tumors, SO% of normal prostate, and not detected in other normal tissues tested. Transglutaminase showed 14.84 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 30% of prostate tumors, SO% of normal prostate, and is not detected in other normal tissues tested. High density lipoprotein binding protein.
(HDLBP) showed 28.06 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors, 7S% of normal prostate, and is undetectable in all other normal tissues tested. CGI-69 showed 3.56 fold over-expression in prostate tissues as compared to other normal tissues tested. It is 2S a low abundant gene, detected in more than 90% of prostate tumors, and in 7S% normal prostate tissues. The expression of this gene in normal tissues was very low.
KIAA0122 showed 4.24 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in S7% of prostate tumors, it was undetectable in all normal tissues tested including normal prostate tissues.
19142.2 bangur showed 23.25 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors and 100% of normal prostate. It was undetectable in other normal tissues tested. 5566.1 Wang showed 3.31 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 97% of prostate tumors, 75% normal prostate and was also over-expressed in normal bone marrow, pancreas, and activated PBMC.
Novel clone 23379 (also referred to as P553S) showed 4.86 fold over-expression in prostate tissues as compared to other normal tissues tested. It was detectable in 97% of prostate tumors and 75% normal prostate and is undetectable in all other normal tissues tested. Novel clone 23399 showed 4.09 fold over-expression in prostate tissues as compared to other normal tissues tested. It was over-expressed in 27% of prostate tumors and was undetectable in all normal tissues tested including normal prostate tissues. Novel clone 23320 showed 3.15 fold over-expression in prostate tissues as compared to other normal tissues tested. It was detectable in all prostate tumors and 50% of normal prostate tissues. It was also expressed in normal colon and trachea.
Other normal tissues do not express this gene at high level.
Subsequent full-length cloning studies on P553S, using standard techniques, revealed that this clone is an incomplete spliced form of P501 S.
The determined cDNA sequences for four splice variants of P553S are provided in SEQ ID
NO: 702-705. An amino acid sequence encoded by SEQ ID NO: 705 is provided in SEQ ID NO: 706. The cDNA sequence of SEQ ID NO: 702 was found to contain two open reading frames (ORFs). The amino acid sequences encoded by these two ORFs are provided in SEQ ID NO: 707 and 708.

IDENTIFICATION OF PROSTATE-SPECIFIC ANTIGENS
BY ELECTRONIC SUBTRACTION
This Example describes the use of an electronic subtraction technique to identify prostate-specific antigens.

Potential prostate-specific genes present in the GenBank human EST
database were identified by electronic subtraction (similar to that described by Vasmatizis et al., P~oc. Natl. Acad. Sci. USA 95:300-304, 1998). The sequences of EST
clones (43,482) derived from various prostate libraries were obtained from the GenBank public human EST database. Each prostate EST sequence was used as a query sequence in a BLASTN (National Center for Biotechnology Information) search against the human EST database. All matches considered identical (length of matching sequence >100 base pairs, density of identical matches over this region > 70%) were grouped (aligned) together in a cluster. Clusters containing more than 200 ESTs were discarded since they probably represented repetitive elements or highly expressed genes such as those for ribosomal proteins. If two or more clusters shared common ESTs, those clusters were grouped together into a "supercluster," resulting in 4,345 prostate superclusters.
Records for the 479 human cDNA libraries represented in the GenBank release were downloaded to create a database of these cDNA library records.
These 479 cDNA libraries were grouped into three groups: Plus (normal prostate and prostate tumor libraries, and breast cell line libraries, in which expression was desired), Minus (libraries from other normal adult tissues, in which expression was not desirable), and Other (libraries from fetal tissue, infant tissue, tissues found only in women, non-prostate tumors and cell lines other than prostate cell lines, in which expression was considered to be irrelevant). A summary of these library groups is presented in Table II.

Table II
Prostate cDNA Libraries and ESTs Library # of Libraries# of ESTs Plus 25 43,482 Normal 11 18,875 Tumor 11 21,769 Cell lines 3 2,838 Minus 166 Other 287 Each supercluster was analyzed in terms of the ESTs within the supercluster. The tissue source of each EST clone was noted and used to classify the superclusters into four groups: Type 1- EST clones found in the Plus group libraries only; no expression detected in Minus or Other group libraries; Type 2- EST
clones derived from the Plus and Other group libraries only; no expression detected in the Minus group; Type 3- EST clones derived from the Plus, Minus and Other group libraries, but the number of ESTs derived from the Plus group is higher than in either the Minus or Other groups; and Type 4- EST clones derived from Plus, Minus and Other group libraries, but the number derived from the Plus group is higher than the number derived from the Minus group. This analysis identified 4,345 breast clusters (see Table III). From these clusters, 3,172 EST clones were ordered from Research Genetics, Inc., and were received as frozen glycerol stocks in 96-well plates.

Table III
Prostate Cluster Summary # of # of ESTs Type SuperclustersOrdered Total 4345 3172 The EST clone inserts were PCR-amplified using amino-linked PCR
primers for Synteni microarray analysis. When more than one PCR product was obtained for a particular clone, that PCR product was not used for expression analysis.
In total, 2,528 clones from the electronic subtraction method were analyzed by microarray analysis to identify electronic subtraction breast clones that had high levels of tumor vs. normal tissue mRNA. Such screens were performed using a Synteni (Palo Alto, CA) microaxray, according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Within these analyses, the clones were arrayed on the chip, which was then probed with fluorescent probes generated from normal and tumor prostate cDNA, as well as various other normal tissues. The slides were scanned and the fluorescence intensity was measured.
Clones with an expression ratio greater than 3 (i. e., the level in prostate tumor and normal prostate mRNA was at least three times the level in other normal tissue mRNA) were identified as prostate tumor-specific sequences (Table IV).
The sequences of these clones are provided in SEQ ID NO: 401-453, with certain novel sequences shown in SEQ ID NO: 407, 413, 416-419, 422, 426, 427 and 450.

Table IV
Prostate-tumor Specific Clones SEQ ID NO. Sequence Comments Designation 401 22545 previously identified P 1 OOOC

402 22547 previously identified P704P

403 22548 known 404 22550 known 406 22552 prostate secretory protein 407 22553 novel 408 22558 previously identified P509S

409 22562 glandular kallikrein 410 22565 previously identified P1000C

412 22568 B1006C (breast tumor antigen) 413 22570 novel 415 22572 previously identified P706P

416 22573 novel 417 22574 novel 418 22575 novel 419 22580 novel 421 22582 prostatic secretory protein 422 22583 novel 423 22584 prostatic secretory protein 424 22585 prostatic secretory protein 425 22586 known 426 22587 novel 427 22588 novel 429 22590 known 431 22592 known 432 22593 Previously identified P777P

433 22594 T cell receptor gamma chain 434 22595 Previously identified P705P

435 22596 Previously identified P707P

437 22848 known 438 I 22849 prostatic secretory protein 441 22853 ' PAP

442 22854 previously identified P509S

443 22855 previously identified P705P

444 22856 previously identified P774P

446 23601 previously identified P777P

450 23612 novel 452 23618 previously identified P1000C

453 ~ 23622 ~ previously identified P705P

Further studies on the clone of SEQ ID NO: 407 (also referred to as P1020C) led to the isolation of an extended cDNA sequence provided in SEQ ID
NO:
591. This extended cDNA sequence was found to contain an open reading frame that encodes the predicted amino acid sequence of SEQ ID NO: 592. The P1020C cDNA
and amino acid sequences were found to show some similarity to the human endogenous retroviral HERV-I~ pol gene and protein.

ANALYSIS
This Example describes the isolation of additional prostate-specific polypeptides from a prostate tumor cDNA library.
A human prostate tumor cDNA expression library as described above was screened using microarray analysis to identify clones that display at least a three fold over-expression in prostate tumor and/or normal prostate tissue, as compared to non-prostate normal tissues (not including testis). 142 clones were identified and sequenced. CerEain of these clones are shown in SEQ ID NO: 454-467. Of these sequences, SEQ ID NO: 459-460 represent novel genes. The others (SEQ ID NO:

458 and 461-467) correspond to known sequences. Comparison of the determined cDNA sequence of SEQ ID NO: 461 with sequences in the Genbank database using the BLAST program revealed homology to the previously identified transmembrane protease serine 2 (TMPRSS2). The full-length cDNA sequence for this clone is provided in SEQ ID NO: 894, with the corresponding amino acid sequence being provided in SEQ ID NO: 895. The cDNA sequence encoding the first 209 amino acids of TMPRSS2 is provided in SEQ ID NO: 896, with the first 209 amino acids being provided in SEQ ID NO: 897.
The sequence of SEQ ID NO: 462 (referred to as P835P) was found to correspond to the previously identified clone FLJ13518 (Accession AK023643;
SEQ ID
NO: 917), which had no associated open reading frame (ORF). This clone was used to search the Geneseq DNA database and matched a clone previously identified as a G
protein-coupled receptor protein (DNA Geneseq Accession A09351; amino acid Geneseq Accession Y92365), that is characterized by the presence of seven transmembrane domains. The sequences of fragments between these domains are provided in SEQ ID NO: 921-928, with SEQ ID NO: 921, 923, 925 and 927 representing extracellular domains and SEQ ID NO: 922, 924, 926 and 928 representing intracellular domains. SEQ ID NO: 921-928 represent amino acids 1-28, 53-61, 103, 124-143, 165-201, 226-238, 263-272 and 297-381, respectively, of P835P.
The full-length cDNA sequence for P835P is provided in SEQ ID NO: 916. The cDNA
sequence of the open reading frame for P835P, including stop codon, is provided in SEQ ID NO: 918, with the open reading frame without stop codon being provided in SEQ ID NO: 919 and the corresponding amino acid sequence being provided in SEQ
ID
NO: 920.

This Example describes the full length cloning of P710P.
The prostate cDNA library described above was screened with the P710P
fragment described above. One million colonies were plated on LB/Ampicillin plates.

Nylon membrane filters were used to lift these colonies, and the cDNAs picked up by these filters were then denatured and cross-linked to the filters by LTV
light. The P71 OP
fragment was radiolabeled and used to hybridize with the filters. Positive cDNA clones were selected and their cDNAs recovered and sequenced by an automatic Perkin Elmer/Applied Biosystems Division Sequencer. Four sequences were obtained, and are presented in SEQ ID NO: 468-471. These sequences appear to represent different splice variants of the P710P gene. Subsequent comparison of the cDNA sequences of with those in Genbank releaved homology to the DD3 gene (Genbank accession numbers AF103907 & AF103908). The cDNA sequence of DD3 is provided in SEQ ID
NO: 690.

PROTEIN EXPRESSION OF PROSTATE-SPECIFIC ANTIGENS
I S This example describes the expression and purification of prostate-specific antigens in E. coli, baculovirus and mammalian cells.
a) Expression of P501 S in E. coli Expression of the full-length form of P501 S was attempted by first cloning P501 S without the leader sequence (amino acids 36-553 of SEQ ID NO:
113) downstream of the first 30 amino acids of the M. tubes°culosis antigen Ral2 (SEQ ID
NO: 484) in pETl7b. Specifically, PSO1S DNA was used to perform PCR using the primers AW025 (SEQ ID NO: 485) and AW003 (SEQ ID NO: 486). AW025 is a sense cloning primer that contains a HindIII site. AW003 is an antisense cloning primer that contains an EcoRI site. DNA amplification was performed using 5 ~,1 l OX Pfu buffer, 1 X120 mM dNTPs, 1 ~l each of the PCR primers at 10 ~M concentration, 40 ~.l water, 1 ~l Pfu DNA polymerase (Stratagene, La Jolla, CA) and 1 ~,1 DNA at 100 ng/~,1.
Denaturation at 95°C was performed for 30 sec, followed by 10 cycles of 95°C for 30 sec, 60°C for 1 min and by 72°C for 3 min. 20 cycles of 95°C for 30 sec, 65°C for 1 min and by 72°C for 3 min, and lastly by 1 cycle of 72°C for 10 min.
The PCR product was cloned to Ral2m/pETl7b using HindIII and EcoRI. . The sequence of the resulting fusion construct (referred to as Ral2-P501 S-F) was confirmed by DNA
sequencing.
The fusion construct was transformed into BL21(DE3)pLysE, pLysS and CodonPlus E. coli (Stratagene) and grown overnight in LB broth with kanamycin.
The resulting culture was induced with IPTG. Protein was transferred to PVDF
membrane and blocked with 5% non-fat milk (in PBS-Tween buffer), washed three times and incubated with mouse anti-His tag antibody (Clontech) for 1 hour. The membrane was washed 3 times and probed with HRP-Protein A (Zymed) fox 30 min. Finally, the membrane was washed 3 times and developed with ECL (Amersham). No expression was detected by Western blot. Similarly, no expression was detected by Western blot when the Ral2-P501 S-F fusion was used for expression in BL21 CodonPlus by CE6 phage (Invitrogen).
An N-terminal fragment of P501 S (amino acids 36-325 of SEQ ID NO:
113) was cloned down-stream of the first 30 amino acids of the M. tuberculosis antigen Ral2 in pETI7b as follows. P501 S DNA was used to perform PCR using the primers AW025 (SEQ ID NO: 485) and AW027 (SEQ ID NO: 487). AW027 is an antisense cloning primer that contains an EcoRI site and a stop codon. DNA amplification was performed essentially as described above. The resulting PCR product was cloned to Ral2 in pETl7b at the HindIII and EcoRI sites. The fusion construct (referred to as Ral2-P501 S-N) was confirmed by DNA sequencing.
The Ral2-P501 S-N fusion construct was used for expression in BL21 (DE3)pLysE, pLysS and CodonPlus, essentially as described above. Using Western blot analysis, protein bands were observed at the expected molecular weight of 36 kDa. Some high molecular weight bands were also observed, probably due to aggregation of the recombinant protein. No expression was detected by Western blot when the Ral2-P501 S-F fusion was used for expression in BL21 CodonPlus by CE6 phage.
A fusion construct comprising a C-terminal portion of P501 S (amino acids 257-553 of SEQ ID NO: 113) located down-stream of the first 30 amino acids of the M. tuberculosis antigen Ral2 (SEQ ID NO: 484) was prepared as follows.

DNA was used to perform PCR using the primers AW026 (SEQ ID NO: 488) and AW003 (SEQ ID NO: 486). AW026 is a sense cloning primer that contains a HindIII
site. DNA amplification was performed essentially as described above. The resulting PCR product was cloned to Ral2 in pETl7b at the HindIII and EcoRI sites. The sequence for the fusion construct (referred to as Ral2-P501 S-C) was confimned.
The Ral2-P501 S-C fusion construct was used for expression in BL21 (DE3)pLysE, pLysS and CodonPlus, as described above. A small amount of protein was detected by Western blot, with some molecular weight aggregates also being observed. Expression was also detected by Western blot when the Ral2-fusion was used for expression in BL21 CodonPlus induced by CE6 phage.
A fusion construct comprising a fragment of P501 S (amino acids 36-298 of SEQ ID NO: 113) located down-stream of the M. tuberculosis antigen Ral2 (SEQ ID
NO: 848) was prepared as follows. P501 S DNA was used to perform PCR using the primers AW042 (SEQ ID NO: 849) and AW053 (SEQ ID NO: 850). AW042 is a sense cloning primer that contains a EcoRI site. AW053 is an antisense primer with stop and Xho I sites. DNA amplification was performed essentially as described above.
The resulting PCR product was cloned to Ral2 in pETl7b at the EcoRI and Xho I
sites. The resulting fusion construct (referred to as Ral2-P501 S-E2) was expressed in B834 (DE3) pLys S E. coli host cells in TB media for 2 h at room temperature. Expressed protein was purified by washing the inclusion bodies and running on a Ni-NTA column.
The purified protein stayed soluble in buffer containing 20 mM Tris-HCl (pH 8), 100 mM
NaCI, 10 mM (3-Me and 5% glycerol. The determined cDNA and amino acid sequences for the expressed fusion protein are provided in SEQ ID NO: 851 and 852, respectfully.
b) Expression of P501 S in Baculovirus The Bac-to-Bac baculovirus expression system (BRL Life Technologies, Inc.) was used to express P501 S protein in insect cells. Full-length P501 S
(SEQ ID
NO: 113) was amplified by PCR and cloned into the XbaI site of the donor plasmid pFastBacI. The recombinant bacmid and baculovirus were prepared according to the manufacturer's instructions. The recombinant baculovirus was amplified in Std cells and the high titer viral stocks were utilized to infect High Five cells (Invitrogen) to make the recombinant protein. The identity of the full-length protein was confirmed by N-terminal sequencing of the recombinant protein and by Western blot analysis (Figure 7). Specifically, 0.6 million High Five cells in 6-well plates were infected with either the unrelated control virus BV/ECD PD (lane 2), with recombinant baculovirus for P501 S at different amounts or MOIs (lanes 4-8), or were uninfected (lane 3).
Cell lysates were run on SDS-PAGE under reducing conditions and analyzed by Western blot with the anti-P501 S monoclonal antibody P501 S-10E3-G4D3 (prepared as described below). Lane 1 is the biotinylated protein molecular weight marker (BioLabs).
The localization of recombinant P501 S in the insect cells was investigated as follows. The insect cells overexpressing P501 S were fractionated into fractions of nucleus, mitochondria, membrane and cytosol. Equal amounts of protein from each fraction were analyzed by Western blot with a monoclonal antibody against PSOIS. Due to the scheme of fractionation, both nucleus and mitochondria fractions contain some plasma membrane components. However, the membrane fraction is basically free from mitochondria and nucleus. P501 S was found to be present in all fractions that contain the membrane component, suggesting that PSOlS may be associated with plasma membrane of the insect cells expressing the recombinant protein.
c) Expression of P501 S in mammalian cells Full-length P501 S (553 amino acids; SEQ ID NO: 113) was cloned into various mammalian expression vectors, including pCEP4 (Invitrogen), pVR1012 (Vical, San Diego, CA) and a modified form of the retroviral vector pBMN, referred to as pBIB. Transfection of P501 S/pCEP4 and P501 S/pVR1012 into HEK293 fibroblasts was carried out using the Fugene transfection reagent (Boehringer Mannheim).
Briefly, 2 u1 of Fugene reagent was diluted into 100 u1 of serum-free media and incubated at room temperature for 5-10 min. This mixture was added to 1 ug of PSO1S plasmid DNA, mixed briefly and incubated for 30 minutes at room temperature. The Fugene/DNA mixture was added to cells and incubated for 24-48 hours.
Expression of recombinant P501 S in transfected HEK293 fibroblasts was detected by means of Western blot employing a monoclonal antibody to P501 S.
Transfection of p501 S/pCEP4 into CHO-K cells (American Type Culture Collection, Rockville, MD) was carried out using GenePorter transfection reagent (Gene Therapy Systems, San Diego, CA). Briefly, 15 ~l of GenePorter was diluted in 500 p,1 of serum-free media and incubated at room temperature for 10 min.
The GenePorter/media mixture was added to 2 ~,g of plasmid DNA that was diluted in 500 q1 of serum-free media, mixed briefly and incubated for 30 min at room temperature. CHO-K cells were rinsed in PBS to remove serum proteins, and the GenePorter/DNA mix was added and incubated for 5 hours. The transfected cells were then fed an equal volume of 2x media and incubated for 24-48 hours.
FRCS analysis of PSOIS transiently infected CHO-K cells, demonstrated surface expression of PSO1S. Expression was detected using rabbit polyclonal antisera raised against a P501 S peptide, as described below. Flow cytometric analysis was performed using a FaCScan (Becton Dickinson), and the data were analyzed using the Cell Quest program.
d) Expression of P703P in Baculovirus The cDNA for full-length P703P-DES (SEQ ID NO: 326), together with several flanking restriction sites, was obtained by digesting the plasmid pCDNA703 with restriction endonucleases Xba I and Hind III. The resulting restriction fragment (approx. 800 base pairs) was ligated into the transfer plasmid pFastBacI which was digested with the same restriction enzymes. The sequence of the insert was confirmed by DNA sequencing. The recombinant transfer plasmid pFBP703 was used to make recombinant bacmid DNA and baculovirus using the Bac-To-Bac Baculovirus expression system (BRL Life Technologies). High Five cells were infected with the recombinant virus BVP703, as described above, to obtain recombinant P703P
protein:

e) Expression of P788P in E. Coli A truncated, N-terminal portion, of P788P (residues I-644 of SEQ ID
NO: 777; refereed to as P788P-N) fused with a C-terminal 6xHis Tag was expressed in E. coli as follows. P788P cDNA was amplified using the primers AW080 and AW081 S (SEQ ID NO: 81 S and 816). AW080 is a sense cloning primer with an NdeI
site.
AW081 is an antisense cloning primer with a XhoI site. The PCR-amplified P788P, as well as the vector pCRXl, were digested with NdeI and XhoI. Vector and insert were ligated and transformed into NovaBlue cells. Colonies were randomly screened for insert and then sequenced. P788P-N clone #6 was confirmed to be identical to the designed construct. The expression construct P788P-N #6/pCRX1 was transformed into E. coli BL21 CodonPlus-RIL competent cells. After induction, most of the cells grew well, achieving OD600 of greater than 2.0 after 3 hr. Coomassie stained SDS-PAGE showed an over-expressed band at about 7S kD. Western blot analysis using a 6xHisTag antibody confirmed the band was P788P-N. The determined cDNA sequence I S for P788P-N is provided in SEQ ID NO: 817, with the corresponding amino acid sequence being provided in SEQ ID NO: 818.
f) Expression of PS l OS in E. coli The PS l OS protein has 9 potential transmembrane domains and is predicted to be located at the plasma membrane. The C-terminal protein of this protein, as well as the predicted third extracellular domain of PS 1 OS were expressed in E. coli as follows.
The expression construct referred to as Ral2-PSO1 S-C was designed to have a 6 HisTag at the N-terminal enc, followed by the M. tuberculosis antigen Ral2 (SEQ ID NO: 819) and then the C-terminal portion of PS 1 OS (amino residues 1261 of SEQ ID NO: 538). Full-length PS10S was used to amplify the PS10S-C
fragment by PCR using the primers AWOS6 and AWOS7 (SEQ ID NO: 820 and 821, respectively). AWOS6 is a sense cloning primer with an EcoRI site. AWOS7 is an antisense primer with stop and XhoI sites. The amplified PSO1 S fragment and Ral2/pCRXI were digested with EcoRI and XhoI and then purified. The insert and vector were ligated together and transformed into NovaBlue. Colonies were randomly screened for insert and sequences. For protein expression, the expression construct was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. A mini-induction screen was performed to optimize the expression conditions. After induction the cells grew well, achieving OD 600 nm greater than 2.0 after 3 hours.
Coomassie stain SDS-PAGE showed a highly over-expressed band at approx. 30 kD. Though this is higher than the expected molecular weight, western blot analysis was positive, showing this band to be the His tag-containing protein. The optimized culture conditions are as follows. Dilute overnight culture/daytime culture (LB +
kanamycin +
chloramphenicol) into 2xYT (with kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1 liter 2xYT. Allow to grow at 37 °C until OD600 = 0.6. Take an aliquot out as TO sample. Add 1 mM IPTG and allow to grow at 30 °C for 3 hours.
Take out a T3 sample, spin down cells and store at -80 °C. The determined cDNA and amino acid sequences for the Ral2-PS l OS-C construct are provided in SEQ ID NO: 822 and 825, respectively.
The expression construct PS l OS-C was designed to have a 5' added start codon and a glycine (GGA) codon and then the PS l OS C terminal fragment followed by the in frame 6x histidine tag and stop codon from the pET28b vector. The cloning strategy is similar to that used for Ral2-PS l OS-C, except that the PCR
primers employed were those shown in SEQ ID NO: 828 and 829, respectively and the NcoI/XhoI cut in pET28b was used. The primer of SEQ ID NO: 828 created a 5' NcoI
site and added a start codon. The antisense primer of SEQ ID NO: 829 creates a XhoI
site on PS l OS C terminal fragment. Clones were confirmed by sequencing. For protein expression, the expression construct was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. An OD600 of greater than 2.0 was obtained 30 hours after induction. Coomassie stained SDS-PAGE showed an over-expressed band at about 11 kD. Western blot analysis confirmed that the band was PS l OS-C, as did N-terminal protein sequencing. The optimized culture conditions are as follows:
dilute overnight culture/daytime culture (LB + kanamycin + chloramphenicol) into 2x YT (+
kanamycin and chloramphenicol) at a ratio of 25 mL culture to 1 liter 2x YT, and allow to grow at 37 °C until an OD 600 of about 0.5 is reached. Take out an aliquot as TO
sample. Add 1 mM IPTG and allow to grow at 30 °C for 3 hours. Spin down the cells and store at -80 °C until purification. The determined cDNA and amino acid sequences for the P510S-C construct are shown in SEQ ID NO: 823 and 826, respectively.
The predicted third extracellular domain of P510S (P510S-E3; residues 328-676 of SEQ ID NO: 538) was expressed in E. coli as follows. The P510S
fragment was amplified by PCR using the primers shown in SEQ ID NO: 830 and 831. The primer of SEQ ID NO: 830 is a sense primer with an NdeI site for use in ligating into pPDM. The primer of SEQ ID NO: 831 is an antisense primer with an added XhoI
site for use in ligating into pPDM. The resulting fragment was cloned to pPDM at the Ndel and XhoI sites. Clones were confirmed by sequencing. For protein expression, the clone ws transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells.
After induction, an OD600 of greater than 2.0 was achieved after 3 hours. Coomassie stained SDS-PAGE showed an over-expressed band at about 39 kD, and N-terminal sequencing confirmed the N-terminal to be that of PS l OS-E3. Optimized culture conditions are as follows: dilute overnight culture/daytime culture (LB + kanamycin +
chloramphenicol) into 2x YT (kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1 liter 2x YT. Allow to grow at 37 °C until OD 600 equals 0.6. Take out an aliquot as TO
sample. Add 1 mM IPTG and allow to grow at 30 °C for 3 hours. Take out a T3 sample, spin down the cells and store at -80 °C until purification. The determined cDNA and amino acid sequences for the P501 S-E3 construct are provided in SEQ
ID
NO: 824 and 827, respectively.
g) Expression of P775S in E. Coli The antigen P775P contains multiple open reading frames (ORF). The third ORF, encoding the protein of SEQ ID NO: 483, has the best emotif score.
An expression fusion construct containing the M. tuberculosis antigen Ral2 (SEQ
ID NO:
819) and P775P-ORF3 with an N-terminal 6x HisTag was prepared as follows.

ORF3 was amplified using the sense PCR primers of SEQ ID NO: 832 and the anti-sense PCR primer of SEQ ID NO: 833. The PCR amplified fragment of P775P and Ral2/pCRXl were digested with the restriction enzymes EcoRI and XhoI. Vector and insert were ligated and then transformed into NovaBlue cells. Colonies were randomly screened for insert and then sequenced. A clone having the desired sequence was transformed into E. coli BL21 (DE3) CodonPlus-RIL competent cells. Two hours after S induction, the cell density peaked at OD600 of approximately 1.8. Coomassie stained' SDS-PAGE showed an over-expressed band at about 31 kD. Western blot using 6x HisTag antibody confirmed that the band was Ral2-P77SP-ORF3. The determined cDNA and amino acid sequences for the fusion construct are provided in SEQ ID
NO:
834 and 835, respectively.
H) Expression of a P703P His t~ fusion protein in E. coli The cDNA for the coding region of P703P was prepared by PCR using the primers of SEQ ID NO: 836 and 837. The PCR product was digested with EcoRI
restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag 1 S in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The correct construct was confirmed by DNA sequence analysis and then transformed into E coli BL21 (DE3) pLys S expression host cells. The determined amino acid and cDNA sequences for the expressed recombinant P703P are provided in SEQ ID NO:
838 and 839, respectively.
I) Expression of a P70SP His tai fusion protein in E. coli The cDNA for the coding region of P70SP was prepared by PCR using the primers of SEQ ID NO: 840 and 841. The PCR product was digested with EcoRI
restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag 2S in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The correct construct was confirmed by DNA sequence analysis and then transformed into E. coli BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. The determined amino acid and cDNA sequences for the expressed recombinant P70SP
are provided in SEQ ID NO: 842 and 843, respectively.

J) Expression of a P711P His tai fusion protein in E. coli The cDNA for the coding region of P711 P was prepared by PCR using the primers of SEQ ID NO: 844 and 845. The PCR product was digested with EcoRI
restriction enzyme, gel purified and cloned into a modified pET28 vector with a His tag in frame, which had been digested with Eco72I and EcoRI restriction enzymes.
The correct construct was confirmed by DNA sequence analysis and then transformed into E. coli BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells. The determined amino acid and cDNA sequences for the expressed recombinant P711P
are provided in SEQ ID NO: 846 and 847, respectively.
I~) Expression of P767P in E. coli The full-length coding region of P767P (amino acids 2-374 of SEQ ID
NO: 590) was amplified by PCR using the primers PDM-468 and PDM-469 (SEQ ID
NO: 935 and 936, respectively). DNA amplification was perfornned using 10 ~,I
lOX
Pfu buffer, 1 ~,I 10 mM dNTPs, 2 ~1 each of the PCR primers at I O ~.M
concentration, 83 ~,1 water, 1.5 ~1 Pfu DNA polymerase (Stratagene, La Jolla, CA) and 1 ~,1 DNA at 100 ng/~.1. Denaturation at 96°C was performed for 2 min, followed by 40 cycles of 96°C for 20 sec, 66°C for 15 sec and by 72°C for 2 min., and lastly by 1 cycle of 72°C
for 4 min. The PCR product was digested with XhoI and cloned into a modified pET28 vector with a histidine tag in frame on the 5' end that was digested with Eco72I and XhoI. The construct was confirmed to be correct through sequence analysis and transformed into E. coli BL21 pLysS and BL21 CodonPlus RP cells. The cDNA
coding region for the recombinant B767P protein is provided in SEQ ID NO: 938, with the corresponding amino acid sequence being provided in SEQ ID NO: 941. The full length P767P did not express at high enough levels for detection or purification.
A truncated coding region of P767P (referred to as B767P-B; amino acids 47-374 of SEQ ID NO: 590) was amplified by PCR using the primers PDM-573 and PDM-469 (SEQ ID NO: 937 and 936, respectively) and the PCR conditions described above for full-length P767P. The PCR product was digested with XhoI
and cloned into the modified pET28 vector that was digested with Eco72I and XhoI.
The construct was confirmed to be correct through sequence analysis and transformed into E. coli BL21 pLysS and BL21 CodonPlus RP cells. The protein was found to be expressed in the inclusion body pellet. The coding region for the expressed protein is provided in SEQ ID NO: 939, with the corresponding amino acid sequence being provided in SEQ ID NO: 940.

PREPARATION AND CHARACTERIZATION OF ANTIBODIES
AGAINST PROSTATE-SPECIFIC POLYPEPTIDES
a) Preparation and Characterization of Polyclonal Antibodies against P703P, P504S and P509S
Polyclonal antibodies against P703P, P504S and P509S were prepared as follows.
Each prostate tumor antigen expressed in an E. coli recombinant expression system was grown overnight in LB broth with the appropriate antibiotics at 37°C in a shaking incubator. The next morning, 10 ml of the overnight culture was added to 500 ml to 2x YT plus appropriate antibiotics in a 2L-baffled Erlenmeyer flask.
When the Optical Density (at 560 nm) of the culture reached 0.4-0.6, the cells were induced with IPTG (1 mM). Four hours after induction with IPTG, the cells were harvested by centrifugation. The cells were then washed with phosphate buffered saline and centrifuged again. The supernatant was discarded and the cells were either frozen for future use or immediately processed. Twenty ml of lysis buffer was added to the cell pellets and vortexed. To break open the E. coli cells, this mixture was then run through the French Press at a pressure of 16,000 psi. The cells were then centrifuged again and the supernatant and pellet were checked by SDS-PAGE for the partitioning of the recombinant protein. For proteins that localized to the cell pellet, the pellet was resuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusion body pellet was washed and centrifuged again. This procedure was repeated twice more. The washed inclusion body pellet was solubilized with either 8 M urea or 6 M guanidine containing 10 mM Tris pH 8.0 plus 10 mM imidazole. The solubilized protein was added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 min to 1 hour at room temperature with continuous agitation. After incubation, the resin and protein mixture were poured through a disposable column and the flow through was collected.
. The column was then washed with 10-20 column volumes of the solubilization buffer.
The antigen was then eluted from the column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. A SDS-PAGE gel was run to determine which fractions to pool for further purification.
As a final purification step, a strong anion exchange resin such as HiPrepQ (Biorad) was equilibrated with the appropriate buffer and the pooled fractions from above were loaded onto the column. Each antigen was eluted off the column with a increasing salt gradient. Fractions were collected as the column was run and another SDS-PAGE gel was run to determine which fractions from the column to pool. The 1 S pooled fractions were dialyzed against 10 mM Tris pH 8Ø The proteins were then vialed after filtration through a 0.22 micron filter and the antigens were frozen until needed for immunization.
Four hundred micrograms of each prostate antigen was combined with 100 micrograms of muramyldipeptide (MDP). Every four weeks rabbits were boosted with 100 micrograms mixed with an equal volume of Incomplete Freund's Adjuvant (IFA). Seven days following each boost, the animal was bled. Sera was generated by incubating the blood at 4°C for 12-4 hours followed by centrifugation.
Ninety-six well plates were coated with antigen by incubating with 50 microliters (typically 1 microgram) of recombinant protein at 4 °C for 20 hours. 250 microliters of BSA blocking buffer was added to the wells and incubated at room temperature fox 2 hours. Plates were washed 6 times with PBS/0.01% Tween.
Rabbit sera was diluted in PBS. Fifty microliters of diluted sera was added to each well and incubated at room temperature for 30 min. Plates were washed as described above before 50 microliters of goat anti-rabbit horse radish peroxidase (HRP) at a 1:10000 dilution was added and incubated at room temperature for 30 min. Plates were again washed as described above and 100 microliters of TMB microwell peroxidase substrate was added to each well. Following a 15 min incubation in the dark at room temperature, the colorimetric reaction was stopped with 100 microliters of 1N

and read immediately at 450 nm. All polyclonal antibodies showed immunoreactivity to the appropriate antigen.
b) Preparation and Characterization of Antibodies against P501 S
A murine monoclonal antibody directed against the carboxy-terminus of the prostate-specific antigen P501 S was prepared as follows.
A truncated fragment of P501 S (amino acids 355-526 of SEQ ID NO:
113) was generated and cloned into the pET28b vector (Novagen) and expressed in E.
coli as a thioredoxin fusion protein with a histidine tag. The trx-PSO1S
fusion protein was purified by nickel chromatography, digested with thrombin to remove the trx fragment and further purified by an acid precipitation procedure followed by reverse phase HPLC.
Mice were immunized with truncated P501 S protein. Serum bleeds from mice that potentially contained anti-P501 S polyclonal sera were tested for specific reactivity using ELISA assays with purified P501 S and trx-P501 S
proteins.
Serum bleeds that appeared to react specifically with P501 S were then screened for P501 S reactivity by Western analysis. Mice that contained a P501 S-specific antibody component were sacrificed and spleen cells were used to generate anti-P501 S
antibody producing hybridomas using standard techniques. Hybridoma supernatants were tested for P501 S-specific reactivity initially by ELISA, and subsequently by FAGS
analysis of reactivity with P501 S transduced cells. Based on these results, a monoclonal hybridoma referred to as 10E3 was chosen for further subcloning. A number of subclones were generated, tested for specific reactivity to P501 S using ELISA and typed for IgG
isotype. The results of this analysis are shown below in Table V. Of the 16 subclones tested, the monoclonal antibody 1 OE3-G4-D3 was selected for further study.

Table V
Isotype analysis of murine anti-P501 S monoclonal antibodies Hybridoma clone Isotype Estimated [Ig] in supernatant (~,g/ml) 4D 11 IgG l 14.6 1 G1 IgGl 0.6 4F6 IgGl 72 4H5 IgGl 13.8 4H5-E 12 IgG 1 10.7 4H5-EH2 IgG 1 9.2 4H5-H2-A10 IgGI 10 4H5-H2-A3 IgG 1 12.8 4H5-H2-A10-G6 IgGl 13.6 4H5-H2-B 11 IgG l 12.3 1 OE3 IgG2a 3.4 10E3-D4 IgG2a 3.8 1 OE3-D4-G3 IgG2a 9.5 1 OE3-D4-G6 IgG2a 10.4 10E3-E7 IgG2a 6.5 8H12 IgG2a 0.6 S The specificity of 10E3-G4-D3 for P501 S was examined by FACS
analysis. Specifically, cells were fixed (2% formaldehyde, 10 minutes), permeabilized (0.1% saponin, 10 minutes) and stained with 1OE3-G4-D3 at 0.5 -1 ~g/ml, followed by incubation with a secondary, FITC-conjugated goat anti-mouse Ig antibody (Pharmingen, San Diego, CA). Cells were then analyzed for FITC fluorescence using an Excalibur fluorescence activated cell sorter. For FACS analysis of transduced cells, B-LCL were retrovirally transduced with P501 S. For analysis of infected cells, B-LCL
were infected with a vaccinia vector that expresses P501 S. To demonstrate specificity in these assays, B-LCL transduced with a different antigen (P703P) and uninfected B-LCL vectors were utilized. 10E3-G4-D3 was shown to bind with P501 S-transduced B-LCL and also with P501 S-infected B-LCL, but not with either uninfected cells or P703P-transduced cells.
To determine whether the epitope recognized by 10E3-G4-D3 was found on the surface or in an intracellular compartment of cells, B-LCL were transduced with P501 S or HLA-B8 as a control antigen and either fixed and permeabilized as described above or directly stained with 10E3-G4-D3 and analyzed as above. Specific recognition of P501 S by 10E3-G4-D3 was found to require permeabilization, suggesting that the epitope recognized by this antibody is intracellular.
The reactivity of 10E3-G4-D3 with the three prostate tumor cell lines Lncap, PC-3 and DU-145, which are known to express high, medium and very low levels of P501 S, respectively, was examined by permeabilizing the cells and treating them as described above. Higher reactivity of 10E3-G4-D3 was seen with Lncap than with PC-3, which in turn showed higher reactivity that DU-145. These results are in agreement with the real time PCR and demonstrate that the antibody specifically recognizes P501 S in these tumor cell Lines and that the epitope recognized in prostate tumor cell lines is also intracellular.
Specificity of 1 OE3-G4-D3 for P501 S was also demonstrated by Western blot analysis. Lysates from the prostate tumor cell Lines Lncap, DU-145 and PC-3, from P501 S-transiently transfected HEK293 cells, and from non-transfected HEK293 cells were generated. Western blot analysis of these lysates with 10E3-G4-D3 revealed a 46 kDa immunoreactive band in Lncap, PC-3 and P501 S-transfected HEK cells, but not in DU-145 cells or non-transfected HEK293 cells. P501 S mRNA expression is consistent with these results since semi-quantitative PCR analysis revealed that P50I S
mRNA is expressed in Lncap, to a lesser but detectable level in PC-3 and not at all in cells. Bacterially expressed and purified recombinant PSO1S (referred to as PSOISStr2) was recognized by 10E3-G4-D3 (24 kDa), as was full-length P501 S that was transiently expressed in HEK293 cells using either the expression vector VR1012 or pCEP4.
Although the predicted molecular weight of P501 S is 60.5 kDa, both transfected and "native" P501 S run at a slightly lower mobility due to its hydrophobic nature.
Immunohistochemical analysis was performed on prostate tumor and a panel of normal tissue sections (prostate, adrenal, breast, cervix, colon, duodenum, gall bladder, ileum, kidney, ovary, pancreas, paxotid gland, skeletal muscle, spleen and testis). Tissue samples were fixed in formalin solution for 24 hours and embedded in paraffin before being sliced into 10 micron sections. Tissue sections were permeabilized and incubated with 10E3-G4-D3 antibody for 1 hr. HRP-labeled anti-mouse followed by incubation with DAB chromogen was used to visualize P501 S
immunoreactivity. P501 S was found to be highly expressed in both normal prostate and prostate tumor tissue but was not detected in any of the other tissues tested.
To identify the epitope recognized by 10E3-G4-D3, an epitope mapping approach was pursued. A series of 13 overlapping 20-21 mers (5 amino acid overlap;
SEQ ID NO: 489-501) was synthesized that spanned the fragment of PSOlS used to generate 10E3-G4-D3. Flat bottom 96 well microtiter plates were coated with either the peptides or the P501 S fragment used to immunize mice, at 1 microgram/ml for 2 hours at 37 °C. Wells were then aspirated and blocked with phosphate buffered saline containing 1% (w/v) BSA for 2 hours at room temperature, and subsequently washed in PBS containing 0.1 % Tween 20 (PBST). Purified antibody 10E3-G4-D3 was added at 2 fold dilutions (1000 ng - 16 ng) in PBST and incubated for 30 minutes at room temperature. This was followed by washing 6 times with PBST and subsequently incubating with HRP-conjugated donkey anti-mouse IgG (H+L)Affmipure F(ab') fragment (Jackson Immunoresearch, West Grove, PA) at 1:20000 , for 30 minutes.
Plates were then washed and incubated for 15 minutes in tetramethyl benzidine.
Reactions were stopped by the addition of 1N sulfuric acid and plates were read at 450 nm using an ELISA plate reader. As shown in Fig. 8, reactivity was seen with the peptide of SEQ ID NO: 496 (corresponding to amino acids 439-459 of P501 S) and with the PSO1S fragment but not with the remaining peptides, demonstrating that the epitope recognized by 10E3-G4-D3 is localized to amino acids 439-459 of SEQ ID NO:
113.
In order to further evaluate the tissue specificity of P501 S, mufti-array immunohistochemical analysis was performed on approximately 4700 different human tissues encompassing all the major normal organs as well as neoplasias~
derived from these tissues. Sixty-five of these human tissue samples were of prostate origin. Tissue sections 0.6 mm in diameter were formalin-fixed and paraffin embedded. Samples were pretreated with HIER using 10 mM citrate buffex pH 6.0 and boiling for 10 min.
Sections were stained with 10E3-G4-D3 and P501 S immunoreactivity was visualized with HRP. All the 65 prostate tissues samples (5 normal, 55 untreated prostate tumors, hormone refractory prostate tumors) were positive, showing distinct perinuclear staining. All other tissues examined were negative for P501 S expression.
c) Preparation and Characterization of Antibodies against P503S
5 A fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) was expressed and purified from bacteria essentially as described above for P501 S
and used to immunize both rabbits and mice. Mouse monoclonal antibodies were isolated using standard hybridoma technology as described above. Rabbit monoclonal antibodies were isolated using Selected Lymphocyte Antibody Method (SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada). Table VI, below, lists the monoclonal antibodies that were developed against P503S. .
Table VI
Antibody Species 20D4 Rabbit JA1 Rabbit 1 A4 ~ Mouse 1 C3 Mouse 1 C9 Mouse 1 D 12 Mouse 2A11 Mouse 2H9 Mouse 4H7 Mouse 8A8 Mouse 8D 10 Mouse 9C 12 Mouse 6D 12 - ~ Mouse The DNA sequences encoding the complementarity determining regions (CDRs) for the rabbit monoclonal antibodies 20D4 and JA1 were determined and are provided in SEQ ID NO: 502 and 503, respectively.

In order to better define the epitope binding region of each of the antibodies, a series of overlapping peptides were generated that span amino acids 109-213 of SEQ ID NO: 114. These peptides were used to epitope map the anti-PS03S
monoclonal antibodies by ELISA as follows. The recombinant fragment of PS03S
that S was employed as the immunogen was used as a positive control. Ninety-six well microtiter plates were coated with either peptide or recombinant antigen at 20 ng/well overnight at 4 °C. Plates were aspirated and blocked with phosphate buffered saline containing 1% (w/v) BSA for 2 hours at room temperature then washed in PBS
containing 0.I% Tween 20 (PBST). Purified rabbit monoclonal antibodies diluted in PBST were added to the wells and incubated for 30 min at room temperature.
This was followed by washing 6 times with PBST and incubation with Protein-A HRP
conjugate at a 1:2000 dilution for a fiuther 30 min. Plates were washed six times in PBST and incubated with tetramethylbenzidine (TMB) substrate for a further 1 S min. The reaction was stopped by the addition of 1N sulfuric acid and plates were read at 4S0 nm using at 1 S ELISA plate reader. ELISA with the mouse monoclonal antibodies was performed with supernatants from tissue culture run neat in the assay.
All of the antibodies bound to the recombinant PS03S fragment, with the exception of the negative control SP2 supernatant. 20D4, JA1 and 1D12 bound strictly to peptide #2101 (SEQ ID NO: S04), which corresponds to amino acids 1S1-169 of SEQ ID NO: 114. 1C3 bound to peptide #2102 (SEQ ID NO: SOS), which corresponds to amino acids 16S-I84 of SEQ ID NO: I14. 9C12 bound to peptide #2099 (SEQ ID
NO: S22), which corresponds to amino acids 120-139 of SEQ ID NO: 114. The other antibodies bind to regions that were not examined in these studies.
Subsequent to epitope mapping, the antibodies were tested by FAGS
2S analysis on a cell line that stably expressed PS03S to confirm that the antibodies bind to cell surface epitopes. Cells stably transfected with a control plasmid were employed as a negative control. Cells were stained live with no fixative. 0.S ug of anti-monoclonal antibody was added and cells were incubated on ice for 30 min before being washed twice and incubated with a FITC-labelled goat anti-rabbit or mouse secondary antibody for 20 min. After being washed twice, cells were analyzed with an Excalibur fluorescent activated cell sorter. The monoclonal antibodies 1C3, 1D12, 9C12, and JAl, but not 8D3, were found to bind to a cell surface epitope of PS03S.
In order to determine which tissues express P503S, immunohistochemical analysis was performed, essentially as described above, on a S panel of normal tissues (prostate, adrenal, breast, cervix, colon, duodenum, gall bladder, ileum, kidney, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis). HRP-labeled anti-mouse or anti-rabbit antibody followed by incubation with TMB was used to visualize PS03S immunoreactivity. PS03S was found to be highly expressed in prostate tissue, with lower levels of expression being observed in cervix, colon, ileum and kidney, and no expression being observed in adrenal, breast, duodenum, gall bladder, ovary, pancreas, parotid gland, skeletal muscle, spleen and testis.
Western blot analysis was used to characterize anti-PS03S monoclonal antibody specificity. SDS-PAGE was performed on recombinant (rec) PS03S
expressed in and purified from bacteria and on lysates from HEK293 cells transfected with full 1S length PS03S. Protein was transferred to nitrocellulose and then Western blotted with each of the anti-PS03 S monoclonal antibodies (20D4, JA 1, 1 D 12, 6D 12 and 9C 12) at an antibody concentration of 1 ug/ml. Protein was detected using horse radish peroxidase (HRP) conjugated to either a goat anti-mouse monoclonal antibody or to protein A-sepharose. The monoclonal antibody 20D4 detected the appropriate molecular weight 14 kDa recombinant PS03S (amino acids 113-241) and the 23.5 kDa species in the HEI~293 cell lysates transfected with full length PS03S. Other anti-PS03S monoclonal antibodies displayed similar specificity by Western blot.
d) Preparation and Characterization of Antibodies against P703P
Rabbits were immunized with either a truncated (P703Ptr1; SEQ ID NO:
2S 172) or full-length mature form (P703Pfl; SEQ ID NO: S23) of recombinant protein was expressed in and purified from bacteria as described above.
Affinity purified polyclonal antibody was generated using ixnmunogen P703Pfl or P703Ptrl attached to a solid support. Rabbit monoclonal antibodies were isolated using SLAM

technology at Imrngenics Pharmaceuticals. Table VII below lists both the polyclonal and monoclonal antibodies that were generated against P703P.
Table VII
Antibody Immunogen Species/type Aff. Purif. P703P (truncated);P703Ptr1 Rabbit polyclonal #2594 Aff. Purif. P703P (full length);P703Pfl Rabbit polyclonal #9245 2D4 P703Ptr1 Rabbit monoclonal 8H2 P703Ptr1 Rabbit monoclonal 7H8 ~ P703Ptr1 ~ Rabbit monoclonal The DNA sequences encoding the complementarity determining regions (CDRs) for the rabbit monoclonal antibodies 8H2, 7H8 and 2D4 were determined and are provided in SEQ ID NO: 506-508, respectively.
Epitope mapping studies were performed as described above.
Monoclonal antibodies 2D4 and 7H8 were found to specifically bind to the peptides of SEQ ID NO: 509 (corresponding to amino acids 145-159 of SEQ ID NO: 172) and SEQ
ID NO: 510 (corresponding to amino acids 11-25 of SEQ ID NO: 172), respectively.
The polyclonal antibody 2594 was found to bind to the peptides of SEQ ID NO:

514, with the polyclonal antibody 9427 binding to the peptides of SEQ ID NO:
515-517.
The specificity of the anti-P703P antibodies was determined by Western blot analysis as follows. SDS-PAGE was performed on (1) bacterially expressed recombinant antigen; (2) lysates of HEK293 cells and Ltk-/- cells either untransfected or transfected with a plasmid expressing full length P703P; and (3) supernatant isolated from these cell cultures. Protein was transferred to nitrocellulose and then Western blotted using the anti-P703P polyclonal antibody #2594 at an antibody concentration of 1 ug/ml. Protein was detected using horse radish peroxidase (HRP) conjugated to an anti-rabbit antibody. A 35 kDa immunoreactive band could be observed with recombinant P703P. Recombinant P703P runs at a slightly higher molecular weight since it is epitope tagged. In lysates and supernatants from cells transfected with full length P703P, a 30 kDa band corresponding to P703P was observed. To assure specificity, lysates from HEK293 cells stably transfected with a control plasmid were also tested and were negative for P703P expression. Other anti-P703P
antibodies showed similar results.
Immunohistochemical studies were performed as described above, using anti-P703P monoclonal antibody. P703P was found to be expressed at high levels in normal prostate and prostate tumor tissue but was not detectable in all other tissues tested (breast tumor, lung tumor and normal kidney).
e) Preparation and Characterization of Antibodies against P504S
Full-length P504S (SEQ ID NO: 108) was expressed and purified from bacteria essentially as described above for P501 S and employed to raise rabbit monoclonal antibodies using Selected Lymphocyte Antibody Method (SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada). The anti-monoclonal antibody 13H4 was shown by Western blot to bind to both expressed recombinant P504S and to native P504S in tumor cells.
Immunohistochemical studies using 13H4 to assess P504S expression in various prostate tissues were performed as described above. A total of 104 cases, including 65 cases of radical prostatectomies with prostate cancer (PC), 26 cases of prostate biopsies and 13 cases of benign prostate hyperplasia (BPH), were stained with the anti-P504S monoclonal antibody 13H4. P504S showed strongly cytoplasmic granular staining in 64/65 (98.5%) of PCs in prostatectomies and 26/26 (100% ) of PCs in prostatic biopsies. P504S was stained strongly and diffusely in carcinomas (4+ in 91.2% of cases of PC; 3+ in 5.5%; 2+ in 2.2% and 1+ in 1.1%) and high grade prostatic intraepithelial neoplasia (4+ in all cases). The expression of P504S did not vary with Gleason score. Only 17/91 (I8.7%) of cases of NP/BPH around PC and 2113 (15.4%) of BPH cases were focally (1+, no 2+ to 4+ in all cases) and weakly positive for P504S in large glands. Expression of P504S was not found in small atrophic glands, postatrophic hyperplasia, basal cell hyperplasia and transitional cell metaplasia in either biopsies or prostatectomies. P504S was thus found to be over-expressed in all Gleason scores of prostate cancer (98.5 to 100% of sensitivity) and exhibited only focal positivities in large normal glands in 19/104 of cases (82.3% of specificity). These findings indicate that P504S may be usefully employed for the diagnosis of prostate cancer.

S CHARACTERIZATION OF CELL SURFACE EXPRESSION AND

This example describes studies demonstrating that the prostate-specific antigen PSO1S is expressed on the surface of cells, together with studies to determine the probable chromosomal location of P501 S.
The protein P501 S (SEQ ID NO: 113) is predicted to have 11 transmembrane domains. Based on the discovery that the epitope recognized by the anti-P501 S monoclonal antibody 10E3-G4-D3 (described above in Example 17) is intracellular, it was predicted that following transmembrane determinants would allow the prediction of extracellular domains of P501 S. Fig. 9 is a schematic representation of the P501 S protein showing the predicted location of the transmembrane domains and the intracellular epitope described in Example 17. Underlined sequence represents the predicted transmembrane domains, bold sequence represents the predicted extracellular domains, and italicized sequence represents the predicted intracellular domains.
Sequence that is both bold and underlined represents sequence employed to generate polyclonal rabbit serum. The location of the transmembrane domains was predicted using HHMTOP as described by Tusnady and Simon (Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction, J. Mol. Biol. 283:489-506, 1998).
Based on Fig. 9, the P501 S domain flanked by the transmembrane domains corresponding to amino acids 274-295 and 323-342 is predicted to be extracellular. The peptide of SEQ ID NO: 518 corresponds to amino acids 306-320 of P501 S and lies in the predicted extracellular domain. The peptide of SEQ ID
NO: 519, which is identical to the peptide of SEQ ID NO: 518 with the exception of the substitution of the histidine with an asparginine, was synthesized as described above. A

Cys-Gly was added to the C-terminus of the peptide to facilitate conjugation to the carrier protein. Cleavage of the peptide from the solid support was carned out using the following cleavage mixture: trifluoroacetic acid:ethanediolahioanisol:water:phenol (40:1:2:2:3). After cleaving for two hours, the peptide was precipitated in cold ether.
The peptide pellet was then dissolved in 10% vlv acetic acid and lyophilized prior to purification by C18 reverse phase hplc. A gradient of 5-60% acetonitrile (containing 0.05% TFA) in water (containing 0.05% TFA) was used to elute the peptide. The purity of the peptide was verified by hplc and mass spectrometry, and was determined to be >95%. The purified peptide was used to generate rabbit polyclonal antisera as described above.
Surface expression of P501 S was examined by FACS analysis. Cells were stained with the polyclonal anti-PSO1S peptide serum at 10 ~,g/ml, washed, incubated with a secondary FITC-conjugated goat anti-rabbit Ig antibody (ICN), washed and analyzed for FITC, fluorescence using an Excalibur fluorescence activated cell sorter. For FACS analysis of transduced cells, B-LCL were retrovirally transduced with P501 S. To demonstrate specificity in these assays, B-LCL transduced with an irrelevant antigen (P703P) or nontransduced were stained in parallel. For FACS analysis of prostate tumor cell lines, Lncap, PC-3 and DU-145 were utilized. Prostate tumor cell lines were dissociated from tissue culture plates using cell dissociation medium and stained as above. All samples were treated with propidium iodide (PI) prior to FAGS
analysis, and data was obtained from PI-excluding (i. e., intact and non-permeabilized) cells. The rabbit polyclonal serum generated against the peptide of SEQ ID NO:

was shown to specifically recognize the surface of cells transduced to express P501 S, demonstrating that the epitope recognized by the polyclonal serum is extracellular.
To determine biochemically if P501 S is expressed on the cell surface, peripheral membranes from Lncap cells were isolated and subjected to Western blot analysis. Specifically, Lncap cells were lysed using a Bounce homogenizer in 5 ml of homogenization buffer (250 mM sucrose, 10 mM HEPES, 1mM EDTA, pH 8.0, 1 complete protease inhibitor tablet (Boehringer Mannheim)). Lysate samples were spun at 1000 g for 5 min at 4 °C. The supernatant was then spun at 8000g for 10 min at 4 °C.

Supernatant from the 8000g spin was recovered and subjected to a 100,000g spin for 30 min at 4 °C to recover peripheral membrane. Samples were then separated by SDS-PAGE and Western blotted with the mouse monoclonal antibody 10E3-G4-D3 (described above in Example 17) using conditions described above. Recombinant purified P501 S, as well as HEK293 cells transfected with and over-expressing were included as positive controls for P501 S detection. LCL cell lysate was included as a negative control. P501 S could be detected in Lncap total cell lysate, the 8000g (internal membrane) fraction and also in the IOO,OOOg (plasma membrane) fraction.
These results indicate that P501 S is expressed at, and localizes to, the peripheral membrane.
To demonstrate that the rabbit polyclonal antiserum generated to the peptide of SEQ ID NO: 519 specifically recognizes this peptide as well as the corresponding native peptide of SEQ ID NO: 518, ELISA analyses were performed.
For these analyses, flat-bottomed 96 well microtiter plates were coated with either the I S peptide of SEQ ID NO: 519, the longer peptide of SEQ ID NO: 520 that spans the entire predicted extracellular domain, the peptide of SEQ ID NO: 521 which represents the epitope recognized by the P501 S-specific antibody 1 OE3-G4-D3, or a P501 S
fragment (corresponding to amino acids 355-526 of SEQ ID NO: 1 I3) that does not include the immunizing peptide sequence, at 1 ~g/ml for 2 hours at 37 °C. Wells were aspirated, blocked with phosphate buffered saline containing 1% (w/v) BSA for 2 hours at room temperature and subsequently washed in PBS containing 0.1% Tween 20 (PBST).
Purified anti-PSO1S polyclonal rabbit serum was added at 2 fold dilutions (1000 ng -125 ng) in PBST and incubated for 30 min at room temperature. This was followed by washing 6 times with PBST and incubating with HRP-conjugated goat anti-rabbit IgG
(H+L) Affinipure F(ab') fragment at 1:20000 for 30 min. Plates were then washed and incubated for 15 min in tetramethyl benzidine. Reactions were stopped by the addition of 1N sulfuric acid and plates were read at 450 nm using an ELISA plate reader. As shown in Fig. 11, the anti-PSOlS polyclonal rabbit serum specifically recognized the peptide of SEQ ID NO: 519 used in the immunization as well as the longer peptide of SEQ ID NO: 520, but did not recognize the irrelevant P501 S-derived peptides and fragments.
In further studies, rabbits were immunized with peptides derived from the P501 S sequence and predicted to be either extracellular or intracellular, as shown in Fig. 9. Polyclonal rabbit sera were isolated and polyclonal antibodies in the serum were purified, as described above. To determine specific reactivity with P501 S, FACS
analysis was employed, utilizing either B-LCL transduced with P501 S or the irrelevant antigen P703P, of B-LCL infected with vaccinia virus-expressing P501 S. For surface expression, dead and non-intact cells were excluded from the analysis as described above. For intracellular staining, cells were fixed and permeabilized as described above. Rabbit polyclonal serum generated against the peptide of SEQ ID NO:
548, which corresponds to amino acids 181-198 of PSOlS, was found to recognize a surface epitope of P501 S. Rabbit polyclonal serum generated against the peptide SEQ
ID NO:
551, which corresponds to amino acids 543-553 of P501 S, was found to recognize an epitope that was either potentially extracellular or intracellular since in different experiments intact or permeabilized cells were recognized by the polyclonal sera.
Based on similar deductive reasoning, the sequences of SEQ ID NO: 541-547, 549 and 550, which correspond to amino acids 109-122, 539-553, 509-520, 37-54, 342-359, 295-323, 217-274, 143-160 and 75-88, respectively, of P501 S, can be considered to be potential surface epitopes of P501 S recognized by antibodies.
In further studies, mouse monoclonal antibodies were raised against amino acids 296 to 322 to P501 S, which are predicted to be in an' extracellular domain.
A/J mice were immunized with P501 S/adenovirus, followed by subsequent boosts with an E. coli recombinant protein, referred to as PSOlN, that contains amino acids 296 to 322 of PSOlS, and with peptide 296-322 (SEQ ID NO: 898) coupled with KLH. The mice were subsequently used for splenic B cell fusions to generate anti-peptide hybridomas. The resulting 3 clones, referred to as 4F4 (IgGl,kappa), 4G5 (IgG2a,kappa) and 9B9 (IgGl,kappa), were grown for antibody production. The mAb was purified by passing the supernatant over a Protein A-sepharose column, followed by antibody elution using 0.2M glycine, pH 2.3. Purified antibody was neutralized by the addition of 1M Tris, pH 8, and buffer exchanged into PBS.
For ELISA analysis, 96 well plates were coated with P501 S peptide 296 322 (referred to as P501-long), an irrelevant P775 peptide, PSO1S-N, PSOlTR2, long-KLH, PSO1S peptide 306-319 (referred to as P501-short)-KLH, or the irrelevant peptide 2073-KLH, all at a concentration of 2 ug/ml and allowed to incubate for 60 minutes at 37 °C. After coating, plates were washed SX with PBS + 0.1%
Tween and then blocked with PBS, 0.5% BSA, 0.4% Tween20 for 2 hours at room temperature.
Following the addition of supernatants or purified mAb, the plates were incubated for 60 minutes at room temperature. Plates were washed as above and donkey anti-mouse IgHRP-linked secondary antibody was added and incubated for 30 minutes at room temperature, followed by a final washing as above. TMB peroxidase substrate was added and incubated 15 minutes at room temperature in the dark. The reaction was stopped by the addition of 1N H2S04 and the OD was read at 450 nM. All three hybrid , clones secreted mAb that recognized peptide 296-322 and the recombinant protein PSO1N.
For FACS analysis, HEI~293 cells were transiently transfected with a PSOI S/VRI O I2 expression constructs using Fugene 6 reagent. After 2 days of culture, cells were harvested and washed, then incubated with purified 4G5 mAb for 30 minutes on ice. After several washes in PBS, 0.5% BSA, 0.01% azide, goat anti-mouse Ig-FITC
was added to the cells and incubated for 30 minutes on ice. Cells were washed and resuspended in wash buffer including 1% propidium iodide and subjected to FACS
analysis. The FACS analysis confirmed that amino acids 296-322 of P501 S are in an extracellular domain and are cell surface expressed.
The chromosomal location of P501 S was determined using the GeneBridge 4 Radiation Hybrid panel (Research Genetics). The PCR primers of SEQ
ID NO: 528 and. 529 were employed in PCR with DNA pools from the hybrid panel according to the manufacturer's directions. After 38 cycles of amplification, the reaction products were separated on a 1.2% agarose gel, and the results were analyzed through the Whitehead Institute/MIT Center for Genome Research web server (http://www-genome.wi.mit.edu/cgi-bin/contig/rlunapper.pl) to determine the probable chromosomal location. Using this approach, P501 S was mapped to the long arm of chromosome 1 at WI-9641 between q32 and q42. This region of chromosome 1 has been linked to prostate cancer susceptibility in hereditary prostate cancer (Smith et al.
Science 274:1371-1374, 1996 and Berthon et al. Am. J. Hum. Genet. 62:1416-1424, 1998). These results suggest that P501 S may play a role in prostate cancer malignancy.

Steroid (androgen) hormone modulation is a common treatment modality in prostate cancer. The expression of a number of prostate tissue-specific antigens have previously been demonstrated to respond to androgen. The responsiveness of the prostate-specific antigen P501 S to androgen treatment was examined in a tissue culture system as follows.
Cells from the prostate tumor cell line LNCaP were plated at 1.5 x 106 cells/T75 flask (for RNA isolation) or 3 x 105 cells/well of a 6-well plate (for FACS
analysis) and grown overnight in RPMI 1640 media containing 10% charcoal-stripped fetal calf serum (BRL Life Technologies, Gaithersburg, MD). Cell culture was continued for an additional 72 hours in RPMI 1640 media containing 10%
charcoal-stripped fetal calf serum, with 1 nM of the synthetic androgen Methyltrienolone (R1881; New England Nuclear) added at various time points. Cells were then harvested for RNA isolation and FAGS analysis at 0, 1, 2, 4, 8, 16, 24, 28 and 72-hours post androgen addition. FACS analysis was performed using the anti-P501 S antibody G4-D3 and permeabilized cells.
For Northern analysis, 5-10 micrograms of total RNA was run on a formaldehyde denaturing gel, transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, P.iscataway, NJ), cross-linked and stained with methylene blue. The filter was then prehybridized with Church's Buffer (250 mM Na2HP04, 70 mM
H3P04, 1 mM EDTA, 1 % SDS, 1 % BSA in pH 7.2) at 65 °C for 1 hour. P501 S DNA
was labeled with 32P using High Prime random-primed DNA labeling kit (Boehringer Mamlheim). Unincorporated label was removed using MicroSpin 5300-HR columns (Amersham Pharmacia Biotech). The RNA filter was then hybridized with fresh Church's Buffer containing labeled cDNA overnight, washed with 1X SCP (0.1 M
NaCI, 0.03 M NaaHP04.7H20, 0.001 M Na2EDTA), 1 % sarkosyl (n-lauroylsarcosine) and exposed to X-ray film.
Using both FACS and Northern analysis, P501 S message and protein levels were found in increase in response to androgen treatment.
' EXAMPLE 21 PREPARATION OF FUSION PROTEINS OF PROSTATE-SPECIFIC ANTIGENS
The example describes the preparation of a fusion protein of the prostate-specific antigen P703P and a truncated form of the known prostate antigen PSA.
The I S truncated form of PSA has a 21 amino acid deletion around the active serine site. The expression construct for the fusion protein also has a restriction site at 3' end, immediately prior to the termination codon, to aid in adding cDNA for additional antigens.
The full-length cDNA for PSA was obtained by RT-PCR from a pool of RNA from human prostate tumor tissues using the primers of SEQ ID NO: 607 and 608, and cloned in the vector pCR-Blunt II-TOPO. The resulting cDNA was employed as a template to make two different fragments of PSA by PCR with two sets of primers (SEQ ID NO: 609 and 610; and SEQ ID NO: 611 and 612). The PCR products having the expected size were used as templates to make truncated forms of PSA by PCR
with the primers of SEQ ID NO: 61 l and 613, which generated PSA (delta 208-218 in amino acids). The cDNA for the mature form of P703P with a 6X histidine tag at the 5' end, was prepared by PCR with P703P and the primers of SEQ ID NO: 614 and 615. The cDNA for the fusion of P703P with the truncated form of PSA (referred to as FOPP) was then obtained by PCR using the modified P703P cDNA and the truncated form of PSA cDNA as templates and the primers of SEQ ID NO: 614 and 615. The FOPP

cDNA was cloned into the NdeI site and XhoI site of the expression vector pCRXl, and confirmed by DNA sequencing. The determined cDNA sequence for the fusion construct FOPP is provided in SEQ ID NO: 616, with the amino acid sequence being provided in SEQ ID NO: 617.
The fusion FOPP was expressed as a single recombinant protein in E.
coli as follows. The expression plasmid pCRXIFOPP was transformed into the E.
coli strain BL21-CodonPlus RIL. The transformant was shown to express FOPP protein upon induction with 1 mM IPTG. The culture of the corresponding expression clone was inoculated into 25 ml LB broth containing 50 ug/ml kanamycin and 34 ug/ml chloramphenicol, grown at 37 °C to OD600 of about l, and stored at 4 °C overnight.
The culture was diluted into 1 liter of TB LB containing 50 ug/ml kanamycin and 34 ug/ml chloramphenicol, and grown at 37 °C to OD600 of 0.4. IPTG was added to a final concentration of 1 mM, and the culture was incubated at 30 °C for 3 hours. The cells were pelleted by centrifugation at 5,000 RPM for 8 min. To purify the protein, the cell pellet was suspended in 25 ml of 10 mM Tris-Cl pH 8.0, 2mM PMSF, complete protease inhibitor and 15 ug lysozyme. The cells were lysed at 4 °C for 30 minutes, sonicated several times and the lysate centrifuged for 30 minutes at 10,000 x g. The precipitate, which contained the inclusion body, was washed twice with 10 mM
Tris-Cl pH 8.0 and 1 % CHAPS. The inclusion body was dissolved in 40 ml of 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 M urea. The solution was bound to 8 ml Ni-NTA (Qiagen) for one hour at room temperature. The mixture .was poured into a 25 ml column and washed with 50 ml of IO mM Tris-Cl pH 6.3, 100 mM sodium phosphate, 0.5% DOC and 8M urea. The bound protein was eluted with 350 mM imidazole, 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 M urea. The fractions containing FOPP proteins were combined and dialyzed extensively against 10 mM Tris-Cl pH
4.6, aliquoted and stored at - 70 °C.

PERIPHERAL BLOOD OF PROSTATE CANCER PATIENTS
S Circulating epithelial cells were isolated from fresh blood of normal individuals and metastatic prostate cancer patients, mRNA isolated and cDNA
prepared using real-time PCR procedures. Real-time PCR was performed with the TaqmanTM
procedure using both gene specific primers and probes to determine the levels of gene expression.
Epithelial cells were enriched from blood samples using an immunomagnetic bead separation method (Dynal A.S., Oslo, Norway). Isolated cells were lysed and the magnetic beads removed. The lysate was then processed for poly A+
mRNA isolation using magnetic beads coated with Oligo(dT)2S. After washing the beads in buffer, bead/poly A+ RNA samples were suspended in 10 mM Tris HCl pH
8.0 1S and subjected to reversed transcription. The resulting cDNA was subjected to real-time PCR using gene specific primers. Beta-actin content was also determined and used for normalization. Samples with PSO1S copies greater than the mean of the normal samples + 3 standard deviations were considered positive. Real time PCR on blood samples was performed using the TaqmanTM procedure but extending to SO cycles using forward and reverse primers and probes specific for PSO1 S. Of the eight samples tested, 6 were positive for PSO1 S and (3-actin signal. The remaining 2 ~ samples had no detectable (3-actin or PSO1S. No PSO1S signal was observed in the four normal blood samples tested.

SCID MOUSE-PASSAGED PROSTATE TUMORS
When considering the effectiveness of antigens in the treatment of prostate cancer, the continued presence of the antigens in tumors during androgen ablation therapy is important. The presence of the prostate-specific antigens P703P and P50I S in prostate tumor samples grown in SLID mice in the presence of testosterone was evaluated as follows.
Two prostate tumors that had metastasized to the bone were removed from patients, implanted into SCID mice and grown in the presence of testosterone.
Tumors were evaluated for mRNA expression of P703P, P501 S and PSA using quantitative real time PCR with the SYBR green assay method. Expression of and P501 S in a prostate tumor was used as a positive control and the absence in normal intestine and normal heart as negative controls. In both cases, the specific mRNA was present in late passage tumors. Since the bone metastases were grown in the presence of testosterone, this implies that the presence of these genes would not be lost during androgen ablation therapy.

I5 ANTI-P503 S MONOCLONAL ANTIBODY INHIBITS TUMOR GROWTH IN T111~0 The ability of the anti-P503S monoclonal antibody 20D4 to suppress tumor formation in mice was examined as follows.
Ten SCID mice were injected subcutaneously with HEK293 cells that expressed P503S. Five mice received 150 micrograms of 20D4 intravenously at day 0 (time of tumor cell injection), day 5 and day 9. Tumor size was measured for 50 days.
Of the five animals that received no 20D4, three formed detectable tumors after about 2 weeks which continued to enlarge throughout the study. In contrast, none of the five mice that received 20D4 formed tumors. These results demonstrate that the anti-Mab 20D4 displays potent anti-tumor activity irz vivo.

CHARACTERIZATION OF A T CELL RECEPTOR CLONE FROM A

T cells have a limited lifespan. However, cloning of T cell receptor (TCR) chains and subsequent transfer essentially enables infinite propagation of the T

cell specificity. Cloning of tumor-antigen TCR chains allows the transfer of the specificity into T cells isolated from patients that share the TCR MHC-restricting allele.
Such T cells could then be expanded and used in adoptive transfer settings to introduce the tumor antigen specificity into patients carrying tumors that express the antigen. T
S cell receptor alpha and beta chains from a CD8 T cell clone specific for the prostate-specific antigen PSO1S were isolated and sequenced as follows.
Total mRNA from 2 x 106 cells from CTL clone 4ES (described above in Example 12) was isolated using Trizol reagent and cDNA was synthesized. To determine Va and Vb sequences in this clone, a panel of Va and Vb subtype-specific primers was synthesized and used in RT-PCR reactions with cDNA generated from each of the clones. The RT-PCR reactions demonstrated that each of the clones expressed a common Vb sequence that corresponded to the Vb7 subfamily.
Futhermore, using cDNA generated from the clone, the Va sequence expressed was determined to be Va6. To clone the full TCR alpha and beta chains from clone 4ES, 1 S primers were designed that spanned the initiator and terminator-coding TCR
nucleotides. The primers were as follows: TCR Valpha-6 S'(sense): GGATCC---GCCGCCACC-ATGTCACTTTCTAGCCTGCT (SEQ ID NO: 899) BamHI site Kozak TCR alpha sequence TCR alpha 3' (antisense): GTCGAC---TCAGCTGGACCACAGCCGCAG (SEQ ID NO: 900) SaII site TCR alpha constant sequence TCR Vbeta-7. S'(sense): GGATCC---GCCGCCACC--ATGGGCTGCAGGCTGCTCT (SEQ ID NO: 901) BamHI site Kozak TCR alpha sequence TCR beta 3' (antisense): GTCGAC---TCAGAAATCCTTTCTCTTGAC (SEQ
ID NO: 902) SaII site TCR beta constant sequence. Standard 3S cycle RT-PCR
reactions were established using cDNA synthesized from the CTL clone and the above 2S primers, employing the proofreading thermostable polymerase PWO (Roche, Nutley, NJ).
The resultant specific bands (approx. 8S0 by fox alpha and approx. 9S0 for beta) were ligated into the PCR blunt vector (Invitrogen) and transformed into E.
coli. E . coli transformed with plasmids containing full-length alpha and beta chains were identified, and large scale preparations of the corresponding plasmids were generated. Plasmids containing full-length TCR alpha and beta chains were submitted for sequencing. The sequencing reactions demonstrated the cloning of full-length TCR
alpha and beta chains with the determined cDNA sequences for the Vb and Va chains being shown in SEQ ID NO: 903 and 904, respectively. The corresponding amino acid sequences are shown in SEQ ID NO: 905 and 906, respectively. The Va sequence was shown by nucleotide sequence alignment to be 99% identical (347/348) to Va6.2, and the Vb to be 99% identical to Vb7 (336/338).

CAPTURE OF PROSTATE SPECTFIC CELLS USING

As described above, P503S is found on the surface of prostate cells.
Secondary coated microsphere beads specific for mouse IgG were coupled with the purified P503S-specific monoclonal antibody 1D12. The bound P503S antibody was then used to capture HEK cells expressing recombinant PS03S. This provides a model system for prostate-specific cell capture which may be usefully employed in the detection of prostate cells in blood, and therefore in the detection of prostate cancer.
P503S-transfected HEK cells were harvested and redissolved in wash buffer (PBS, 0.1% BSA, 0.6% sodium citrate) at an appropriate volume to give at least 54 cells per sample. Round bottom Eppendorf tubes were used for all procedures involving beads. The stock concentrations were as shown below in Table VIII.
Table VIII
Stock concentrations Sample concentrationAmount needed Epithelial enrich 1' beads/ml 125 u1 stock per beads 4 5 ml beads/ml (Dynal Biotech volume Inc. Lake Success, NY) 1D12 ascites antibody0.1 ug/ml (0.1X) 0.05 u1 to 2.5 u1 2 to 5 ug/ml stock per mg/ml (5X) titrations sample a- Mamma Mu 0.9 mg/m11 ug/ml ( 1 X) 1.1 u1 stock per sample Pan-mouse IgG beads 1' beads/ml 125 u1 stock per 4 5 ml beads/ml (Dynal Biotech) volume Blocked immunomagnetic beads were pre-washed as follows: all beads needed were pooled and washed once with 1 ml wash buffer. The . beads were resuspended din a 3X volume of 1% BSA (v/v) in wash buffer and incubated for 1S min rotating at 4 °C. The beads were then washed three times with 2X volume of wash S buffer and resuspended to original volume. Non-blocked beads were pooled, washed three times with 2X volume of wash buffer and resuspended to original volume.
Primary antibody was incubated with secondary beads in a fresh Eppendorf for 30 minutes, rotating at 4 °C. Approximately 200 u1 wash buffer was added to increase the total volume for even mixing of the sample. The antibody-bead solution was transferred to a fresh Eppendorf, washed twice with an equal volume of wash buffer and resuspended to original volume. Target cells were added to each sample and incubated for 4S minutes, rotating at 4 °C. The tubes were transferred to a magnet, the supernatant removed, taking care not the agitate the beads, and the samples were washed twice with 1 ml wash buffer. The samples were then ready for RT-PCR
1 S using a Dynabeads mRNA direct microkit (Dynal Biotech).
Epithelial cell enrichment was placed in a magnet and supernant was removed. The epithelial enrichment beads were then resuspendedin 100 u1 lysis/binding buffer fortified with Rnasin (2 U/ul per sample), and sotred at -70 °C
until use. Oligo (dT25) Dynabeads were pre-washed as follows: all beads needed were pooled (23 ul/sample), washed three times with an excess volume of lysis/binding buffer, and resuspsended of original volume. The lysis supernant was separated with a magnet and transferred to a fresh Eppendorf. 20 u1 oligo(dT2S) Dynabeads were added per samplem ad rolled for S min at room temperature. Supernant was separated using a magnet and discarded, leaving the mRNA annealed of the beads. The bead/mRNA
2S complex was washed with buffer and resuspended in cold Tris-HCl.
For RT-PCR, the Tris-HCl supernatant was separated and discarded using MPS. For each sample containing 15 cells or less, the following was added to give a total volume of 30 u1: 14.25 u1 H20; 1.S u1 BSA; 6 u1 first strand buffer; 0.75 mL
10 mM dNTP mix; 3 u1 Rnasin; 3 uI O.IM dTT; and 1.S uI Superscript II. The resulting solution was incubated for 1 hour at 42 °C, diluted 1:S in H20, heated at 80°C for 2 min to detach cDNA from the beads, and immediately placed on MPS. The supernatant containing cDNA was transferred to a new tube and stored at -20 °C.
Table IX shows the percentage of capture of PS03S-transfected HEK
cells as determined by RT-PCR.
S
Tahla Tk capture P503S- % capture LnCAP cells transfected HEK cells 0.1 uglml PS03S Mab 36.90 0.00 O.S ug/ml PS03S Mab 67.40 2.93 1 ug/ml PS03S Mab 40.22 0.00 S ug/ml PS03S Mab I3.1 I 0.00 Anti-Mu beads only, 1.42 0.00 non-blocked Anti-Mu beads only, 1S.6S 20.2I
blocked Absolute control, 100.00 100.00 non-capture cells From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE hISTING
<110> Corixa Corporation Xu, Jiangchun Dillon, Davin C.
Mitcham, Jennifer Z.
Harlocker, Susan Z.
Yuqui, Jiang Kalos, Michael D.
Fanger, Gary R.
Retter, Marc W.
Stolk, John A.
Day, Craig H.
Vedvick, Thomas S.
Carter, Darrick Li, Samuel Wang, Aijun Skeiky, Yasir A.W.
Hepler, William Henderson, Robert A.
<120> COMPOSTTIONS AND METHODS FOR THE THERAPY AND
DIAGNOSIS OF PROSTATE CANCER
<130> 210121.42723PC
<140> PCT
<141> 2001-03-27 <160> 943 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 814 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(814) <223> n = A,T,C or G
<400> 1 tttttttttttttttcacagtataacagctctttatttctgtgagttctactaggaaatc60 atcaaatctgagggttgtctggaggacttcaatacacctccccccatagtgaatcagctt120 ccagggggtccagtccctctccttacttcatccccatcccatgccaaaggaagaccctcc180 ctccttggctcacagccttctctaggcttcccagtgcctccaggacagagtgggttatgt240 tttcagctccatccttgctgtgagtgtctggtgcgttgtgcctccagcttctgctcagtg300 cttcatggacagtgtccagcacatgtcactctccactctctcagtgtggatccactagtt360 ctagagcggccgccaccgcggtggagctccagcttttgttccctttagtg.agggttaatt420 gcgcgcttggcgtaatcatggtcataactgtttcctgtgtgaaattgttatccgctcaca480 attccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtg540 anctaactcacattaattgcgttgcgctcactgnccgctttccagtcnggaaaactgtcg600 tgccagctgcattaatgaatcggccaacgcncggggaaaagcggtttgcgttttgggggc660 tcttccgcttctcgctcactnantcctgcgctcggtcnttcggctgcggggaacggtatc720 actcctcaaaggnggtattacggttatccnnaaatcnggggatacccnggaaaaaanttt780 aacaaaaggg cancaaaggg cngaaacgta aaaa 814 <210> 2 <211> 816 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (816) <223> n = A,T,C or G
<400> 2 acagaaatgttggatggtggagcaoctttctatacgacttacaggacagcagatggggaa60 ttcatggctgttggagcaatagaaccccagttctacgagctgctgatcaaaggacttgga120 ctaaagtctgatgaacttcccaatcagatgagcatggatgattggccagaaatgaagaag180 aagtttgcagatgtatttgcaaagaagacgaaggcagagtggtgtcaaatctttgacggc240 acagatgcctgtgtgactccggttctgacttttgaggaggttgttcatcatgatcacaac300 aaggaacggggctcgtttatcaccagtgaggagcaggacgtgagcccccgccctgcacct360 ctgctgttaaacaccccagccatcccttctttcaaaagggatccactagttctagaagcg420 gccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgctt480 ggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccccc540 aacatacgagccggaacataaagtgttaagcctggggtgcctaatgantgagctaactcn600 cattaattgcgttgcgctcactgcccgctttccagtcgggaaaactgtcgtgccactgcn'660 ttantgaatcrigcoaccccccgggaaaaggcggttgcnttttgggcctcttccgctttcc720 tcgctcattgatcctngcncccggtcttcggctgcggngaacggttcactcctcaaaggc780 ggtntnccggttatccccaaacnggggatacccnga 816 <210> 3 <211> 773 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(773) <223> n = A,T,C or G
<400>

cttttgaaagaagggatggctggggtgtttaacagcagaggtgcagggcgggggctcacg60 tcctgctcctcactggtgataaacgagccccgttccttgttgtgatcatgatgaacaacc120 tcctcaaaagtcagaaccggagtcacacaggcatctgtgccgtcaaagatttgacaccac180 tctgccttcgtcttctttgcaaatacatctgcaaacttcttcttcatttctggccaatca240 tccatgctcatctgattgggaagttcatcagactttagtccanntcctttgatcagcagc300 tcgtagaactggggttctattgctccaacagccatgaattccccatctgctgtcctgtaa360 gtcgtatagaaaggtgctccaccatecaacatgttctgtcctcgagggggggcccggtac420 ccaattcgccctatantgagtcgtattacgcgcgctcactggccgtcgttttacaacgtc480 gtgactgggaaaaccctgggcgttaccaacttaatcgccttgcagcacatccccctttcg540 ccagctgggcgtaatancgaaaaggcccgcaccgatcgcccttccaacagttgcgcacct600 gaatgggnaaatgggacccccctgttaccgcgcattnaacccccgcngggtttngttgtt660 acccccacntnnaccgcttacactttgccagcgccttancgcccgctccctttcnccttt720 cttcccttcctttcncnccnctttcccccggggtttcccccntcaaaccccna 773 <210> 4 <211> 828 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(828) <223> n = A,T,C or G
<400>

cctcctgagtcctactgacctgtgctttctggtgtggagtccagggctgctaggaaaagg60 aatgggcagacacaggtgtatgccaatgtttctgaaatgggtataatttcgtcctctcct120 tcggaacactggctgtctctgaagacttctcgctcagtttcagtgaggacacacacaaag180 acgtgggtgaccatgttgtttgtggggtgcagagatgggaggggtggggcccaccctgga240 agagtggacagtgacacaaggtggacactctctacagatcactgaggataagctggagcc300 acaatgcatgaggcacacacacagcaaggatgacnctgtaaacatagcccacgctgtcct360 gngggcactgggaagcctanatnaggccgtgagcanaaagaaggggaggatccactagtt420 ctanagcggccgccaccgcggtgganctccancttttgttccctttagtgagggttaatt480 gcgcgcttggcntaatcatggtcatanctntttcctgtgtgaaattgttatccgctcaca540 attccacacaacatacganccggaaacataaantgtaaacctggggtgcctaatgantga600 ctaactcacattaattgcgttgcgctcactgcccgctttccaatcnggaaacctgtcttg660 ccncttgcattnatgaatcngccaacccccggggaaaagcgtttgcgttttgggcgctct720 tccgcttcctcnctcanttantccctncnctcggtcattccggctgcngcaaaccggttc780 accncctccaaagggggtattccggtttccccnaatccgggganancc 828 <210> 5 <211> 834 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(834) <223> n = A,T,C or G
<400>

tttttttttttttttactgatagatggaatttattaagcttttcacatgtgatagcacat60 agttttaattgcatccaaagtactaacaaaaactctagcaatcaagaatggcagcatgtt120 attttataacaatcaacacctgtggcttttaaaatttggttttcataagataatttatac180 tgaagtaaatctagccatgcttttaaaaaatgctttaggtcactccaagcttggcagtta240 acatttggcataaacaataataaaacaatcacaatttaataaataacaaatacaacattg300 taggccataatcatatacagtataaggaaaaggtggtagtgttgagtaagcagttattag360 aatagaataccttggcctctatgcaaatatgtctagacactttgattcactcagccctga420 cattcagttttcaaagtaggagacaggttctacagtatcattttacagtttccaacacat480 tgaaaacaagtagaaaatgatgagttgatttttattaatgcattacatcctcaagagtta540 tcaccaacccctcagttataaaaaattttcaagttatattagtcatataacttggtgtgc600 ttattttaaattagtgctaaatggattaagtgaagacaacaatggtcccctaatgtgatt660 gatattggtcatttttaccagcttctaaatctnaactttcaggcttttgaactggaacat720 tgnatnacagtgttccanagttncaacctactggaacattacagtgtgcttgattcaaaa780 tgttattttgttaaaaattaaattttaacctggtggaaaaataatttgaaatna 834 <210> 6 <211> 818 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(818) <223> n = A,T,C or G
<400> 6 ttttttttttttttttttttaagaccctcatcaatagatggagacatacagaaatagtca60 aaccacatctacaaaatgccagtatcaggcggcggcttcgaagccaaagtgatgtttgga120 tgtaaagtgaaatattagttggcggatgaagcagatagtgaggaaagttgagccaataat180 gacgtgaagtccgtggaagcctgtggctacaaaaaatgttgagccgtagatgccgtcgga240 aatggtgaagggagactcgaagtactctgaggcttgtaggagggtaaaatagagacccag300 taaaattgtaataagcagtgcttgaattatttggtttcggttgttttctattagactatg360 gtgagctcaggtgattgatactcctgatgcgagtaatacggatgtgtttaggagtgggac420 ttctaggggatttagcggggtgatgcctgttgggggccagtgccctcctagttggggggt480 aggggctaggctggagtggtaaaaggctcagaaaaatcctgcgaagaaaaaaacttctga540 ggtaataaataggattatcccgtatcgaaggcctttttggacaggtggtgtgtggtggcc600 ttggtatgtgctttctcgtgttacatcgcgccatcattggtatatggttagtgtgttggg660 ttantanggcctantatgaagaacttttggantggaattaaatcaatngcttggccggaa720 gtcattanganggctnaaaaggccctgttangggtctgggctnggttttacccnacccat780 ggaatncnccccccggacnantgnatccctattcttaa g1g <210> 7 <211> 817 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(817) <223> n = A,T,C or G
<400>

tttttttttttttttttttttggctctagagggggtagagggggtgctatagggtaaata60 cgggccctatttcaaagatttttaggggaattaattctaggacgatgggtatgaaactgt120 ggtttgctccacagatttcagagcattgaccgtagtatacccccggtcgtgtagcggtga180 aagtggtttggtttagacgtccgggaattgcatctgtttttaagcctaatgtggggacag240 ctcatgagtgcaagacgtcttgtgatgtaattattatacnaatgggggcttcaatcggga300 gtactactcgattgtcaacgtcaaggagtcgcaggtcgcctggttctaggaataatgggg360 gaagtatgtaggaattgaagattaatccgccgtagtcggtgttctcctaggttcaatacc420 attggtggccaattgatttgatggtaaggggagggatcgttgaactcgtctgttatgtaa480 aggatnccttngggatgggaaggcnatnaaggactanggatnaatggcgggcangatatt540 tcaaacngtctctanttcctgaaacgtctgaaatgttaataanaattaantttngttatt600 gaatnttnnggaaaagggcttacaggactagaaaccaaatangaaaantaatnntaangg660 cnttatcntnaaaggtnataaccnctcctatnatcccacccaatngnattccccacncnn720 acnattggatnccccanttccanaaanggccnccccccggtgnannccnccttttgttcc780 cttnantganggttattcncccctngcnttatcancc 817 <210> 8 <211> 799 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(799) <223> n = A,T,C or G
<400> 8 catttccggg tttactttct aaggaaagcc gagcggaagc tgctaacgtg ggaatcggtg 60 cataaggaga actttctgct ggcacgcgct agggacaagc gggagagcga ctccgagcgt 120 ctgaagcgca cgtcccagaa ggtggacttg gcactgaaac agctgggaca catccgcgag 180 tacgaacagc gcctgaaagt gctggagcgg gaggtccagc agtgtagccg cgtcctgggg 240 tgggtggccg angcctganc cgctctgcct tgctgccccc angtgggccg ccaccccctg 300 acctgcctgg gtccaaacac tgagccctgc tggcggactt caagganaac ccccacangg 360 ggattttgctcctanantaaggctcatctgggcctcggcccccccacctggttggccttg420 tctttgangtgagccccatgtccatctgggccactgtcnggaccacctttngggagtgtt480 ctccttacaaccacannatgcccggctcctcccggaaaccantcccancctgngaaggat540 caagncctgnatccactnntnctanaaccggccnccnccgcngtggaacccnccttntgt600 tccttttcnttnagggttaatnncgccttggccttnccanngtcctncncnttttccnnt660 gttnaaattgttangcncccnccnntcccncnncnncnancccgacccnnannttnnann720 ncctgggggtnccnncngattgacccnnccnccctntanttgcnttngggnncnntgccc780 ctttccctctnggganncg 799 <210> 9 <211> 801 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(801) <223> n = A,T,C or G
<400> 9 acgccttgatcctcccaggctgggactggttctgggaggagccgggcatgctgtggtttg60 taangatgacactcccaaaggtggtcctgacagtggcccagatggacatggggctcacct120 caaggacaaggccaccaggtgcgggggccgaagcccacatgatccttactctatgagcaa180 aatcccctgtgggggcttctccttgaagtccgccancagggctcagtctttggacccang240 caggtcatggggttgtngnccaactgggggccncaacgcaaaanggcncagggcctcngn300 cacccatcccangacgcggctacactnctggacctcccnctccaccactttcatgcgctg360 ttcntacccgcgnatntgtcccanctgtttcngtgccnactccancttctnggacgtgcg420 ctacatacgcccggantcncnctcccgctttgtccctatccacgtnccancaacaaattt480 cnccntantgcaccnattcccacntttnncagntttccncnncgngcttccttntaaaag540 ggttganccccggaaaatnccccaaagggggggggccnggtacccaactnccccctnata600 gctgaantccccatnaccnngnctcnatgganccntccnttttaannacnttctnaactt660 gggaananccctcgnccntncccccnttaatcccnccttgcnangnncntcccccnntcc720 ncccnnntnggcntntnanncnaaaaaggcccnnnancaatctcctnncncctcanttcg780 ccanccctcgaaatcggccnc 801 <210> 10 <211> 789 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(789) <223> n = A,T,C or G
<400>

cagtctatntggccagtgtggcagctttccctgtggctgccggtgccacatgcctgtccc60 acagtgtggccgtggtgacagcttcagccgccctcaccgggttcaccttctcagccctgc120 agatcctgccctacacactggcctccctctaccaccgggagaagcaggtgttcctgccca180 aataccgaggggacactggaggtgctagcagtgaggacagcctgatgaccagcttcctgc240 caggccctaagcctggagctcccttccctaatggacacgtgggtgctggaggcagtggcc300 tgctcccacctccacccgcgctctgcggggcctctgcctgtgatgtctccgtacgtgtgg360 tggtgggtgagcccaccgangccagggtggttccgggccggggcatctgcctggacctcg420 ccatcctggatagtgcttcctgctgtcccangtggccccatccctgtttatgggctccat480 tgtccagctcagccagtctgtcactgcctatatggtgtctgccgcaggcctgggtctggt540 cccatttactttgctacacaggtantatttgacaagaacganttggccaaatactcagcg600 ttaaaaaattccagcaacattgggggtggaaggcctgcctcactgggtccaactccccgc660 tcctgttaaccccatggggctgccggcttggccgccaatttctgttgctgccaaantnat720 gtggctctct gctgccacct gttgctggct gaagtgcnta cngcncanct nggggggtng 780 ggngttccc 789 <210> 11 <211> 772 <212> DNA
<213> Homo sapien, <220>
<221> misc_feature <222> (1)...(772) <223> n = A,T,C or G
<400> 11 cccaccctacccaaatattagacaccaacacagaaaagctagcaatggattcccttctac60 tttgttaaataaataagttaaatatttaaatgcctgtgtctctgtgatggcaacagaagg120 accaacaggccacatcctgataaaaggtaagaggggggtggatcagcaaaaagacagtgc180 tgtgggctgaggggacctggttcttgtgtgttgcccctcaggactcttcccctacaaata240 actttcatatgttcaaatcccatggaggagtgtttcatcctagaaactcccatgcaagag300 ctacattaaacgaagctgcaggttaaggggcttanagatgggaaaccaggtgactgagtt360 tattcagctcccaaaaacccttctctaggtgtgtctcaactaggaggctagctgttaacc420 ctgagcctgggtaatccacctgcagagtccccgcattccagtgcatggaacccttctggc480 ctccctgtataagtccagactgaaacccccttggaaggnctccagtcaggcagccctana540 aactggggaaaaaagaaaaggacgccccancccccagctgtgcanctacgcacctcaaca600 gcacagggtggcagcaaaaaaaccactttactttggcacaaacaaaaactngggggggca660 accccggcaccccnangggggttaacaggaancngggnaacntggaacccaattnaggca720 ggcccnccaccccnaatnttgctgggaaatttttcctcccctaaattntttc 772 <210> 12 <211> 751 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(751) <223> n = A,T,C or G
<400>

gccccaattccagctgccacaccacccacggtgactgcattagttcggatgtcatacaaa60 agctgattgaagcaaccctctactttttggtcgtgagccttttgcttggtgcaggtttca120 ttggctgtgttggtgacgttgtcattgcaacagaatgggggaaaggcactgttctctttg180 aagtanggtgagtcctcaaaatccgtatagttggtgaagccacagcacttgagccctttc240 atggtggtgttccacacttgagtgaagtcttcctgggaaccataatctttcttgatggca300 ggcactaccagcaacgtcagggaagtgctcagccattgtggtgtacaccaaggcgaccac360 agcagctgcnacctcagcaatgaagatgangaggangatgaagaagaacgtcncgagggc420 acacttgctctcagtcttancaccatancagcccntgaaaaccaanancaaagaccacna480 cnccggctgcgatgaagaaatnaccccncgttgacaaacttgcatggcactggganccac540 agtggcccnaaaaatcttcaaaaaggatgccccatcnattgaccccccaaatgcccactg600 ccaacaggggctgccccacncncnnaacgatganccnattgnacaagatctncntggtct660 tnatnaacntgaaccctgcntngtggctcctgttcaggnccnnggcctgacttctnaann720 aangaactcngaagnccccacnggananncg 751 <210> 13 <211> 729 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .('729) <223> n = A,T,C or G
<400> 13 gagccaggcgtccctctgcctgcccactcagtggcaacacccgggagctgttttgtcctt60 tgtggancctcagcagtnccctctttcagaactcantgccaaganccctgaacaggagcc120 accatgcagtgcttcagcttcattaagaccatgatgatcctcttcaatttgctcatcttt180 ctgtgtggtgcagccctgttggcagtgggcatctgggtgtcaatcgatggggcatccttt240 ctgaagatcttcgggccactgtcgtccagtgccatgcagtttgtcaacgtgggctacttc300 ctcatcgcagccggcgttgtggtcttagctctaggtttcctgggctgctatggtgctaag360 actgagagcaagtgtgccctcgtgacgttcttcttcatcctcctcctcatcttcattgct420 gaggttgcaatgctgtggtcgccttggtgtacaccacaatggctgagcacttcctgacgt480 tgctggtaatgcctgccatcaanaaaagattatgggttcccaggaanacttcactcaagt540 gttggaacaccaccatgaaagggctcaagtgctgtggcttcnnccaactatacggatttt600 gaagantcacctacttcaaagaaaanagtgcctttcccccatttctgttgcaattgacaa660 acgtccccaacacagccaattgaaaacctgcacccaacccaaangggtccccaaccanaa720 attnaaggg 729 <210> 14 <211> 816 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(816) <223> n = A,T,C or G
<400>

tgctcttcctcaaagttgttcttgttgccataacaaccaccataggtaaagcgggcgcag60 tgttcgctgaaggggttgtagtaccagcgcgggatgctctccttgcagagtcctgtgtct120 ggcaggtccacgcagtgccctttgtcactggggaaatggatgcgctggagctcgtcaaag180 ccactcgtgtatttttcacaggcagcctcgtccgacgcgtcggggcagttgggggtgtct240 tcacactccaggaaactgtcnatgcagcagccattgctgcagcggaactgggtgggctga300 cangtgccagagcacactggatggcgcctttccatgnnangggccctgngggaaagtccc360 tganccccananctgcctctcaaangccccaccttgcacaccccgacaggctagaatgga420 atcttcttcccgaaaggtagttnttcttgttgcccaanccanccccntaaacaaactctt480 gcanatctgctccgngggggtcntantaccancgtgggaaaagaaccccaggcngcgaac540 caancttgtttggatncgaagcnataatctnctnttctgcttggtggacagcaccantna600 ctgtnnanctttagnccntggtcctcntgggttgnncttgaacctaatcnccnntcaact660 gggacaaggtaantngccntcctttnaattcccnancntnccccctggtttggggttttn720 cncnctcctaccccagaaannccgtgttcccccccaactaggggccnaaaccnnttnttc780 cacaaccctnccccacccacgggttcngntggttng 816 <210> 15 <211> 783 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(783) <223> n = A,T,C or G
<400> 15 ccaaggcctg ggcaggcata nacttgaagg tacaacccca ggaacccctg gtgctgaagg 60 atgtggaaaacacagattggcgcctactgcggggtgacacggatgtcagggtagagagga120 aagacccaaaccaggtggaactgtggggactcaaggaangcacctacctgttccagctga180 cagtgactagctcagaccacccagaggacacggccaacgtcacagtcactgtgctgtcca240 ccaagcagacagaagactactgcctcgcatccaacaangtgggtcgctgccggggctctt300 tcccacgctggtactatgaccccacggagcagatctgcaagagtttcgtttatggaggct360 gcttgggcaacaagaacaactaccttcgggaagaagagtgcattctancctgtcngggtg420 tgcaaggtgggcctttganangcanctctggggctcangcgactttcccccagggcccct480 ccatggaaaggcgccatccantgttctctggcacctgtcagcccacccagttccgctgca540 ncaatggctgctgcatcnacantttcctngaattgtgacaacaccccccantgcccccaa600 ccctcccaacaaagcttccctgttnaaaaatacnccanttggcttttnacaaacncccgg660 cncctccnttttccccnntnaacaaagggcnctngcntttgaactgcccnaacccnggaa720 tctnccnnggaaaaantnccccccctggttcctnnaancccctccncnaaanctnccccc780 ccc 783 <210> 16 <211> 801 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(801) <223> n = A,T,C or G
<400>

gccccaattccagctgccacaccacccaoggtgactgcattagttcggatgtoatacaaa60 agctgattgaagcaaccctctactttttggtcgtgagccttttgcttggtgcaggtttca120 ttggctgtgttggtgacgttgtcattgcaacagaatgggggaaaggcactgttctctttg180 aagtagggtgagtcctcaaaatccgtatagttggtgaagccacagcacttgagccctttc240 atggtggtgttccacacttgagtgaagtcttcctgggaaccataatctttcttgatggca300 ggcactaccagcaacgtcaggaagtgctcagccattgtggtgtacaccaaggcgaccaca360 gcagctgcaacctcagcaatgaagatgaggaggaggatgaagaagaacgtcncgagggca420 ~

cacttgctctccgtcttagcaccatag cccangaaaccaagagcaaagaccacaacg480 cag ccngctgcgaatgaaagaaantacccacgttgacaaactgcatggccactggacgacagt540 tggcccgaanatcttcagaaaagggatgccccatcgattgaacacccanatgcccactgc600 cnacagggctgcnccncncngaaagaatgagccattgaagaaggatcntcntggtcttaa660 tgaactgaaaccntgcatggtggcccctgttcagggctcttggcagtgaattctganaaa720 aaggaacngcntnagcccccccaaanganaaaacacccccgggtgttgccctgaattggc780 ggccaagganccctgccccng 801 <210> 17 <211> 740 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(740) <223> n = A,T,C or G
<400> 17 gtgagagccaggcgtccctctgcctgcccactcagtggcaacacccgggagctgttttgt60 cctttgtggagcctcagcagttccctctttcagaactcactgccaagagccctgaacagg120 agccaccatgcagtgcttcagcttcattaagaccatgatgatcctcttcaatttgctcat180 ctttctgtgtggtgcagccctgttggcagtgggcatctgggtgtcaatcgatggggcatc240 ctttctgaagatcttcgggccactgtcgtccagtgccatgcagtttgtcaacgtgggcta300 cttcctcatcgcagccggcgttgtggtctttgctcttggtttcctgggctgctatggtgc360 taagacggagagcaagtgtgccctcgtgacgttcttcttcatcctcctcctcatcttcat420 tgctgaagttgcagctgctgtggtcgccttggtgtacaccacaatggctgaaccattcct480 gacgttgctggtantgcctgccatcaanaaagattatgggttcccaggaaaaattcactc540 aantntggaacaccnccatgaaaagggctccaatttctgntggcttccccaactataccg600 gaattttgaaagantcnccctacttccaaaaaaaaananttgcctttncccccnttctgt660 tgcaatgaaaacntcccaanacngccaatnaaaacctgcccnnnca.aaaaggntcncaaa720 caaaaaaantnnaagggttn 740 <210> 18 <211> 802 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(802) <223> n = A,T,C or G
<400> 18 ccgctggttg.cgctggtccagngnagccacgaagcacgtcagcatacacagcctcaatca60 caaggtcttccagctgccgcacattacgcagggcaagagcctccagcaacactgcatatg120 ggatacactttactttagcagccagggtgacaactgagaggtgtcgaagcttattcttct180 gagcctctgttagtggaggaagattccgggcttcagctaagtagtcagcgtatgtcccat240 aagcaaacactgtgagcagccggaaggtagaggcaaagtcactctcagccagctctctaa300 cattgggcatgtccagcagttctccaaacacgtagacaccagnggcctccagcacctgat360 ggatgagtgtggccagcgctgcccccttggccgacttggctaggagcagaaattgctcct420 ggttctgccctgtcaccttcacttccgcactcatcactgcactgagtgtgggggacttgg480 gctcaggatgtccagagacgtggttccgccccctcncttaatgacaccgnccanncaacc540 gtcggctcccgccgantgngttcgtcgtncctgggtcagggtctgctggccnctacttgc600 aancttcgtcnggcccatggaattcaccncaccggaactngtangatccactnnttctat660 aaccggncgccaccgcnnntggaactccactcttnttncctttacttgagggttaaggtc720 acccttnncgttaccttggtccaaaccntnccntgtgtcganatngtnaatcnggnccna780 tnccanccncatangaagccng 802 <210> 19 <211> 731 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1j...(731) <223> n = A,T,C or G
<400>

cnaagcttccaggtnacgggccgcnaancctgacccnaggtancanaangcagncngcgg60 gagcccaccgtcacgnggnggngtctttatnggagggggcggagccacatcnctggacnt120 cntgaccccaactccccnccncncantgcagtgatgagtgcagaactgaaggtnacgtgg180 caggaaccaagancaaannctgctccnntccaagtcggcnnagggggcggggctggccac240 gcncatccntcnagtgctgnaaagccccnncctgtctacttgtttggagaacngcnnnga300 catgcccagngttanataacnggcngagagtnantttgcctctcccttccggctgcgcan360 cgngtntgcttagnggacataacctgactacttaactgaacccnngaatctnccncccct420 ccactaagccagaacaaaaaacttcgacat gtcacctgnctgctcaagta480 ccactcantt aagtgtaccccatncccaatgtntgctngangctctgncctgcnttangttcggtcctgg540 gaagacctatcaattnaagctatgtttctgactgcctcttgctccctgnaacaancnacc600 cnncnntccaagggggggncggcccccaatccccccaaccntnaattnantttanccccn660 cccccnggcccggccttttacnancntcnnnnacngggnaaaaccnnngctttncccaac720 nnaatccncct 731 <210> 20 <211> 754 <212> DNA
<213>.Homo sapien <220>
<221> misc_feature <222> (1)...(754) <223> n = A,T,C or G
<400>

tttttttttttttttttttttaaaaaccccctccattnaatgnaaacttccgaaattgtc60 caaccccctcntccaaatnnccntttccgggngggggttccaaacccaanttanntttgg120 annttaaattaaatnttnnttggnggnnnaanccnaatgtnangaaagttnaacccanta180 tnancttnaatncctggaaaccngtngnttccaaaaatntttaacccttaantccctccg240 aaatngttnanggaaaacccaanttctcntaaggttgtttgaaggntnaatnaaaanecc300 nnccaattgtttttngccacgcctgaattaattggnttccgntgttttccnttaaaanaa360 ggnnanccccggttantnaatccccccnnccccaattataccgantttttttngaattgg420 gancccncgggaattaacggggnnnntccctnttggggggcnggnnccccccccntcggg480 ggttngggncaggncnnaattgtttaagggtccgaaaaatccctccnagaaaaaaanctc540 ccaggntgagnntngggtttnccccccccccanggcccctctcgnanagttggggtttgg600 ggggcctgggattttntttcccctnttncctcccccccccccnggganagaggttngngt660 tttgntcnncggccccnccnaaganctttnocganttnanttaaatccntgcctnggcga720 agtccnttgnagggntaaanggccccctnncggg 754 <210> 21 <211> 755 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(755) <223> n = A,T,C or G
<400>

atcancccatgaccccnaacnngggaccnctcanccggncnnncnaccnccggccnatca60 nngtnagnncactncnnttnnatcacnccccnccnactacgcccncnanccnacgcncta120 nncanatnccactganngcgcgangtnganngagaaanctnataccanagncaccanacn180 ccagctgtccnanaangcctnnnatacnggnnnatccaatntgnancctccnaagtattn240 nncnncanatgattttcctnanccgattacccntnccccctancccctcccccccaacna300 cgaaggcnctggnccnaaggnngcgncnccccgctagntccccnncaagtcncncnccta360 aactcanccnnattacncgcttcntgagtatcactccccgaatctcaccctactcaactc420 aaaaanatcngatacaaaataatncaagcctgnttatnacactntgactgggtctctatt480 ttagnggtccntnaancntcctaatacttccagtctnccttcnccaatttccnaanggct540 ctttcngacagcatnttttggttcccnnttgggttcttanngaattgcccttcntngaac600 gggctcntcttttccttcggttancctggnttcnnccggccagttattatttcccntttt660 aaattcntnccntttanttttggcnttcnaaacccccggccttgaaaacggccccctggt720 aaaaggttgttttganaaaatttttgttttgttcc 755 <210> 22 <211> 849 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(849) <223> n = A,T,C or G
<400> 22 tttttttttttttttangtgtngtcgtgcaggtagaggcttactacaantgtgaanacgt60 acgctnggantaangcgacccganttctagganncnccctaaaatcanactgtgaagatn120 atcctgnnnacggaanggtcaccggnngatnntgctagggtgnccnctcccannncnttn180 cataactcngnggccctgcccaccaccttcggcggcccngngnccgggcccgggtcattn240 gnnttaaccncactnngcnancggtttccnnccccnncngacccnggcgatccggggtnc30.0 tctgtcttcccctgnagncnanaaantgggccncggncccctttacccctnnacaagcca360 , cngccntctanccncngccccccctccantnngggggactgccnanngctccgttnctng420 nnaccccnnngggtncctcggttgtcgantcnaccgnangccanggattccnaaggaagg480 tgcgttnttggcccctacccttcgctncggnncacccttcccgacnanganccgctcccg540 cncnncgnngcctcncctcgcaacacccgcnctcntcngtncggnnncccccccacccgc600 nccctcncncngncgnancnctccnccnccgtctcanncaccaccccgccccgccaggcc660 ntcanccacnggnngacnngnagcncnntcgcnccgcgcngcgncnccctcgccncngaa720 ctncntcnggccantnncgctcaanccnnacnaaacgccgctgcgcggcccgnagcgncc780 ncctccncgagtcctcccgncttccnacccangnnttccncgaggacacnnnaccccgcc840 nncangcgg 849 <210> 23 <211> 872 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(872) <223> n = A,T,C or G
<400>

gcgcaaactatacttcgctcgnactcgtgcgcctcgctnctcttttcctccgcaaccatg60 tctgacnancccgattnggcngatatcnanaagntcgancagtccaaactgantaacaca120 cacacncnanaganaaatccnctgccttccanagtanacnattgaacnngagaaccangc180 nggcgaatcgtaatnaggcgtgcgccgccaatntgtcnccgtttattntnccagcntcnc240 ctnccnaccctacntcttcnnagctgtcnnacccctngtncgnaccccccnaggtcggga300 tcgggtttnnnntgaccgngcnncccctccccccntccatnacganccncccgcaccacc360 nanngcncgcnccccgnnctcttcgccnccctgtcctntncccctgtngcctggcncngn420 accgcattgaccctcgccnnctncnngaaancgnanacgtccgggttgnnannancgctg480 tgggnnngcgtctgcnccgcgttccttccnncnncttccaccatcttcnttacngggtct540 ccncgccntctcnnncacnccctgggacgctntcctntgccccccttnactccccccctt600 cgncgtgncccgnccccaccntcatttncanacgntcttcacaannncctggntnnctcc660 cnancngncngtcanccnagggaagggnggggnnccnntgnttgacgttgnggngangtc720 cgaanantcctcnccntcancnctacccctcgggcgnnctctcngttnccaacttancaa780 ntctcccccgngngcncntctcagcctcncccnccccnctctctgcantgtnctctgctc840 tnaccnntacgantnttcgncnccctctttcc ~ 872 <2l0> 24 <211> 815 <212> DNA
<2l3> Homo sapien <220>
<221> misc_feature <222> (1)...(815) <223> n = A,T,C or G
<400> 24 gcatgcaagc ttgagtattc tatagngtca cctaaatanc ttggcntaat catggtcnta 60 nctgncttcctgtgtcaaatgtatacnaantanatatgaatctnatntgacaaganngta120 tcntncattagtaacaantgtnntgtccatcctgtcngancanattcccatnnattncgn180 cgcattcncngcncantatntaatngggaantcnnntnnnncaccnncatctatcntncc240 gcnccctgactggnagagatggatnanttctnntntgaccnacatgttcatcttggattn300 aananccccccgcngnccaccggttngnngcnagccnntcccaagacctcctgtggaggt360 aacctgcgtcaganncatcaaacntgggaaacccgcnnccangtnnaagtngnnncanan420 gatcccgtccaggnttnaccatcccttcncagcgccccctttngtgccttanagngnagc480 gtgtccnanccnctcaacatganacgcgccagnccanccgcaattnggcacaatgtcgnc540 gaaccccctagggggantnatncaaanccccaggattgtccncncangaaatcccncanc600 cccnccctacccnnctttgggacngtgaccaantcccggagtnccagtccggccngnctc660 ccccaccggtnnccntgggggggtgaanctcngnntcanccngncgaggnntcgnaagga720 accggncctnggncgaanngancnntcngaagngccncntcgtataaccccccctcncca780 nccnacngntagntcccccccngggtncggaangg 815 <210> 25 <211> 775 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(775) <223> n = A,T,C or G
<400> 25 ccgagatgtctcgctccgtggccttagctgtgctcgcgctactctctctttctggcctgg60 aggctatccagcgtactccaaagattcaggtttactcacgtcatccagcagagaatggaa120 agtcaaatttcctgaattgctatgtgtctgggtttcatccatccgacattgaanttgact180 tactgaagaatgganagagaattgaaaaagtggagcattcagacttgtctt.tcagcaagg240 actggtctttotatctcntgtactacactgaattcacccccactgaaaaagatgagtatg300 cctgccgtgtgaaccatgtgactttgtcacagcccaagatagttaagtgggatcgagaca360 tgtaagcagncnncatggaagtttgaagatgccgcatttggattggatgaattccaaatt420 ctgcttgcttgcnttttaatantgatatgcntatacaccctaccctttatgnccccaaat480 tgtaggggttacatnantgttcncntnggacatgatcttcctttataantccnccnttcg540 aattgcccgtcncccngttnngaatgtttccnnaaccacggttggctcccccaggtcncc600 tcttacggaagggcctgggccnctttncaaggttgggggaaccnaaaatttcncttntgc660 ccncccnccacnntcttgngnncncantttggaacccttccnattccccttggcctcnna720 nccttnnctaanaaaacttnaaancgtngcnaaanntttnacttccccccttacc 775 <210> 26 <211> 820 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(820) <223> n = A,T,C or G
<400>

anattantacagtgtaatcttttcccagaggtgtgtanagggaacggggcctagaggcat60 cccanagatancttatancaacagtgctttgaccaagagctgctgggcacatttcctgca120 gaaaaggtggcggtccccatcactcctcctctcccatagccatcccagaggggtgagtag180 ccatcangccttcggtgggagggagtcanggaaacaacanaccacagagcanacagacca240 ntgatgaccatgggcgggagcgagcctcttccctgnaccggggtggcananganagccta300 nctgaggggtcacactataaacgttaacgaccnagatnancacctgcttcaagtgcaccc360 ttcctacctgacnaccagngaccnnnaactgcngcctggggacagcnctgggancagcta420 acnnagcactcacctgcccccccatggccgtncgcntccctggtcctgncaagggaagct480 ccctgttggaattncgggganaccaaggganccccctcctccanctgtgaaggaaaaann540 gatggaattttncccttccggccnntcccctcttcctttacacgccccctnntactcntc600 tccctctnttntcctgncncacttttnaccccnnnatttcccttnattgatcggannctn660 ganattccactnncgcctnccntcnatcngnaanacnaaanactntctnacccnggggat720 gggnncctcgntcatcctctctttttcnctaccnccnnttctttgcctctccttngatca780 tccaaccntcgntggccntncccccccnnntcctttnccc 820 <210> 27 <211> 818 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(818) <223> n = A,T,C or G
<400> 27 tctgggtgatggcctcttcctcctcagggacctctgactgctctgggccaaagaatctct60 tgtttcttctccgagccccaggcagcggtgattcagccctgcccaacctgattctgatgal20 ctgcggatgctgtgacggacccaaggggcaaatagggtcccagggtccagggaggggcgc180 ctgctgagcacttccgcccctcaccctgcccagcccctgccatgagctctgggctgggtc240 tccgcctccagggttctgctcttccangcangccancaagtggcgctgggccacactggc300 ttcttcctgccccntccctggctctgantctctgtcttcctgtcctgtgcangcnccttg360 gatctcagtttccctcnctcanngaactctgtttctganntcttcanttaactntgantt420 tatnaccnantggnctgtnctgtcnnactttaatgggccngaccggctaatccctccctc480 nctcccttccanttcnnn~aaccngcttnccntcntctccccntancccgccngggaanc540 ctcctttgccctnaccangggccnnnaccgcccntnnctnggggggcnnggtnnctncnc600 ctgntnnccccnctcncnnttncctcgtcccnncnncgcnnngcannttcncngtcccnn660 tnnctcttcnngtntcgnaangntcncntntnnnnngncnngntnntncntccctctcnc720 cnnntgnangtnnttnnnncncngnnccccnnnncnnnnnnggnnntnnntctncncngc780 cccnncccccngnattaaggcctccnntctccggccnc 818 <210> 28 <211> 731 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(731) <223> n = A,T,C or G
<400> 28 aggaagggcggagggatattgtangggattgagggataggagnataangggggaggtgtg60 tcccaacatganggtgnngttctcttttgaangagggttgngtttttannccnggtgggt120 gattnaaccccattgtatggagnnaaaggntttnagggatttttcggctcttatcagtat180 ntanattcctgtnaatcggaaaatnatntttcnncnggaaaatnttgctcccatccgnaa240 attnctcccgggtagtgcatnttngggggncngccangtttcccaggctgctanaatcgt300 actaaagnttnaagtgggantncaaatgaaaacctnncacagagnatccntacccgactg360 tnnnttnccttcgccctntgactctgcnngagcccaatacccnngngnatgtcncccngn420 nnngcgncnctgaaannnnctcgnggctnngancatcanggggtttcgcatcaaaagcnn480 cgtttcncatnaaggcactttngcctcatccaaccnctngccctcnnccatttngccgtc540 nggttcncctacgctnntngcncctnnntnganattttncccgcctngggnaancctcct600 gnaatgggtagggncttntcttttnaccnngnggtntactaatcnnctncacgcntnctt660 tctcnaccccccccctttttcaatcccancggcnaatggggtctccccnncgangggggg720 nnncccanncc 731 <210> 29 <211> 822 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(822) <223> n = A,T,C or G
<400> 29 actagtccagtgtggtggaattccattgtgttggggncncttctatgantantnttagat60 cgctcanacctcacancctcccnacnangcctataangaanannaataganctgtncnnt120 atntntacnctcatanncctcnnnacccactccctcttaacccntactgtgcctatngcn180 tnnctantctntgccgcctncnanccaccngtgggccnaccncnngnattctcnatctcc240 tcnccatntngcctanantangtncataccctatacctacnccaatgctannnctaancn300 tccatnanttannntaactaccactgacntngactttcncatnanctcctaatttgaatc360 tactctgactcccacngcctannnattagcancntcccccnacnatntctcaaccaaatc420 ntcaacaacctatctanctgttcnccaaccnttncctccgatccccnnacaacccccctc480 ccaaatacccnccacctgacncctaacccncaccatcccggcaagccnanggncatttan540 ccactggaatcacnatngganaaaaaaaacccnaactctctancncnnatctccctaana600 aatnctcctnnaatttactnncantnccatcaancccacntgaaacnnaacccctgtttt660 tanatcccttctttcgaaaaccnaccctttannncccaacctttngggcccccccnctnc720 ccnaatgaaggncncccaatcnangaaacgnccntgaaaaancnaggcnaanannntccg780 canatcctatcccttanttnggggncccttncccngggcccc 822 <210> 30 <211> 787 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(787) <223> n = A,T,C or <400>

cggccgcctgctctggcacatgcctcctgaatggcatcaaaagtgatggactgcccattg60 ctagagaagaccttctctcctactgtcattatggagccctgcagactgagggctcccctt120 gtctgcaggatttgatgtctgaagtcgtggagtgtggcttggagctcctcatctacatna180 gctggaagccctggagggcctctctcgccagcctcccccttctctccacgctctccangg240 acaccaggggctccaggcagcccattattcccagnangacatggtgtttctccacgcgga300 cccatggggcctgnaaggccagggtctcctttgacaccatctctcccgtcctgcctggca360 ggccgtgggatccactanttctanaacggncgccaccncggtgggagctccagcttttgt420 tcccnttaatgaaggttaattgcncgcttggcgtaatcatnggtcanaactntttcctgt480 gtgaaattgtttntcccctcncnattccncncnacatacnaacccggaancataaagtgt540 taaagcctgggggtngcctnnngaatnaactnaactcaattaattgcgttggctcatggc600 ccgctttccnttcnggaaaactgtcntcccctgcnttnntgaatcggccaccccccnggg660 aaaagcggtttgcnttttngggggntccttccncttcccccctcnctaanccctncgcct720 cggtcgttncnggtngcggggaangggnatnnnctcccncnaagggggngagnnngntat780 ccccaaa 787 <210> 31 <211> 799 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1). .(799) <223> n = A,T,C or G
<400> 31 tttttttttttttttttggcgatgctactgtttaattgcaggaggtgggggtgtgtgtac60 catgtaccagggctattagaagcaagaaggaaggagggagggcagagcgccctgctgagc120 aacaaaggactcctgcagccttctctgtctgtctcttggcgcaggcacatggggaggcct180 cccgcagggtgggggccaccagtccaggggtgggagcactacanggggtgggagtgggtg240 gtggctggtncnaatggcctgncacanatccctacgattcttgacacctggatttcacca300 ggggaccttctgttctcccanggnaacttcntnnatctcnaaagaacacaactgtttctt360 cngcanttctggctgttcatggaaagcacaggtgtccnatttnggctgggacttggtaca420 tatggttccggcccacctctcccntcnaanaagtaattcacccccccccnccntctnttg480 cctgggcccttaantacccacaccggaactcanttanttattcatcttnggntgggcttg540 ntnatcnccncctgaangcgccaagttgaaaggccacgccgtncccnctccccatagnan600 nttttnncntcanctaatgcccccccnggcaacnatccaatccccccccntgggggcccc660 agcccanggcccccgnctcgggnnnccngncncgnantccccaggntctcccantcngnc720 ccnnngcncccccgcacgcagaacanaaggntngagccnccgcannnnnnnggtnncnac780 ctcgccccccccnncgnng 799 <210> 32 <211> 789 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(789) <223> n = A,T,C or G
<400> 32 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt60 ttttnccnagggcaggtttattgacaacctcncgggacacaancaggctggggacaggac120 ggcaacaggctccggcggcggcggcggcggccctacctgcggtaccaaatntgcagcctc180 cgctcccgcttgatnttcctctgcagctgcaggatgccntaaaacagggcctcggccntn240 ggtgggcaccctgggatttnaatttccacgggcacaatgcggtcgcancccctcaccacc300 nattaggaatagtggtnttacccnccnccgttggcncactccccntggaaaccacttntc360 gcggctccggcatctggtcttaaaccttgcaaacnctggggccctctttttggttantnt420 nccngccacaatcatnactcagactggcncgggctggccccaaaaaancnccccaaaacc480 ggnccatgtcttnncggggttgctgcnatntncatcacctcccgggcncancaggncaac540 ccaaaagttcttgnggcccncaaaaaanctccggggggncccagtttcaacaaagtcatc600 ccccttggcccccaaatcctccccccgnttnctgggtttgggaacccacgcctctnnctt660 tggnnggcaagntggntcccccttcgggcccccggtgggcccnnctctaangaaaacncc720 ntcctnnncaccatccccccnngnnacgnctancaangnatccctttttttanaaacggg780 ccccccncg 789 <210> 33 <211> 793 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(793) <223> n = A,T,C or G
<400> 33 gacagaacat gttggatggt ggagcacctt tctatacgac ttacaggaca gcagatgggg 60 aattcatggctgttggagcaatanaaccccagttctacgagctgctgatcaaaggacttgl20 gactaaagtctgatgaacttcccaatcagatgagcatggatgattggccagaaatgaana180 agaagtttgcagatgtatttgcaaagaagacgaaggcagagtggtgtcaaatctttgacg240 gcacagatgcctgtgtgactccggttctgacttttgaggaggttgttcatcatgatcaca300 acaangaacggggctcgtttatcaccantgaggagcaggacgtgagcccccgccctgcac360 ctctgctgttaaacaccccagccatcccttctttcaaaagggatccactacttctagagc420 ggncgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgct480 tggcgtaatcatggtcatanctgtttcctgtgtgaaattgttatccgctcacaattccac540 acaacatacganccggaagcatnaaattttaaagcctggnggtngcctaatgantgaact600 nactcacattaattggctttgcgctcactgcccgctttccagtccggaaaacctgtcctt660 gccagctgccnttaatgaatcnggccaccccccggggaaaaggcngtttgcttnttgggg720 cgcncttcccgctttctcgcttcctgaantccttccccccggtctttcggcttgcggcna780 acggtatcnacct 793 <210> 34 <211> 756 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(756) <223> n = A,T,C or G
<400> 34 gccgcgaccggcatgtacgagcaactcaagggcgagtggaaccgtaaaagccccaatctt60 ancaagtgcggggaanagctgggtcgactcaagctagttcttctggagctcaacttcttg120 ccaaccacagggaccaagctgaccaaacagcagctaattctggcccgtgacatactggag180 atcggggcccaatggagcatcctacgcaangacatcccctccttcgagcgctacatggcc240 cagctcaaatgctactactttgattacaangagcagctccccgagtcagcctatatgcac300 cagctcttgggcctcaacctcctcttcctgctgtcccagaaccgggtggctgantnccac360 acgganttggancggctgcctgcccaangacatacanaccaatgtctacatcnaccacca420 gtgtcctggagcaatactgatgganggcagctaccncaaagtnttcctggccnagggtaa480 catcccccgccgagagctacaccttcttcattgacatcctgctcgacactatcagggatg540 aaaatcgcngggttgctccagaaaggctncaanaanatccttttcnctgaaggcccccgg600 atncnctagtnctagaatcggcccgccatcgcggtggancctccaacctttcgttnccct660 ttactgagggttnattgccgcccttggcgttatcatggtcacnccngttncctgtgttga720 aattnttaaccccccacaattccacgccnacattng 756 <210> 35 <211> 834 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1j...(834) <223> n = A,T,C or G
<400> 35 ggggatctct anatcnacct gnatgcatgg ttgtcggtgt ggtcgctgtc gatgaanatg 60 aacaggatct tgcccttgaa gctctcggct gctgtnttta agttgctcag tctgccgtca 120 tagtcagaca cnctcttggg caaaaaacan caggatntga gtcttgattt cacctccaat - 180 aatcttcngg gctgtctgct cggtgaactc gatgacnang ggcagctggt tgtgtntgat 240 aaantccanc angttctcct tggtgacctc cccttcaaag ttgttccggc cttcatcaaa 300 cttctnnaan angannancc canctttgtc gagctggnat ttgganaaca cgtcactgtt 360 ggaaactgat cccaaatggt atgtcatcca tcgcctctgc tgcctgcaaa aaacttgctt 420 ggcncaaatc cgactccccn tccttgaaag aagccnatca cacccccctc cctggactcc 480 nncaangact ctnccgctnc cccntccnng cagggttggt ggcannccgg gcccntgcgc 540 ttcttcagccagttcacnatnttcatcagcccctctgccagctgttntattccttggggg600 ggaanccgtctctcccttcctgaannaactttgaccgtnggaatagccgcgcntcnccnt660 acntnctgggccgggttcaaantccctccnttgncnntcncctcgggccattctggattt720 nccnaactttttccttcccccnccccncggngtttggntttttcatngggccccaactct780 gctnttggccantcccctgggggcntntancnccccctntggtcccntngggcc 834 <210> 36 <211> 814 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(814) <223> n = A,T,C or G
<400>

cggncgctttccngccgcgccccgtttccatgacnaaggctcccttcangttaaatacnn60 cctagnaaacattaatgggttgctctactaatacatcatacnaaccagtaagcctgccca120 naacgccaactcaggccattcctaccaaaggaagaaaggctggtctctccaccccctgta180 ggaaaggcctgccttgtaagacaccacaatncggctgaatctnaagtcttgtgttttact240 aatggaaaaaaaaaataaacaanaggttttgttctcatggctgcccaccgcagcctggca300 ctaaaacancccagcgctcacttctgcttgganaaatattctttgctcttttggacatca360 ggcttgatggtatcactgccacntttccacccagctgggcncccttcccccatntttgtc420 antganctggaaggcctgaancttagtctccaaaagtctcngcccacaagaccggccacc480 aggggangtcntttncagtggatctgccaaanantacccntatcatcnntgaataaaaag540 gcccctgaacganatgcttccancancctttaagacccataatcctngaaccatggtgcc600 cttccggtctgatccnaaaggaatgttcctgggtcccantccctcctttgttncttacgt660 tgtnttggacccntgctngnatnacccaantganatccccngaagcaccctncccctggc720 atttgantttcntaaattctctgccctacnnctgaaagcacnattccctnggcnccnaan780 ggngaactcaagaaggtctnngaaaaaccacncn 814 <210> 37 <211> 760 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(760) <223> n = A,T,C or G
<400>

gcatgctgctcttcctcaaagttgttcttgttgccataacaaccaccataggtaaagcgg60 gcgcagtgttcgctgaaggggttgtagtaccagcgcgggatgctctccttgcagagtcct120 gtgtctggcaggtccacgcaatgccctttgtcactggggaaatggatgcgctggagctcg180 tcnaanccactcgtgtatttttcacangcagcctcctccgaagcntccgggcagttgggg240 gtgtcgtcacactccactaaactgtcgatncancagcccattgctgcagcggaactgggt300 gggctgacaggtgccagaacacactggatnggcctttccatggaagggcctgggggaaat360 cncctnancccaaactgcctctcaaaggccaccttgcacaccccgacaggctagaaatgc420 actcttcttcccaaaggtagttgttcttgttgcccaagcancctccancaaaccaaaanc480 ttgcaaaatctgctccgtgggggtcatnnntaccanggttggggaaanaaacccggcngn540 ganccnccttgtttgaatgcnaaggnaataatcctcctgtcttgcttgggtggaanagca600 caattgaactgttaacnttgggccgngttccnctngggtggtctgaaactaatcaccgtc660 actggaaaaaggtangtgccttccttgaattcccaaanttcccctngntttgggtnnttt720 ctcctctnccctaaaaatcgtnttccccccccntanggcg 760 <210> 38 <211> 724 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(724) <223> n = A,T,C or G
<400> 38 tttttttttttttttttttttttttttttttttttaaaaaccccctccattgaatgaaaa60 cttccnaaattgtccaaccccctcnnccaaatnnccatttccgggggggggttccaaacc120 caaattaattttggantttaaattaaatnttnattnggggaanaanccaaatgtnaagaa180 aatttaacccattatnaacttaaatncctngaaacccntggnttccaaaaatttttaacc240 cttaaatccctccgaaattgntaanggaaaaccaaattcncctaaggctntttgaaggtt300 ngatttaaacccccttnanttnttttnacccnngnctnaantatttngnttccggtgttt360 tcctnttaancntnggtaactcccgntaatgaannnccctaanccaattaaaccgaattt420 tttttgaattggaaattccnngggaattnaccggggtttttcccntttgggggccatncc480 cccnctttcggggtttgggnntaggttgaatttttnnangncccaaaaaancccccaana540 aaaaaactcccaagnnttaattngaatntcccccttcccaggccttttgggaaaggnggg600 tttntgggggccnggganttcnttcccccnttnccnccccccccccnggtaaanggttat660 ngnntttggtttttgggccccttnanggaccttccggatngaaattaaatccccgggncg720 gccg ' 724 <210> 39 <211> 751 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(751) <223> n = A,T,C or G
<400> 39 tttttttttttttttctttgctcacatttaatttttattttgattttttttaatgctgca60 caacacaatatttatttcatttgtttcttttatttcattttatttgtttgctgctgctgt120 tttatttatttttactgaaagtgagagggaacttttgtggccttttttcctttttctgta180 ggccgccttaagctttctaaatttggaacatctaagcaagctgaanggaaaagggggttt240 cgcaaaatcactcgggggaanggaaaggttgctttgttaatcatgccctatggtgggtga300 ttaactgcttgtacaattacntttcacttttaattaattgtgctnaangctttaattana360 cttgggggttccctccccanaccaaccccnctgacaaaaagtgccngccctcaaatnatg420 tcccggcnntcnttgaaacacacngcngaangttctcattntccccncnccaggtnaaaa480 tgaagggttaccatntttaacnccacctccacntggcnnngcctgaatcctcnaaaancn540 ccctcaancnaattnctnngccccggtcncgcntnngtcccncccgggctcegggaantn600 cacccccngaanncnntnncnaacnaaattccgaaaatattcccnntcnctcaattcccc660 cnnagactntcctcnncnancncaattttcttttnntcacgaacncgnnccnnaaaatgn720 nnnncncctccnctngtccnnaatcnccanc 751 <210> 40 <211> 753 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(753) <223> n = A,T,C or G
<400>

gtggtattttctgtaagatcaggtgttcctccctcgtaggtttagaggaaacaccctcat60 agatgaaaacccccccgagacagcagcactgcaactgccaagcagccggggtaggagggg120 cgccctatgcacagctgggcccttgagacagcagggcttcgatgtcaggctcgatgtcaa180 tggtctggaagcggcggctgtacctgcgtaggggcacaccgtcagggcccaccaggaact240 tctcaaagttccaggcaacntcgttgcgacacaccggagaccaggtgatnagcttggggt300 cggtcataancgcggtggcgtcgtcgctgggagctggcagggcctcccgcaggaaggcna360 ataaaaggtgcgcccccgcaccgttcanctcgcacttctcnaanaccatgangttgggct420 cnaacccaccaccannccggacttccttganggaattcccaaatctcttcgntcttgggc480 ttctnctgatgccctanctggttgcccngnatgccaancanccccaanccccggggtcct540 aaancacccncctcctcntttcatctgggttnttntccccggaccntggttcctctcaag600 ggancccatatctcnaccantactcaccntncccccccntgnnacccanccttctanngn660 ttcccncccgncctctggcccntcaaanangcttncacnacctgggtctgccttcccccc720 tnccctatctgnaccccncntttgtctcantnt 753 <210> 41 <211> 341 <212> DNA
<213> Homo sapien <400> 41 actatatcca tcacaacaga catgcttcat cccatagact tcttgacata gcttcaaatg 60 agtgaaccca tccttgattt atatacatat atgttctcag tattttggga gcctttccac 120 ttctttaaac cttgttcatt atgaacactg aaaataggaa tttgtgaaga gttaaaaagt 180 tatagcttgt ttacgtagta agtttttgaa gtctacattc aatccagaca cttagttgag 240 tgttaaactg tgatttttaa aaaatatcat ttgagaatat tctttcagag gtattttcat 300 ttttactttt tgat'taattg tgttttatat attagggtag t 341 <210> 42 <211> 101 <212> DNA
<213> Homo sapien <400> 42 acttactgaa tttagttctg tgctcttcct tatttagtgt tgtatcataa atactttgat 60 gtttcaaaca ttctaaataa ataattttca gtggcttcat a 101 <210> 43 <221> 305 <212> DNA
<213> Homo sapien <400> 43 acatctttgttacagtctaagatgtgttcttaaatcaccattccttcctggtcctcaccc60 tccagggtggtctcacactgtaattagagctattgaggagtctttacagcaaattaagat120 tcagatgccttgctaagtctagagttctagagttatgtttcagaaagtctaagaaaccca180 cctcttgagaggtcagtaaagaggacttaatatttcatatctacaaaatgaccacaggat240 tggatacagaacgagagttatcctggataactcagagctgagtacctgcccgggggccgc300 tcgaa 305 , <210> 44 <211> 852 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1)...(852) <223> n = A,T,C or G
<400> 44 acataaatatcagagaaaagtagtctttgaaatatttacgtccaggagttctttgtttct60 gattatttggtgtgtgttttggtttgtgtccaaagtattggcagcttcagttttcatttt120 ctctccatcctcgggcattcttcccaaatttatataccagtcttcgtccatccacacgct180 ccagaatttctcttttgtagtaatatctcatagctcggctgagcttttcataggtcatgc240 tgctgttgttcttctttttaccccatagctgagccactgcctctgatttcaagaacctga300 agacgccctcagatcggtcttcccattttattaatcctgggttcttgtctgggttcaaga360 ggatgtcgcggatgaattcccataagtgagtccctctcgggttgtgctttttggtgtggc420 acttggcaggggggtcttgctcctttttcatatcaggtgactctgcaacaggaaggtgac480 tggtggttgtcatggagatctgagcccggcagaaagttttgctgtccaacaaatctactg540 tgctaccatagttggtgtcatataaatagttctngtctttccaggtgttcatgatggaag600 gctcagtttgttcagtcttgacaatgacattgtgtgtggactggaacaggtcactactgc660 actggccgttccacttcagatgctgcaagttgctgtagaggagntgccccgccgtccctg720 ccgcccgggtgaactcctgcaaactcatgctgcaaaggtgctcgccgttgatgtcgaact780 cntggaaagggatacaattggcatccagctggttggtgtccaggaggtgatggagccact840 cccacacctggt g52 <210> 45 <211> 234 <212> DNA
<213> Homo sapien <400> 45 acaacagacc cttgctcgct aacgacctca tgctcataaa gttggacgaa tccgtgtccg 60 agtctgacac catccggagc atcagcattg cttcgcagtg cc'ctaccgcg gggaactctt 120 gcctcgtttc tggctggggt ctgctggcga acggcagaat gcctaccgtg ctgcagtgcg 180 tgaacgtgtc ggtggtgtct gaggaggtct gcagtaagct ctatgacccg ctgt 234 <210> 46 <211> 590 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(590) <223> n = A, T, C or G
<400> 46 actttttatttaaatgtttataaggcagatctatgagaatgatagaaaacatggtgtgta60 atttgatagcaatattttggagattacagagttttagtaattaccaattacacagttaaa120 aagaagataatatattccaagcanatacaaaatatctaatgaaagatcaaggcaggaaaa180 tgantataactaattgacaatggaaaatcaattttaatgtgaattgcacattatccttta240 aaagctttcaaaanaaanaattattgcagtctanttaattcaaacagtgttaaatggtat300 caggataaanaactgaagggcanaaagaattaattttcacttcatgtaacncacccanat360 ttacaatggcttaaatgcanggaaaaagcagtggaagtagggaagtantcaaggtctttc420 tggtctctaatctgccttactctttgggtgtggctttgatcctctggagacagctgccag480 ggctcctgttatatccacaatcccagcagcaagatgaagggatgaaaaaggacacatgct540 gccttcctttgaggagacttcatctcactggccaacactcagtcacatgt 590 <220> 47 <211> 774 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> 0) ... (774) <223> n = A,T,C or G
<400>

acaagggggcataatgaaggagtgggganagattttaaagaaggaaaaaaaacgaggccc60 tgaacagaattttcctgnacaacggggcttcaaaataattttcttggggaggttcaagac120 gcttcactgcttgaaacttaaatggatgtgggacanaattttctgtaatgaccctgaggg180 cattacagacgggactctgggaggaaggataaacagaaaggggacaaaggctaatcccaa240 aacatcaaagaaaggaaggtggcgtcatacctcccagcctacacagttctccagggctct300 cctcatccctggaggacgacagtggaggaacaactgaccatgtccccaggctcctgtgtg360 ctggctcctggtcttcagcccccagctctggaagcccaccctctgctgatcctgcgtggc420 ccacactccttgaacacacatccccaggttatattcctggacatggctgaacctcctatt480 cctacttccgagatgccttgctccctgcagcctgtcaaaatcccactcaccctccaaacc540 acggcatgggaagcctttctgacttgcctgattactccagcatcttggaacaatccctga600 ttccccactccttagaggcaagatagggtggttaagagtagggctggaccacttggagcc660 aggctgctggcttcaaattntggctcatttacgagctatgggaccttgggcaagtnatct720 tcacttctatgggcntcattttgttctacctgcaaaatgggggataataatagt 774 <210> 48 <211> 124 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (124) <223> n = A,T,C or G
<400> 48 canaaattga aattttataa aaaggcattt ttctcttata tccataaaat gatataattt 60 ttgcaantat anaaatgtgt cataaattat aatgttcctt aattacagct caacgcaact 120 tggt 124 <210> 49 <212> 147 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(147) <223> n = A,T,C or G
<400> 49 gccgatgcta ctattttatt gcaggaggtg ggggtgtttt tattattctc tcaacagctt 60 tgtggctaca ggtggtgtct gactgcatna aaaanttttt tacgggtgat tgcaaaaatt 120 ttagggcacc catatcccaa gcantgt 147 <210> 50 <211> 107 <212> DNA
<213> Homo sapien <400> 50 acattaaatt aataaaagga ctgttggggt tctgctaaaa cacatggctt gatatattgc 60 atggtttgag gttaggagga gttaggcata tgttttggga gaggggt 107 <210> 51 <211> 204 <212> DNA
<213> Homo sapien <400> 51 gtcctaggaa gtctagggga cacacgactc tggggtcacg gggccgacac acttgcacgg 60 cgggaaggaa aggcagagaa gtgacaccgt cagggggaaa tgacagaaag gaaaatcaag 120 gccttgcaag gtcagaaagg ggactcaggg cttccaccac agccctgccc cacttggcca 180 cctccctttt gggaccagca atgt 204 <210> 52 <211> 491 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(491) <223> n = A,T,C or G
<400>

acaaagataacatttatcttataacaaaaatttgatagttttaaaggttagtattgtgta60 gggtattttccaaaagactaaagagataactcaggtaaaaagttagaaatgtataaaaca120 ccatcagacaggtttttaaaaaacaacatattacaaaattagacaatcatccttaaaaaa180 aaaacttcttgtatcaatttcttttgttcaaaatgactgacttaantatttttaaatatt240 tcanaaacacttcctcaaaaattttcaanatggtagctttcanatgtnccctcagtccca300 atgttgctcagataaataaatctcgtgagaacttaccacccaccacaagctttctggggc360 atgcaacagtgtcttttctttnctttttctttttttttttttacaggcacagaaactcat420 caattttatttggataacaaagggtctccaaattatattgaaaaataaatccaagttaat480 atcactcttgt 491 <210> 53 <211> 484 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(484) <223> n = A,T,C or G
<400> 53 acataatttagcagggctaattaccataagatgctatttattaanaggtntatgatctga60 gtattaacagttgctgaagtttggtatttttatgcagcattttctttttgctttgataac120 actacagaacccttaaggacactgaaaattagtaagtaaagttcagaaacattagctgct180 caatcaaatctctacataacactatagtaattaaaacgttaaaaaaaagtgttgaaatct240 gcactagtatanaccgctcctgtcaggataanactgctttggaacagaaagggaaaaanc300 agctttgantttctttgtgctgatangaggaaaggctgaattaccttgttgcctctccct360 aatgattggcaggtcnggtaaatnccaaaacatattccaactcaacacttcttttccncg420 tancttgantctgtgtattccaggancaggcggatggaatgggccagcccncggatgttc480 cant 484 <210> 54 <211> 151 <212> DNA

<213> Homo sapien <400> 54 actaaacctc gtgcttgtga actccataca gaaaacggtg ccatccctga acacggctgg 60 ccactgggta tactgctgac aaccgcaaca acaaaaacac aaatccttgg cactggctag 120 tctatgtcct ctcaagtgcc tttttgtttg t l51 <210> 55 <211> 91 <212> DNA
<213> Homo sapien <400> 55 acctggcttg tctccgggtg gttcccggcg ccocccacgg tccccagaac ggacactttc 60 gccctccagt ggatactcga gccaaagtgg t 91 <210> 56 <211> 133 <212> DNA
<213> Homo sapien <400> 56 ggcggatgtg cgttggttat atacaaatat gtcattttat gtaagggact tgagtatact 60 tggatttttg gtatctgtgg gttgggggga cggtccagga accaataccc catggatacc 120 aagggacaac tgt ~ 133 <210> 57 <211> 147 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(147) <223> n = A, T, C or G
<400> 57 actctggaga acctgagccg ctgctccgcc tctgggatga ggtgatgcan gcngtggcgc 60 gactgggagc tgagcccttc cctttgcgcc tgcctcagag gattgttgcc gacntgcana 120 tctcantggg ctggatncat gcagggt 147 <210> 58 <211> 198 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(198) <223> n = A,T,C or G
<400> 58 acagggatat aggtttnaag ttattgtnat tgtaaaatac attgaatttt ctgtatactc 60 tgattacata catttatcct ttaaaaaaga tgtaaatctt aatttttatg ccatctatta 120 atttaccaat gagttacctt gtaaatgaga agtcatgata gcactgaatt ttaactagtt 180 ttgacttcta agtttggt 198 <210> 59 <211> 330 <212> DNA
<213> Homo sapien <400> 59 acaacaaatgggttgtgaggaagtcttatcagcaaaactggtgatggctactgaaaagat60 ccattgaaaattatcattaatgattttaaatgacaagttatcaaaaactcactcaatttt120 cacctgtgctagcttgctaaaatgggagttaactctagagcaaatatagtatcttctgaa180 tacagtcaataaatgacaaagccagggcctacaggtggtttccagactttccagacccag240 cagaaggaatctattttatcacatggatctccgtctgtgctcaaaatacctaatgatatt300 tttcgtctttattggacttctttgaagagt 330 <210> 60 <211> 175 <212> DNA
<213> Homo sapien <400> 60 accgtgggtg ccttctacat tcctgacggc tccttcacca acatctggtt ctacttcggc 60 gtcgtgggct ccttcctctt catcctcatc cagctggtgc tgctcatcga ctttgcgcac 120 tcctggaacc agcggtggct gggcaaggcc gaggagtgcg attcccgtgc ctggt 175 <210> 61 <211> 154 <212> DNA
<213> Homo sapien <400> 61 accccacttt tcctcctgtg agcagtctgg acttctcact gctacatgat gagggtgagt 60 ggttgttgct cttcaacagt atcctcccct ttccggatct gctgagccgg acagcagtgc 120 tggactgcac agccccgggg ctccacattg ctgt 154 <210> 62 <211> 30 <212> DNA
<213> Homo sapien <400> 62 cgctcgagcc ctatagtgag tcgtattaga 30 <210> 63 <211> 89 <212> DNA
<213> Homo sapien <400> 63 acaagtcatt tcagcaccct ttgctcttca aaactgacca tcttttatat ttaatgcttc 60 ctgtatgaat aaaaatggtt atgtcaagt 89 <210> 64 <211> 97 <212> DNA
<213> Homo sapien <400> 64 accggagtaa ctgagtcggg acgctgaatc tgaatccacc aataaataaa ggttctgcag 60 aatcagtgca tccaggattg gtccttggat ctggggt 97 <210> 65 <211> 377 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(377) <223> n = A,T,C or G
<400> 65 acaacaanaantcccttctttaggccactgatggaaacctggaacccccttttgatggca60 gcatggcgtcctaggccttgacacagcggctggggtttgggctntcccaaaccgcacacc120 ccaaccctggtctacccacanttctggctatgggctgtctctgccactgaacatcagggt180 tcggtcataanatgaaatcccaanggggacagaggtcagtagaggaagctcaatgagaaa240 ggtgctgtttgctcagccagaaaacagctgcctggcattcgccgctgaactatgaacccg300 tgggggtgaactacccccangaggaatcatgcctgggcgatgcaanggtgccaacaggag360 gggcgggaggagcatgt 377 <210> 66 <211> 305 <212> DNA
<213> Homo sapien <400> 66 acgcctttccctcagaattcagggaagagactgtcgcctgccttcctccgttgttgcgtg60 agaacccgtgtgccccttcccaccatatccaccctcgctccatctttgaactcaaacacg120 aggaactaactgcaccctggtcctctccccagtccccagttcaccctccatccctcacct180 tcctccactctaagggatatcaacactgcccagcacaggggccct.gaatttatgtggttt240 ttatatattttttaataagatgcactttatgtcattttttaataaagtctgaagaattac300 tgttt 305 <210> 67 <211> 385 <212> DNA
<213> Homo sapien <400> 67 actacacacactccacttgcccttgtgagacactttgtcccagcactttaggaatgctga60 ggtcggaccagccacatctcatgtgcaagattgcccagcagacatcaggtctgagagttc120 cccttttaaaaaaggggacttgcttaaaaaagaagtctagccacgattgtgtagagcagc180 tgtgctgtgctggagattcacttttgagagagttctcctctgagacctgatctttagagg240 ctgggcagtcttgcacatgagatggggctggtctgatctcagcactccttagtctgcttg300 cctctcccagggccccagcctggccacacctgcttacagggcactctcagatgcccatac360 catagtttctgtgctagtggaccgt 385 <210> 68 <211> 73 <212> DNA
<213> Homo sapien <400> 68 acttaaccag atatattttt accccagatg gggatattct ttgtaaaaaa tgaaaataaa 60 gtttttttaa tgg 73 <210> 69 <211> 536 <212> DNA

<213> Homo sapien <220>
<221> misc_feature <222> (1)...(536) <223> n = A,T,C or G
<400> 69 actagtccagtgtggtggaattccattgtgttgggggctctcaccctcctctcctgcagc60 tccagctttgtgctctgcctctgaggagaccatggcccagcatctgagtaccctgctgct120 cctgctggccaccctagctgtggccctggcctggagccccaaggaggaggataggataat180 cccgggtggcatctataacgcagacctcaatgatgagtgggtacagcgtgcccttcactt240 cgccatcagcgagtataacaaggccaocaaagatgactactacagacgtccgctgcgggt300 actaagagccaggcaacagaccgttgggggggtgaattacttcttcgacgtagaggtggg360 ccgaaccatatgtaccaagtcccagcccaacttggacacctgtgccttccatgaacagcc420 agaactgcagaagaaacagttgtgctctttcgagatctacgaagttccctggggagaaca480 gaangtccctgggtgaaatccaggtgtcaagaaatcctanggatctgttgccaggc 536 <210> 70 <211> 477 <212> DNA
<213> Homo sapien <400>

atgacccctaacaggggccctctcagccctcctaatgacctccggcctagccatgtgatt60 tcacttccactccataacgctcctcatactaggcctactaaccaacacactaaccatata120 ccaatgatggcgcgatgtaacacgagaaagcacataccaaggccaccacacaccacctgt180 ccaaaaaggccttcgatacgggataatcctatttattacctcagaagtttttttcttcgc240 agggatttttctgagccttttaccactccagcctagccccta'ccccccaactaggagggc300 actggcccccaacaggcatcaccccgctaaatcccctagaagtcccactcctaaacacat360 ccgtattactcgcatcaggagtatcaatcacctgagctcaccatagtctaatagaaaaca420 accgaaaccaaattattcaaagcactgcttattacaattttactgggtctctatttt 477 <210> 71 <211> 533 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(533) <223> n = A,T,C or G
<400> 71 agagctataggtacagtgtgatctcagctttgcaaacacattttctacatagatagtact60 aggtattaatagatatgtaaagaaagaaatcacaccattaataatggtaagattggttta120 tgtgattttagtggtatttttggcacccttatatatgttttccaaactttcagcagtgat180 attatttccataacttaaaaagtgagtttgaaaaagaaaatctccagcaagcatctcatt240 taaataaaggtttgtcatctttaaaaatacagcaatatgtgactttttaaaaaagctgtc300 aaataggtgtgaccctactaataattattagaaatacatttaaaaacatcgagtacctca360 agtcagtttgccttgaaaaatatcaaatataactcttagagaaatgtacataaaagaatg420 cttcgtaattttggagtangaggttccctcctcaattttgtatttttaaaaagtacatgg480 taaaaaaaaaaattcacaacagtatataaggctgtaaaatgaagaattctgcc 533 <210> 72 <211> 511 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(511) <223> n = A,T,C or G
<400> 72 tattacggaaaaacacaccacataattcaactancaaagaanactgcttcagggcgtgta60 aaatgaaaggcttccaggcagttatctgattaaagaacactaaaagagggacaaggctaa120 aagccgcaggatgtctacactatancaggcgctatttgggttggctggaggagctgtgga180 aaacatgganagattggtgctgganatcgccgtggctattcctcattgttattacanagt240 gaggttctctgtgtgcccactggtttgaaaaccgttctncaataatgatagaatagtaca300 cacatgagaactgaaatggcccaaacccagaaagaaagcccaactagatcctcagaanac360 gcttctagggacaataaccgatgaagaaaagatggcctccttgtgcccccgtctgttatg420 atttctctccattgcagcnanaaacccgttcttctaagcaaacncaggtgatgatggcna480 aaatacaccccctcttgaagnaccnggagga 511 <210> 73 <211> 499 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(499) <223> n = A,T,C or G
<400>

cagtgccagcactggtgccagtaccagtaccaataacagtgccagtgccagtgccagcac60 cagtggtggcttcagtgctggtgccagcctgaccgccactctcacatttgggctcttcgc120 tggccttggtggagctggtgccagcaccagtggcagctctggtgcctgtggtttctccta180 caagtgagattttagatattgttaatcctgccagtctttctcttcaagccagggtgcatc240 ctcagaaacctactcaacacagcactctaggcagccactatcaatcaattgaagttgaca300 ctctgcattaaatctatttgccatttctgaaaaaaaaaaaaaaaaaagggcggccgctcg360 antctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctanttgccagc420 catctgttgtttgcccctcccccgntgccttccttgaccctggaaagtgccactcccact480 gtcctttcctaantaaaat 499 <210> 74 <211> 537 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(537) <223> n = A,T,C or G
<400> 74 tttcataggagaacacactgaggagatacttgaagaatttggattcagccgcgaagagat60 ttatcagcttaactcagataaaatcattgaaagtaataaggtaaaagctagtctctaact120 tccaggcccacggctcaagtgaatttgaatactgcatttacagtgtagagtaacacataa180 cattgtatgcatggaaacatggaggaacagtattacagtgtcctaccactctaatcaaga240 aaagaattacagactctgattctacagtgatgattgaattctaaaaatggtaatcattag300 ggcttttgatttataanactttgggtacttatactaaattatggtagttatactgccttc360 cagtttgcttgatatatttgttgatattaagattcttgacttatattttgaatgggttct420 actgaaaaangaatgatatattcttgaagacatcgatatacatttatttacactcttgat480 tctacaatgtagaaaatgaaggaaatgccccaaattgtatggtgataaaagtcccgt 537 2~
<210> 75 <211> 467 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(467) <223> n = A,T,C or G
<400> 75 caaanacaattgttcaaaagatgcaaatgatacactactgctgcagctcacaaacacctc60 tgcatattacacgtacctcctcctgctcctcaagtagtgtggtctattttgccatcatca120 cctgctgtctgcttagaagaacggctttctgctgcaanggagagaaatcataacagacgg180 tggcacaaggaggccatcttttcctcatcggttattgtccctagaagcgtcttctgagga240 tctagttgggctttctttctgggtttgggccatttcanttctcatgtgtgtactattcta300 tcattattgtataacggttttcaaaccngtgggcacncagagaacctcactctgtaataa360 caatgaggaatagccacggtgatctccagcaccaaatctctccatgttnttccagagctc420 ctccagccaacccaaatagccgctgctatngtgtagaacatccctgn 467 <210> 76 <211> 400 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(400) <223> n = A,T,C or G
<400> 76 aagctgacagcattcgggccgagatgtctcgctccgtggccttagctgtgctcgcgctac60 tctctctttctggcctggaggctatccagcgtactccaaagattcaggtttactcacgtc120 atccagcagagaatggaaagtcaaatttcctgaattgctatgtgtctgggtttcatccat180 ccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcag240 acttgtctttcagcaaggactggtctttctatctcttgtactacactgaattcaccccca300 ctgaaaaagatgagtatgcctgccgtgtgaaccatgtgactttgtcacagcccaagatng360 ttnagtgggatcganacatgtaagcagcancatgggaggt 400 I
<210> 77 <211> 248 <212> DNA

<213> Homo sapien <400> 77 ctggagtgccttggtgtttcaagcccctgcaggaagcagaatgcaccttctgaggcacct60 ccagctgccccggcgggggatgcgaggctcggagcacccttgcccggctgtgattgctgc120 caggcactgttcatctcagcttttctgtccctttgctcccggcaagcgcttctgctgaaa180 gttcatatctggagcctgatgtcttaacgaataaaggtcccatgctccacccgaaaaaaa240 aaaaaaaa 248 <210> 78 <211> 201 <212> DNA
<213> Homo sapien <400> 78 actagtccag tgtggtggaa ttccattgtg ttgggcccaa cacaatggct acctttaaca 60 tcacccagac cccgccctgc ccgtgcccca cgctgctgct aacgacagta tgatgcttac 120 tctgctactc ggaaactatt tttatgtaat taatgtatgc tttcttgttt ataaatgcct 180 gatttaaaaa aaaaaaaaaa a 201 <210> 79 <211> 552 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(552) <223> n = A,T,C or G
<400>

tccttttgttaggtttttgagacaaccctagacctaaactgtgtcacagacttctgaatg60 tttaggcagtgctagtaatttcctcgtaatgattctgttattactttcctattctttatt120 cctctttcttctgaagattaatgaagttgaaaattgaggtggataaatacaaaaaggtag180 tgtgatagtataagtatctaagtgcagatgaaagtgtgttatatatatccattcaaaatt240 atgcaagttagtaattactcagggttaactaaattactttaatatgctgttgaacctact300 ctgttccttggctagaaaaaattataaacaggactttgttagtttgggaagccaaattga360 taatattctatgttctaaaagttgggctatacataaantatnaagaaatatggaatttta420 ttcccaggaatatggggttcatttatgaatantacccggganagaagttttgantnaaac480 cngttttggttaatacgttaatatgtcctnaatnaacaaggcntgacttatttccaaaaa540 aaaaaaaaaaas 552 <210> 80 <211> 476 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(476) <223> n = A,T,C or G
<400>

acagggatttgagatgctaaggccccagagatcgtttgatccaaccctcttattttcaga60 ggggaaaatggggcctagaagttacagagcatctagctggtgcgctggcacccctggcct120 cacacagactcccgagtagctgggactacaggcacacagtcactgaagcaggccctgttt180 gcaattcacgttgccacctccaacttaaacattcttcatatgtgatgtccttagtcacta240 aggttaaactttcccacccagaaaaggcaacttagataaaatcttagagtactttcatac300 tcttctaagtcctcttccagcctcactttgagtcctccttgggggttgataggaantntc360 tcttggctttctcaataaaatctctatccatctcatgtttaatttggtacgcntaaaaat420 gctgaaaaaattaaaatgttctggtttcnctttaaaaaaaaaaaaaaaaaaaaaaa 476 <210> 81 <211> 232 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(232) <223> n = A,T,C or G
<400> 81 tttttttttg tatgccntcn ctgtggngtt attgttgctg ccaccctgga ggagcccagt 60 ttcttctgta tctttctttt ctgggggatc ttcctggctc tgcccctcca ttcccagcct 120 ctcatcccca tcttgcactt ttgctagggt tggaggcgct ttcctggtag cccctcagag 180 actcagtcag cgggaataag tcctaggggt ggggggtgtg gcaagccggc ct 232 <210> 82 <211> 383 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(383) <223> n = A,T,C or G
<400> 82 aggcgggagcagaagctaaagccaaagcccaagaagagtggcagtgccagcactggtgcc60 agtaccagtaccaataacatgccagtgccagtgccagcaccagtggtggcttcagtgctg120 gtgccagcctgaccgc'cactctcacatttgggctcttcgctggccttggtggagctggtg180 ccagcaccagtggcagctctggtgcctgtggtttctcctacaagtgagattttagatatt240 gttaatcctgccagtctttctcttcaagccagggtgcatcctcagaaacctactcaacac300 agcactctnggcagccactatcaatcaattgaagttgacactctgcattaaatctatttg360 ccatttcaaaaaaaaaaaaaaaa 383 <210> 83 <211> 494 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(494) <223> n = A,T,C or G
<400>

accgaattgggaccgctggcttataagcgatcatgtcctccagtattacctcaacgagca60 gggagatcgagtctatacgctgaagaaatttgacccgatgggacaacagacctgctcagc120 ccatcctgctcggttctccccagatgacaaatactctcgacaccgaatcaccatcaagaa180 acgcttcaaggtgctcatgacccagcaaccgcgccctgtcctctgagggtccttaaactg240 atgtcttttctgccacctgttacccctcggagactccgtaaccaaactcttcggactgtg300 agccctgatgcctttttgccagccatactctttggcntccagtctctcgtggcgattgat360 tatgcttgtgtgaggcaatcatggtggcatcacccatnaagggaacacatttganttttt420 tttcncatattttaaattacnaccagaatanttcagaataaatgaattgaaaaactctta480 aaaaaaaaaaaaaa 494 <210> 84 <211> 380 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(380) <223> n = A,T,C or G
<400> 84 gctggtagcc tatggcgtgg ccacggangg gctcctgagg cacgggacag tgacttccca 60 agtatcctgc gccgcgtctt ctaccgtccc tacctgcaga tcttcgggca gattccccag 120 gaggacatggacgtggccctcatggagcacagcaactgctcgtcggagcccggcttctgg180 gcacaccctcctggggcccaggcgggcacctgcgtctcccagtatgccaactggctggtg240 gtgctgctcctcgtcatcttcctgctcgtggccaacatcctgctggtcacttgctcattg300 ccatgttcagttacacattcggcaaagtacagggcaacagcnatctctactgggaaggcc360 agcgttnccgcctcatccgg 380 <210> 85 <211> 481 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(481) <223> n = A,T,C or G
<400>

gagttagctcctccacaaccttgatgaggtcgtctgcagtggcctctcgcttcataccgc60 tnccatcgtcatactgtaggtttgccaccacctcctgcatcttggggcggctaatatcca120 ggaaactctcaatcaagtcaccgtcnatnaaacctgtggctggttctgtcttccgctcgg180 tgtgaaaggatctccagaaggagtgctcgatcttccccacacttttgatgactttattga240 gtcgattctgcatgtccagcaggaggttgtaccagctctctgacagtgaggtcaccagcc300 ctatcatgccnttgaacgtgccgaagaacaccgagccttgtgtggggggtgnagtctcac360 ccagattctgcattaccaganagccgtggcaaaaganattgacaactcgcccaggnngaa420 aaagaacacctcctggaagtgctngccgctcctcgtccnttggtggnngcgcntnccttt480 t 481 <210> 86 <211> 472~~
<212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(472) <223> n = A,T,C or G
<400> 86 aacatcttcctgtataatgctgtgtaatatcgatccgatnttgtctgctgagaattcatt60 acttggaaaagcaacttnaagcctggacactggtattaaaattcacaatatgcaacactt120 taaacagtgtgtcaatctgctcccttactttgtcatcaccagtctgggaataagggtatg180 ccctattcacacctgttaaaagggcgctaagcatttttgattcaacatctttttttttga240 cacaagtccgaaaaaagcaaaagtaaacagttnttaatttgttagccaattcactttctt300 catgggacagagccatttgatttaaaaagcaaattgcataatattgagctttgggagctg360 atatntgagcggaagantagcctttctacttcaccagacacaactcctttcatattggga420 tgttnacnaaagttatgtctcttacagatgggatgcttttgtggcaattctg 472 <210> 87 <211> 413 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(413) <223> n = A,T,C or G
<400> 87 agaaaccagt atctctnaaa acaacctctc ataccttgtg gacctaattt tgtgtgcgtg 60 tgtgtgtgcg cgcatattat atagacaggc acatcttttt tacttttgta aaagcttatg 120 cctctttggt atctatatct gtgaaagttt taatgatctg ccataatgtc ttggggacct 180 ttgtcttctg tgtaaatggt actagagaaa acacctatnt tatgagtcaa tctagttngt 240 tttattcgac atgaaggaaa tttccagatn acaacactna caaactctcc cttgactagg 300 ggggacaaag aaaagcanaa ctgaacatna gaaacaattn cctggtgaga aattncataa 360 acagaaattg ggtngtatat tgaaananng catcattnaa acgttttttt ttt 413 <210> 88 <211> 448 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(4480 <223> n = A,T,C or G
<400>

cgcagcgggtcctctctatctagctccagcctctcgcctgccccactccccgcgtcccgc60 gtcctagccnaccatggccgggcccctgcgcgccccgctgctcctgctggccatcctggc120 cgtggccctggccgtgagccccgcggccggctccagtcccggcaagccgccgcgcctggt180 gggaggcccatggaccccgcgtggaagaagaaggtgtgcggcgtgcactggactttgccg240 tcggcnantacaacaaacccgcaacnacttttaccriagcncgcgctgcaggttgtgccgc300 cccaancaaattgttactnggggtaantaattcttggaagttgaacctgggccaaacnng360 tttaccagaaccnagccaattngaacaattncccctccataacagccccttttaaaaagg420 gaancantcctgntcttttccaaatttt 448 <210> 89 <211> 463 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(463) <223> n = A,T,C or G
<400>

gaattttgtgcactggccactgtgatggaaccattgggccaggatgctttgagtttatca60 gtagtgattctgccaaagttggtgttgtaacatgagtatgtaaaatgtcaaaaaattagc120 agaggtctaggtctgcatatcagcagacagtttgtccgtgtattttgtagccttgaagtt180 ctcagtgacaagttnnttctgatgcgaagttctnattccagtgttttagtcctttgcatc240 tttnatgttnagacttgcctctntnaaattgcttttgtnttctgcaggtactatctgtgg300 tttaacaaaatagaannacttctctgcttngaanatttgaatatcttacatctnaaaatn360 aattctctccccatannaaaacccangcccttggganaatttgaaaaanggntccttcnn420 aattcnnanaanttcagntntcatacaacanaacnggancccc 463 <210> 90 <211> 400 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(400) <223> n = A,T,C or G

<400> 90 agggattgaa ggtctnttnt actgtcggac tgttcancca ccaactctac aagttgctgt 60 cttccactca ctgtctgtaa gcntnttaac ccagactgta tcttcataaa tagaacaaat 120 tcttcaccag tcacatcttc taggaccttt ttggattcag ttagtataag ctcttccact 180 tcctttgtta agacttcatc tggtaaagtc ttaagttttg tagaaaggaa tttaattgct 240 cgttctctaa caatgtcctc tccttgaagt atttggctga acaacccacc tnaagtccct 300 ttgtgcatcc attttaaata tacttaatag ggcattggtn cactaggtta aattctgcaa 360 gagtcatctg tctgcaaaag ttgcgttagt atatctgcca 400 <210> 91 <211> 480 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(480) <223> n = A,T,C or G
<400>

gagctcggatccaataatctttgtctgagggcagcacacatatncagtgccatggnaact60 ggtctaccccacatgggagcagcatgccgtagntatataaggtcattccctgagtcagac120 atgcctctttgactaccgtgtgccagtgctggtgattctcacacacctccnnccgctctt180 tgtggaaaaactggcacttgnctggaactagcaagacatcacttacaaattcacccacga240 gacacttgaaaggtgtaacaaagcgactcttgcattgctttttgtccctccggcaccagt300 tgtcaatactaacccgctggtttgcctccatcacatttgtgatctgtagctctggataca360 tctcctgacagtactgaagaacttcttcttttgtttcaaaagcaactcttggtgcctgtt420 ngatcaggttcccatttcccagtccgaatgttcacatggcatatnttacttcccacaaaa480 <210> 92 <211> 477 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(477) <223> n = A,T,C or G
<400>

atacagcccanatcccaccacgaagatgcgcttgttgactgagaacctgatgcggtcact60 ggtcccgctgtagccccagcgactctccacctgctggaagcggttgatgctgcactcctt120 cccacgcaggcagcagcggggccggtcaatgaactccactcgtggcttggggttgacggt180 taantgcagg~aagaggctgaccacctcgcggtccaccaggatgcccgactgtgcgggacc240 tgcagcgaaactcctcgatggtcatgagcgggaagcgaatgangcccagggccttgccca300 gaaccttccgcctgttctctggcgtcacctgcagctgctgccgctnacactcggcctcgg360 accagcggacaaacggcgttgaacagccgcacctcacggatgcccantgtgtcgcgctcc420 aggaacggcnccagcgtgtccaggtcaatgtcggtgaancctccgcgggtaatggcg 477 <210> 93 <211> 377 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (377) <223> n = A,T,C or G

<400> 93 gaacggctggaccttgcctcgcattgtgctgctggcaggaataccttggcaagcagctcc60 agtccgagcagccccagaccgctgccgcccgaagctaagcctgcctctggccttcccctc120 cgcctcaatgcagaaccantagtgggagcactgtgtttagagttaagagtgaacactgtn180 tgattttacttgggaatttcctctgttatatagcttttcccaatgctaatttccaaacaa240 caacaacaaaataacatgtttgcctgttnagttgtataaaagtangtgattctgtatnta300 aagaaaatattactgttacatatactgcttgcaanttctgtatttattggtnctctggaa360 ataaatatattattaaa 377 <210> 94 <211> 495 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(495) <223> n = A,T,C or G
<400>

ccctttgaggggttagggtccagttcccagtggaagaaacaggccaggagaantgcgtgc60 cgagctgangcagatttcccacagtgaccccagagccctgggctatagtctctgacccct120 ccaaggaaagaccaccttctggggacatgggctggagggcaggacctagaggcaccaagg180 gaaggccccattccggggctgttccccgaggaggaagggaaggggctctgtgtgcccccc240 acgaggaanaggccctgantcctgggatcanacaccccttcacgtgtatccccacacaaa300 tgcaagctcaccaaggtcccctctcagtcccttccctacacoctgaacggncactggccc360 acacccacccagancanccacccgccatggggaatgtnctcaaggaatcgcngggcaacg420 tggactctngtcccnnaagggggcagaatctccaatagangganngaacccttgctnana480 aaaaaaaanaaaaaa 495 <210> 95 <211> 472 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(472) <223> n = A,T,C or G
<400> 95 ggttacttggtttcattgccaccacttagtggatgtcatttagaaccattttgtctgctc60 cctctggaagccttgcgcagagcggactttgtaattgttggagaataactgctgaatttt120 tagctgttttgagttgattcgcaccactgcaccacaactcaatatgaaaactatttnact180 tatttattatcttgtgaaaagtatacaatgaaaattttgttcatactgtatttatcaagt240 atgatgaaaagcaatagatatatattcttttattatgttnaattatgattgccattatta300 atcggcaaaatgtggagtgtatgttcttttcacagtaatatatgccttttgtaacttcac360 ttggttattttattgtaaatgaattacaaaattcttaatttaagaaaatggtangttata420 tttanttcantaatttctttccttgtttacgttaattttgaaaagaatgcat 472 <210> 96 <211> 476 <212> DNA
<213> Homo sapien <220>
<221> misc feature <222> (1)...(476) <223> n = A,T,C or G
<400>

ctgaagcatttcttcaaacttntctacttttgtcattgatacctgtagtaagttgacaat60 gtggtgaaatttcaaaattatatgtaacttctactagttttactttctcccccaagtctt120 ttttaactcatgatttttacacacacaatccagaacttattatatagcctctaagtcttt180 attcttcacagtagatgatgaaagagtcctccagtgtcttgngcanaatgttctagntat240 agctggatacatacngtgggagttctataaactcatacctcagtgggactnaaccaaaat300 tgtgttagtctcaattcctaccacactgagggagcctcccaaatcactatattcttatct360 gcaggtactcctccagaaaaacngacagggcaggcttgcatgaaaaagtnacatctgcgt420 tacaaagtctatcttcctcanangtctgtnaaggaacaatttaatcttctagcttt 476 <210> 97 <211> 479 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(479) <223> n = A,T,C or G
<400>

actctttctaatgctgatatgatcttgagtataagaatg,catatgtcactagaatggata60 aaataatgctgcaaacttaatgttcttatgcaaaatggaacgctaatgaaacacagctta120 caatcgcaaatcaaaactcacaagtgctcatctgttgtagatttagtgtaataagactta180 gattgtgctccttcggatatgattgtttctcanatcttgggcaatnttccttagtcaaat240 caggctactagaattctgttattggatatntgagagcatgaaatttttaanaatacactt300 gtgattatnaaattaatcacaaatttcacttatacctgctatcagcagctagaaaaacat360 ntnntttttanatcaaagtattttgtgtttggaantgtnnaaatgaaatctgaatgtggg420 ttcnatcttattttttcccngacnactanttncttttttagggnctattctganccatc 479 <210> 98 <211> 461 <212> DNA
<213> Homo sapien <400> 98 agtgacttgtcctccaacaaaaccccttgatcaagtttgtggcactgacaatcagaccta60 tgctagttcctgtcatctattcgctactaaatgcagactggaggggaccaaaaaggggca120 tcaactccagctggattattttggagcctgcaaatctattcctacttgtacggactttga180 agtgattcagtttcctctacggatgagagactggctcaagaatatcctcatgcagcttta240 tgaagccactctgaacacgctggttatctagatgagaacagagaaataaagtcagaaaat300 ttacctggagaaaagaggctttggctggggaccatcccattgaaccttctcttaaggact360 ttaagaaaaactaccacatgttgtgtatcctggtgccggccgtttatgaactgaccaccc420 tttggaataatcttgacgctcctgaacttgctcctctgcga 461 <210> 99 <211> 171 <212> DNA
<213> Homo sapien <400> 99 gtggccgcgc gcaggtgttt cctcgtaccg cagggccccc tcccttcccc aggcgtccct 60 cggcgcctct gcgggcccga ggaggagcgg ctggcgggtg gggggagtgt gacccaccct 120 cggtgagaaa agccttctct agcgatctga gaggcgtgcc ttgggggtac c 171 <210> 100 <211> 269 <212> DNA
<213> Homo sapien <400> 100 cggccgcaagtgcaactccagctggggccgtgcggacgaagattctgccagcagttggtc60 cgactgcgacgacggcggcggcgacagtcgcaggtgcagcgcgggcgcctggggtcttgc120 aaggctgagctgacgccgcagaggtcgtgtcacgtcccacgaccttgacgccgtcgggga180 cagccggaacagagcccggtgaagcgggaggcctcggggagcccctcgggaagggcggcc240 cgagagatacgcaggtgcaggtggccgcc 269 <210> 101 <211> 405 <212> DNA
<213> Homo sapien <400>

ttttttttttttttggaatctactgcgagcacagcaggtcagcaacaagtttattttgca60 gctagcaaggtaacagggtagggcatggttacatgttcaggtcaacttcctttgtcgtgg120 ttgattggtttgtctttatgggggcggggtggggtaggggaaacgaagcaaataacatgg180 agtgggtgcaccctccctgtagaacctggttacaaagcttggggcagttcacctggtctg240 tgaccgtcattttcttgacatcaatgttattagaagtcaggatatcttttagagagtcca300 ctgttctggagggagattagggtttcttgccaaatccaacaaaatccactgaaaaagttg360 gatgatcagtacgaataccgaggcatattctcatatcggtggcca 405 <210> 102 <211> 470 <212> DNA
<213> Homo sapien <400>

tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt60 ggcacttaatccatttttatttcaaaatgtctacaaatttaatcccattatacggtattt120 tcaaaatctaaattattcaaattagccaaatccttaccaaataatacccaaaaatcaaaa180 atatacttctttcagcaaacttgttacataaattaaaaaaatatatacggctggtgtttt240 caaagtacaattatcttaacactgcaaacattttaaggaactaaaataaaaaaaaacact300 ccgcaaaggttaaagggaacaacaaattcttttacaacaccattataaaaatcatatctc360 aaatcttaggggaatatatacttcacacgggatcttaacttttactcactttgtttattt420 ttttaaaccattgtttgggcccaacacaatggaatcccccctggactagt 470 <210> 103 <211> 581 <2l2> DNA
<213> Homo sapien <400> 103 ttttttttttttttttttgacccccctcttataaaaaacaagttaccattttattttact60 tacacatatttattttataattggtattagatattcaaaaggcagcttttaaaatcaaac120 taaatggaaactgccttagatacataattcttaggaattagcttaaaatctgcctaaagt180 gaaaatcttctctagctcttttgactgtaaatttttgactcttgtaaaacatccaaattc240 atttttcttgtctttaaaattatctaatctttccattttttccctattccaagtcaattt300 gcttctctagcctcatttcctagctcttatctactattagtaagtggcttttttcctaaa360 agggaaaacaggaagagaaatggcacacaaaacaaacattttatattcatatttctacct420 acgttaataaaatagcattttgtgaagccagctcaaaagaaggcttagatccttttatgt480 ccattttagtcactaaacgatatcaaagtgccagaatgcaaaaggtttgtgaacatttat540 tcaaaagctaatataagatatttcacatactcatctttctg 581 <210> 104 <211> 578 <212> DNA
<213> Homo sapien <400> 104 tttttttttttttttttttttttttctcttctttttttttgaaatgaggatcgagttttt60 cactctctagatagggcatgaagaaaactcatctttccagctttaaaataacaatcaaat120 ctcttatgctatatcatattttaagttaaactaatgagtcactggcttatcttctcctga180 aggaaatctgttcattcttctcattcatatagttatatcaagtactaccttgcatattga240 gaggtttttcttctctatttacacatatatttccatgtgaatttgtatcaaacctttatt300 ttcatgcaaactagaaaataatgtttcttttgcataagagaagagaacaatatagcatta360 caaaactgctcaaattgtttgttaagttatccattataattagttggcaggagctaatac420 aaatcacatttacgacagcaataataaaactgaagtaccagttaaatatccaaaataatt480 aaaggaacatttttagcctgggtataattagctaattcactttacaagcatttattagaa540 tgaattcacatgttattattcctagcccaacacaatgg 578 <210> 105 <211> 538 <212> DNA
<213> Homo sapien <400> 105 tttttttttttttttcagtaataatcagaacaatatttatttttatatttaaaattcata60 gaaaagtgccttacatttaataaaagtttgtttctcaaagtgatcagaggaattagatat120 gtcttgaacaccaatattaatttgaggaaaatacaccaaaatacattaagtaaattattt180 aagatcatagagcttgtaagtgaaaagataaaatttgacctcagaaactctgagcattaa240 aaatccactattagcaaataaattactatggacttcttgctttaattttgtgatgaatat300 ggggtgtcactggtaaaccaacacattctgaaggatacattacttagtgatagattctta360 tgtactttgctaatacgtggatatgagttgacaagtttctctttcttcaatcttttaagg420 ggcgagaaatgaggaagaaaagaaaaggattacgcatactgttctttctatggaaggatt480 agatatgtttcctttgccaatattaaaaaaataataatgtttactactagtgaaaccc 538 <210> 106 <211> 473 <212> DNA
<213> Homo sapien <400> 106 ttttttttttttttttagtcaagtttctatttttattataattaaagtcttggtcatttc60 atttattagctctgcaacttacatatttaaattaaagaaacgttttagacaactgtacaa120 tttataaatgtaaggtgccattattgagtaatatattcctccaagagtggatgtgtccct180 tctcccaccaactaatgaacagcaacattagtttaattttattagtagatatacactgct240 gcaaacgctaattctcttctccatccccatgtgatattgtgtatatgtgtgagttggtag300 aatgcatcacaatctacaatcaacagcaagatgaagctaggctgggctttcggtgaaaat360 agactgtgtctgtctgaatcaaatgatctgacctatcctcggtggcaagaactcttcgaa420 ccgcttcctcaaaggcgctgccacatttgtggctctttgcacttgtttcaaaa 473 <210> 107 <211> 1621 <222> DNA
<213> Homo sapien <400> 107 cgccatggca ctgcagggca tctcggtcat ggagctgtcc ggcctggccc cgggcccgtt 60 ctgtgctatg gtcctggctg acttcggggc gcgtgtggta cgcgtggacc ggcccggctc 120 ccgctacgac gtgagccgct tgggccgggg caagcgctcg ctagtgctgg acctgaagca 180 gccgcgggga gccgccgtgc tgcggcgtct gtgcaagcgg tcggatgtgc tgctggagcc 240 3~
cttccgccgcggtgtcatggagaaactccagctgggcccagagattctgcagcgggaaaa300 tccaaggcttatttatgccaggctgagtggatttggccagtcaggaagcttctgccggtt360 agctggccacgatatcaactatttggctttgtcaggtgttctctcaaaaattggcagaag420 tggtgagaatccgtatgccccgctgaatctcctggctgactttgctggtggtggccttat480 gtgtgcactgggcattataatggctctttttgaccgcacacgcactgacaagggtcaggt540 cattgatgcaaatatggtggaaggaacagcatatttaagttcttttctgtggaaaactca600 gaaatcgagtctgtgggaagcacctcgaggacagaacatgttggatggtggagcaccttt660 ctatacgacttacaggacagcagatggggaattcatggctgttggagcaatagaacccca720 gttctacgagctgctgatcaaaggacttggactaaagtctgatgaacttbccaatcagat780 gagcatggatgattggccagaaatgaagaagaagtttgcagatgtatttgcaaagaagac840 gaaggcagagtggtgtcaaatctttgacggcacagatgcctgtgtgactccggttctgac900 ttttgaggaggttgttcatcatgatcacaacaaggaacggggctcgtttatcaccagtga960 ggagcaggacgtgagcccccgccctgcacctctgctgttaaacaccccagccatcccttc1020 tttcaaaagggatcctttcataggagaacacactgaggagatacttgaagaatttggatt1080 cagccgcgaagagatttatcagcttaactcagataaaatcattgaaagtaataaggtaaa1140 agctagtctctaacttccaggcccacggctcaagtgaatttgaatactgcatttacagtg1200 tagagtaacacataacattgtatgcatggaaacatggaggaacagtattacagtgtccta1260 ccactctaatcaagaaaagaattacagactctgattctacagtgatgattgaattctaaa1320 aatggttatcattagggcttttgatttataaaactttgggtacttatactaaattatggt1380 agttattctgccttccagtttgcttgatatatttgttgatattaagattcttgacttata1440 ttttgaatgggttctagtgaaaaaggaatgatatattcttgaagacatcgatatacattt1500 atttacactcttgattctacaatgtagaaaatgaggaaatgccacaaattgtatggtgat1560 aaaagtcacgtgaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa1620 a 16'21 <210> 108 <211> 382 <212> PRT
<213> Homo sapien <400> 108 Met Ala Leu Gln Gly Ile Ser Val Met Glu Leu Ser Gly Leu Ala Pro Gly Pro Phe Cys Ala Met Val Leu Ala Asp Phe Gly Ala Arg Val Val ~ 20 25 30 Arg Val Asp Arg Pro Gly Ser Arg Tyr Asp Val Ser Arg Leu Gly Arg Gly Lys Arg Ser Leu Val Leu Asp Leu Lys Gln Pro Arg Gly Ala Ala Val Leu Arg Arg Leu Cys Lys Arg Ser..Asp Val Leu Leu Glu Pro Phe Arg Arg Gly Val Met Glu Lys Leu Gln Leu Gly Pro G1u Ile Leu Gln Arg Glu Asn Pro Arg Leu Ile Tyr Ala Arg Leu Ser Gly Phe Gly Gln Ser Gly Ser Phe Cys Arg Leu Ala Gly His Asp Ile Asn Tyr Leu Ala Leu Ser Gly Val Leu 5er Lys Ile Gly Arg Ser Gly Glu Asn Pro Tyr Ala Pro Leu Asn Leu Leu Ala Asp Phe Ala Gly Gly Gly Leu Met Cys Ala Leu Gly Ile Ile Met Ala Leu Phe Asp Arg Thr Arg Thr Asp Lys Gly Gln Val Ile Asp Ala Asn Met Val Glu Gly Thr Ala Tyr Leu Ser Ser Phe Leu Trp Lys Thr Gln Lys Ser Ser Leu Trp Glu Ala Pro Arg Gly Gln Asn Met Leu Asp Gly Gly Ala Pro Phe Tyr Thr Thr Tyr Arg Thr Ala Asp Gly Glu Phe Met Ala Val Gly Ala Ile Glu Pro Gln Phe Tyr Glu Leu Leu Ile Lys Gly Leu Gly Leu Lys Ser Asp Glu Leu Pro Asn Gln Met Ser Met Asp Asp Trp Pro Glu Met Lys Lys Lys Phe Ala Asp Val Phe Ala Lys Lys Thr Lys Ala Glu Trp Cys Gln Ile Phe Asp Gly Thr Asp Ala Cys Val Thr Pro Val Leu Thr Phe Glu Glu Val Val His His Asp His Asn Lys Glu Arg Gly Ser Phe Ile Thr Ser Glu Glu Gln Asp Val Ser Pro Arg Pro Ala Pro Leu Leu Leu Asn Thr Pro Ala Ile Pro Ser Phe Lys Arg Asp Pro Phe Ile Gly Glu His Thr Glu Glu Ile Leu Glu Glu Phe Gly Phe Ser Arg Glu Glu Ile Tyr Gln Leu Asn Ser Asp Lys Ile Ile Glu Ser Asn Lys Val Lys Ala Ser Leu <210> 109 <211> 1524 <212> DNA
<213> Homo sapien <400>

ggcacgaggctgcgccagggcctgagcggaggcgggggcagcctcgccagcgggggcccc60 gggcctggccatgcctcactgagccagcgcctgcgcctctacctcgccgacagctggaac120 cagtgcgacctagtggctctcacctgcttcctcctgggcgtgggctgccggctgaccccg180 ggtttgtaccacctgggccgcactgtcctctgcatcgacttcatggttttcacggtgcgg240 ctgcttcacatcttcacggtcaacaaacagctggggcccaagatcgtcatcgtgagcaag300 atgatgaaggacgtgttcttcttcctcttcttcctcggcgtgtggctggtagcctatggc360 gtggccacggaggggctcctgaggccacgggacagtgacttcccaagtatcctgcgccgc420 gtcttctaccgtccctacctgcagatcttcgggcagattccccaggaggacatggacgtg480 gccctcatggagcacagcaactgctcgtcggagcccggcttctgggcacaccctcctggg540 gcccaggcgggcacctgcgtctcccagtatgccaactggctggtggtgctgctcctcgtc' atcttcctgctcgtggccaacatcctgctggtcaacttgctcattgccatgttcagttac660 acattcggcaaagtacagggcaacagcgatctctactggaaggcgcagcgttaccgcctc720 atccgggaattccactctcggcccgcgctggccccgccctttatcgtcatctcccacttg780 cgcctcctgctcaggcaattgtgcaggcgaccccggagcccccagccgtcctccccggcc840 ctcgagcatttccgggtttacctttctaaggaagccgagcggaagctgctaacgtgggaa900 tcggtgcataaggagaactttctgctggcacgcgctagggacaagcgggagagcgactcc960 gagcgtotgaagcgcacgtcccagaaggtggacttggcactgaaacagctgggacacatc1020 cgcgagtacgaacagcgcctgaaagtgctggagcgggaggtccagcagtgtagccgcgtc1080 ctggggtgggtggccgaggccctgagccgctctgccttgctgcccccaggtgggccgcca1140 ccccctgacctgcctgggtccaaagactgagccctgctggcggacttcaaggagaagccc1200 ccacaggggattttgctcctagagtaaggctcatctgggcctcggcccccgcacctggtg1260 gccttgtccttgaggtgagccccatgtccatctgggccactgtcaggaccacctttggga1320 gtgtcatccttacaaaccacagcatgcccggctcctcccagaaccagtcccagcctggga1380 ggatcaaggcctggatcccgggccgttatccatctggaggctgcagggtccttggggtaa1440 cagggaccacagacccctcaccactcacagattcctcacactggggaaataaagccattt1500 cagaggaaaaaaaaaaaaaaaaaa 1524 <210> 110 <211> 3410 <212> DNA

<213> Homo sapien <400>

gggaaccagcctgcacgcgctggctccgggtgacagccgcgcgcctcggccaggatctga60 gtgatgagacgtgtccccactgaggtgccccacagcagcaggtgttgagcatgggctgag120 aagctggaccggcaccaaagggctggcagaaatgggcgcctggctgattcctaggcagtt180 ggcggcagcaaggaggagaggccgcagcttctggagcagagccgagacgaagcagttctg240 gagtgcctgaacggccccctgagccctacccgcctggcccactatggtccagaggctgtg300 ggtgagccgcctgctgcggcaccggaaagcccagctcttgctggtcaacctgctaacctt360 tggcctggaggtgtgtttggccgcaggcatcacctatgtgccgcctctgctgctggaagt420 gggggtagaggagaagttcatgaccatggtgctgggcattggtccagtgctgggcctggt480 ctgtgtcccgctcctaggctcagccagtgaccactggcgtggacgctatggccgccgccg540 gcccttcatctgggcactgtccttgggcatcctgctgagcctctttctcatcccaagggc600 cggctggctagcagggctgctgtgcccggatcccaggcccctggagctggcactgctcat660 cctgggcgtggggctgctggacttctgtggccaggtgtgcttcactccactggaggccct720 gctctctgacctcttccgggacccggaccactgtcgccaggcctactctgtctatgcctt780 catgatcagtcttgggggctgcctgggctacctcctgcctgccattgactgggacaccag840 tgccctggccccctacctgggcacccaggaggagtgcctctttggcctgctcaccctcat900 cttcctcacctgcgtagcagccacactgctggtggctgaggaggcagcgctgggccccac960 cgagccagcagaagggctgtcggccccctccttgtcgccccactgctgtccatgccgggc1020 ccgcttggctttccggaacctgggcgccctgcttccccggctgcaccagctgtgctgccg1080 catgccccgcaccctgcgccggctcttcgtggctgagctgtgcagctggatggcactcat1140 gaccttcacgctgttttacacggatttcgtgggcgaggggctgtaccagggcgtgcccag1200 agctgagccgggcaccgaggcccggagacactatgatgaaggcgttcggatgggcagcct1260 ggggctgttcctgcagtgcgccatctccctggtcttctctctggtcatggaccggctggt1320 gcagcgattcggcactcgagcagtctatttggccagtgtggcagctttccctgtggctgc1380 cggtgccacatgcctgtcccacagtgtggccgtggtgacagcttcagccgccctcaccgg1440 gttcaccttctcagccctgcagatcctgccctacacactggcctccctctaccaccggga1500 gaagcaggtgttcctgcccaaataccgaggggacactggaggtgctagcagtgaggacag1560 cctgatgaccagcttcctgccaggccctaagcctggagctcccttccctaatggacacgt1620 gggtgctggaggcagtggcctgctcccacctccacccgcgctctgcggggcctctgcctg1680 tgatgtctccgtacgtgtggtggtgggtgagcccaccgaggccagggtggttccgggccg1740 gggcatctgcctggacctcgccatcctggatagtgccttcctgctgtcccaggtggcccc1800 atccctgtttatgggctccattgtccagctcagccagtctgtcactgcctatatggtgtc1860 tgccgcaggcctgggtctggtcgccatttactttgctacacaggtagtatttgacaagag1920 cgacttggccaaatactcagcgtagaaaacttccagcacattggggtggagggcctgcct1980 cactgggtcccagctccccgctcctgttagccccatggggctgccgggctggccgccagt2040 ttctgttgctgccaaagtaatgtggctctctgctgccaccctgtgctgctgaggtgcgta2100 gctgcacagctgggggctggggcgtccctctcctctctccccagtctctagggctgcctg2160 actggaggccttccaagggggtttcagtctggacttatacagggaggccagaagggctcc2220 atgcactggaatgcggggactctgcaggtggattacccaggctcagggttaacagctagc2280 ctcctagttgagacacacctagagaagggtttttgggagctgaataaactcagtcacctg2340 gtttcccatctctaagccccttaacctgcagcttcgtttaatgtagctcttgcatgggag2400 tttctaggatgaaacactcctccatgggatttgaacatatgacttatttgtaggggaaga2460 gtcctgaggggcaacacacaagaaccaggtcccctcagcccacagcactgtctttttgct2520 gatccacccccctcttaccttttatcaggatgtggcctgttggtccttctgttgccatca2580 cagagacacaggcatttaaatatttaacttatttatttaacaaagtagaagggaatccat2640 tgctagcttttctgtgttggtgtctaatatttgggtagggtgggggatccccaacaatca2700 ggtcccctgagatagctggtcattgggctgatcattgccagaatcttcttctcctggggt2760 ctggccccccaaaatgcctaacccaggaccttggaaattctactcatcccaaatgataat2820 tccaaatgctgttacccaaggttagggtgttgaaggaaggtagagggtggggcttcaggt2880 ctcaacggcttccctaaccacccctcttctcttggcccagcctggttccccccacttcca2940 ctcccctctactctctctaggactgggctgatgaaggcactgcccaaaatttcccctacc3000 cccaactttcccctacccccaactttccccaccagctccacaaccctgtttggagctact3060 gcaggaccagaagcacaaagtgcggtttcccaagcctttgtccatctcagcccccagagt3120 atatctgtgcttggggaatctcacacagaaactcaggagcaccccctgcctgagctaagg3180 gaggtcttatctctcagggggggtttaagtgccgtttgcaataatgtcgtcttatttatt3240 tagcggggtgaatattttatactgtaagtgagcaatcagagtataatgtttatggtgaca3300 aaattaaagg ctttcttata tgtttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360 aaaaaaaara aaaaaaaaaa aaaaaaaaaa aaaaaaataa aaaaaaaaaa 3410 <210> 111 <211> 1289 <212> DNA
<213> Homo sapien <400>

agccaggcgtccctctgcctgcccactcagtggcaacacccgggagctgttttgtccttt60 gtggagcctcagcagttccctctttcagaactcactgccaagagccctgaacaggagcca120 ccatgcagtgcttcagcttcattaagaccatgatgatcctcttcaatttgctcatctttc180 tgtgtggtgcagccctgttggcagtgggcatctgggtgtcaatcgatggggcatcctttc240 tgaagatcttcgggccactgtcgtccagtgccatgcagtttgtcaacgtgggctacttcc300 tcatcgcagccggcgttgtggtctttgctcttggtttcctgggctgctatggtgctaaga360 ctgagagcaagtgtgccctcgtgacgttcttcttcatcctcctcctcatcttcattgctg420 aggttgcagctgctgtggtcgccttggtgtacaccacaatggctgagcacttcctgacgt480 tgctggtagtgcctgccatcaagaaagattatggttcccaggaagacttcactcaagtgt540 ggaacaccaccatgaaagggctcaagtgctgtggcttcaccaactatacggattttgagg600 actcaccctacttcaaagagaacagtgcctttcccccattctgttgcaatgacaacgtca660 ccaacacagccaatgaaacctgcaccaagcaaaaggctcacgaccaaaaagtagagggtt720 gcttcaatcagcttttgtatgacatccgaactaatgcagtcaccgtgggtggtgtggcag780 ctggaattgggggcctcgagctggctgccatgattgtgtccatgtatctgtactgcaatc840 tacaataagtccacttctgcctctgccactactgctgccacatgggaactgtgaagaggc900 accctggcaagcagcagtgattgggggaggggacaggatctaacaatgtcacttgggcca960 gaatggacctgccctttctgctccagacttggggctagatagggaccactccttttagcg1020 atgcctgactttccttccattggtgggtggatgggtggggggcattccagagcctctaag1080 gtagccagttctgttgcccattcccccagtctattaaacccttgatatgccccctaggcc1140 tagtggtgatcccagtgctctactgggggatgagagaaaggcattttatagcctgggcat1200 aagtgaaatcagcagagcctctgggtggatgtgtagaaggcacttcaaaatgcataaacc1260 tgttacaatgttaaaaaaaaaaaaaaaaa 1289 <210> 112 <211> 315 <212> PRT
<213> Homo sapien <400> 112 Met Val Phe Thr Val Arg Leu Zeu His Ile Phe Thr Val Asn Lys Gln Leu Gly Pro Lys Ile Val Ile Val Ser Lys Met Met Lys Asp Val Phe Phe Phe Leu Phe Phe Leu Gly Val Trp Leu Val Ala Tyr Gly Val Ala Thr Glu Gly Leu Leu Arg Pro Arg Asp Ser Asp Phe Pro Ser Ile Leu Arg Arg Val Phe Tyr Arg Pro Tyr Leu Gln Ile Phe Gly Gln Ile Pro Gln Glu Asp Met Asp Val Ala Leu Met Glu His Ser Asn Cys Ser Ser Glu Pro Gly Phe Trp~Ala His Pro Pro Gly Ala Gln Ala Gly Thr Cys Val Ser Gln Tyr A1a Asn Trp Leu Val Val Leu Leu Leu Val Ile Phe Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Met Phe Ser Tyr Thr Phe Gly Lys Val Gln Gly Asn Ser Asp Leu Tyr Trp Lys Ala Gln Arg Tyr Arg Leu I1e Arg Glu Phe His Ser Arg Pro Ala Leu Ala Pro Pro Phe Ile Val Ile Ser His Leu Arg Leu Leu Leu Arg Gln Leu Cys Arg Arg Pro Arg Ser Pro Gln Pro Ser Ser Pro Ala Leu Glu His Phe Arg Val Tyr Leu Ser Lys Glu Ala Glu Arg Lys Leu Leu Thr Trp Glu Ser Val His Lys Glu Asn Phe Leu Leu Ala Arg Ala Arg Asp Lys Arg Glu Ser Asp Ser Glu Arg Leu Lys Arg Thr Ser G1n Lys Val Asp Leu Ala Leu Lys Gln Leu G1y His Ile Arg Glu Tyr Glu Gln Arg Leu Lys Val Leu Glu Arg Glu Val Gln Gln Cys Ser Arg Val Leu Gly Trp Val Ala Glu Ala Leu Ser Arg Ser Ala Leu Leu Pro Pro Gly Gly Pro Pro Pro Pro Asp Leu Pro Gly Ser Lys Asp <210> 113 <211> 553 <212> PRT
<213> Homo sapien <400> 113 Met Val Gln Arg Leu Trp Val Ser Arg Leu Leu Arg His Arg Lys Ala Gln Leu Leu Leu Val Asn Zeu Leu Thr Phe Gly Leu Glu Val Cys Leu Ala Ala Gly Ile Thr Tyr Val Pro Pro Leu Leu Leu Glu Val Gly Val Glu Glu Lys Phe Met Thr Met Val Leu Gly Ile Gly Pro Val Leu Gly Leu Val Cys Val Pro Leu Leu Gly Ser Ala Ser Asp His Trp Arg Gly Arg Tyr Gly Arg Arg Arg Pro Phe Ile Trp Ala Leu Ser Leu Gly Ile Leu Leu Ser Leu Phe Leu Ile Pro Arg Ala Gly Trp Leu Ala Gly Leu Leu Cys Pro Asp Pro Arg Pro Leu Glu Leu Ala Leu Leu Ile Leu Gly Val Gly Leu Leu Asp Phe Cys Gly Gln Val Cys Phe Thr Pro Leu Glu Ala Leu Leu Ser Asp Leu Phe Arg Asp Pro Asp His Cys Arg Gln Ala Tyr Ser Val Tyr Ala Phe Met Ile Ser Leu Gly Gly Cys Leu Gly Tyr Leu Leu Pro Ala Ile Asp Trp Asp Thr Ser Ala Leu Ala Pro Tyr Leu Gly Thr Gln Glu Glu Cys Leu Phe Gly Leu Leu Thr Leu Ile Phe Leu Thr Cys Val Ala Ala Thr Leu Leu Val Ala Glu Glu Ala Ala Leu Gly Pro Thr Glu Pro Ala Glu Gly Leu Ser Ala Pro Ser Leu Ser Pro His Cys Cys Pro Cys Arg Ala Arg Leu Ala Phe Arg Asn Leu Gly Ala Leu Leu Pro Arg Leu His Gln Leu Cys Cys Arg Met Pro Arg Thr Leu Arg Arg Leu Phe Val Ala Glu Leu Cys Ser Trp Met Ala Leu Met Thr Phe Thr Leu Phe Tyr Thr Asp Phe Val Gly Glu Gly Leu Tyr Gln Gly Val Pro Arg Ala G1u Pro Gly Thr Glu Ala Arg Arg His Tyr Asp Glu Gly Val Arg Met Gly Ser Leu Gly Leu Phe Leu Gln Cys Ala Ile Ser Leu Val Phe Ser Leu Val Met Asp Arg Leu Val Gln Arg Phe Gly Thr Arg Ala Val Tyr Leu Ala Ser Val Ala Ala Phe Pro Val Ala Ala Gly Ala Thr Cys Leu Ser His Ser Val Ala Val Val Thr Ala Ser Ala Ala Leu Thr Gly Phe Thr Phe Ser Ala Leu Gln Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly Asp Thr Gly Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe Leu Pro Gly Pro Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val Gly Ala Gly Gly Ser Gly Leu Leu Pro Pro Pro Pro Ala Leu Cys Gly Ala Ser Ala Cys Asp Val Ser Val Arg Val Val Val Gly Glu Pro Thr Glu Ala 465 ' 470 475 480 Arg Val Val Pro Gly Arg Gly Ile Cys Leu Asp Leu A1a Ile Leu Asp Ser Ala Phe Leu Leu Ser Gln Val Ala Pro Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser Gln Ser Val Thr Ala Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val Ala Ile Tyr Phe Ala Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala Lys Tyr Ser Ala <210> 114 <211> 241 <212> PRT
<213> Homo sapien <400> 114 Met Gln Cys Phe Ser Phe Ile Lys Thr Met Met Ile Leu Phe Asn Leu 1 ' 5 10 15 Leu Ile Phe Leu Cys Gly Ala Ala Leu Leu Ala Val Gly Ile Trp Val Ser Ile Asp Gly Ala Ser Phe Leu Lys Ile Phe Gly Pro Leu Ser Ser Ser Ala Met Gln Phe Val Asn Val Gly Tyr Phe Leu Ile Ala Ala Gly Val Val Val Phe Ala Leu Gly Phe Leu Gly Cys Tyr Gly Ala Lys Thr Glu Ser Lys Cys A1a Leu Val Thr Phe Phe Phe Ile Leu Leu Leu Ile Phe Ile Ala Glu Val Ala Ala Ala Val Val Ala Leu Val Tyr Thr Thr Met Ala Glu His Phe Leu Thr Leu Leu Val Val Pro Ala Ile Lys Lys Asp Tyr Gly Ser Gln Glu Asp Phe Thr Gln Val Trp Asn Thr Thr Met Lys Gly Leu Lys Cys Cys Gly Phe Thr Asn Tyr Thr Asp Phe Glu Asp Ser Pro Tyr Phe Lys Glu Asn Ser Ala Phe Pro Pro Phe Cys Cys Asn Asp Asn Val Thr Asn Thr Ala Asn Glu Thr Cys Thr Lys Gln Lys Ala His Asp Gln Lys Val Glu Gly Cys Phe Asn Gln Leu Leu Tyr Asp Ile Arg Thr Asn Ala Val Thr Val Gly Gly Val Ala Ala Gly Ile Gly Gly Leu Glu Leu Ala Ala Met Ile Val Ser Met Tyr Leu Tyr Cys Asn Leu Gln <210> 115 <211> 366 <212> DNA
<213> Homo sapien <400>

gctctttctctcccctcctctgaatttaattctttcaacttgcaatttgcaaggattaca60 catttcactgtgatgtatattgtgttgcaaaaaaaaaaaagtgtctttgtttaaaattac120 ttggtttgtgaatccatcttgctttttccccattggaactagtcattaacccatctctga180 actggtagaaaaacatctgaagagctagtctatcagcatctgacaggtgaattggatggt240 tctcagaaccatttcacccagacagcctgtttctatcctgtttaataaattagtttgggt300 tctctacatgcataacaaaccctgctccaatctgtcacataaaagtctgtgacttgaagt360 ttagtc 366 <210> 116 <211> 282 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(282) <223> n = A,T,C or G
<400> 116 acaaagatgaaccatttcctatattatagcaaaattaaaatctacccgtattctaatatt60 gagaaatgagatnaaacacaatnttataaagtctacttagagaagatcaagtgacctcaa120 agactttactattttcatattttaagacacatgatttatcctattttagtaacctggttc180 atacgttaaacaaaggataatgtgaacagcagagaggatttgttggcagaaaatctatgt240 tcaatctngaactatctanatcacagacatttctattccttt 282 <210> 117 <211> 305 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1)...(305) <223> n = A,T,C or G
<400> 117 acacatgtcgcttcactgccttcttagatgcttctggtcaacatanaggaacagggacca60 tatttatcctccctcctgaaacaattgcaaaataanacaaaatatatgaaacaattgcaa120 aataaggcaaaatatatgaaacaacaggtctcgagatattggaaatcagtcaatgaagga180 tactgatccctgatcactgtcctaatgcaggatgtgggaaacagatgaggtcacctctgt240 gactgccccagcttactgcctgtagagagtttctangctgcagttcagacagggagaaat300 tgggt 305 <210> 118 <211> 71 <212> DNA
<213>, Homo sapien <220>
<221> misc_feature <222> (1)...(71) <223> n = A,T,C or G
<400> 118 accaaggtgt ntgaatctct gacgtgggga tctctgattc ccgcacaatc tgagtggaaa 60 aantcctggg t 71 <210> 119 <211> 212 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(212) <223> n = A,T,C or G
<400> 119 actccggttg gtgtcagcag cacgtggcat tgaacatngc aatgtggagc ccaaaccaca 60 gaaaatgggg tgaaattggc caactttcta tnaacttatg ttggcaantt tgccaccaac 120 agtaagctgg cccttctaat aaaagaaaat tgaaaggttt ctcactaanc ggaattaant 180 aatggantca aganactccc aggcctcagc gt 212 <210> 120 <211> 90 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(90) <223> n = A,T,C or G
<400> 120 actcgttgca natcaggggc cccccagagt caccgttgca ggagtccttc tggtcttgcc 60 ctccgccggc gcagaacatg ctggggtggt 90 <210> 121 <211> 218 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(218) <223> n = A,T,C or G
<400> 121 tgtancgtga anacgacaga nagggttgtc aaaaatggag aanccttgaa gtcattttga 60 gaataagatt tgctaaaaga tttggggcta aaacatggtt attgggagac atttctgaag 120 atatncangt aaattangga atgaattcat ggttcttttg ggaattcctt tacgatngcc 180 agcatanact tcatgtgggg atancagcta cccttgta 218 <210> 122 <211> 171 <212> DNA
<213> Homo sapien <400> 122 taggggtgta tgcaactgta aggacaaaaa ttgagactca actggcttaa ccaataaagg 60 catttgttag ctcatggaac aggaagtcgg atggtggggc atcttcagtg ctgcatgagt 120 caccaccccg gcggggtcat ctgtgccaca ggtccctgtt gacagtgcgg t 171 <210> 123 <211> 76 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) ... (76) <223> n = A,T,C or G
<400> 123 tgtagcgtga agacnacaga atggtgtgtg ctgtgctatc caggaacaca tttattatca 60 ttatcaanta ttgtgt 76 <210> 124 <211> 131 <212> DNA
<213> Homo sapien <400> 124 acctttcccc aaggccaatg tcctgtgtgc taactggccg gctgcaggac agctgcaatt 60 caatgtgctg ggtcatatgg aggggaggag actctaaaat agccaatttt attctcttgg 120 ttaagatttg t 131 <210> 125 <211> 432 <212> DNA
<213> Homo sapien <400> 125 actttatcta ctggctatga aatagatggt ggaaaattgc gttaccaact ataccactgg 60 cttgaaaaag aggtgatagc tcttcagagg acttgtgact tttgctcaga tgctgaagaa 120 ctacagtctg catttggcag aaatgaagat gaatttggat taaatgagga tgctgaagat 180 ttgcctcacc aaacaaaagt gaaacaactg agagaaaatt ttcaggaaaa aagacagtgg 240 ctcttgaagt atcagtcact tttgagaatg tttcttagtt actgcatact tcatggatcc 300 catggtgggg gtcttgcatc tgtaagaatg gaattgattt tgcttttgca agaatctcag 360 caggaaacat cagaaccact attttctagc cctctgtcag agcaaacctc agtgcctctc 420 ctctttgctt gt 432 <210> 126 <211> 112 <212> DNA
<213> Homo sapien <400> 126 acacaacttg aatagtaaaa tagaaactga gctgaaattt ctaattcact ttctaaccat 60 agtaagaatg atatttcccc ccagggatca ccaaatattt ataaaaattt gt 112 <210> 127 <211> 54 <212> DNA
<213> Homo sapien <400> 127 accacgaaac cacaaacaag atggaagcat caatccactt gccaagcaca gcag 54 <210> 128 <211> 323 <212> DNA
<213> Homo sapien <400> 128 acctcattag taattgtttt gttgtttcat ttttttctaa tgtctcccct ctaccagctc 60 acctgagata acagaatgaa aatggaagga cagccagatt tctcctttgc tctctgctca 120 ttctctctga agtctaggtt acccattttg gggacccatt'ataggcaata aacacagttc 180 ccaaagcatt tggacagttt cttgttgtgt tttagaatgg ttttcctttt tcttagcctt 240 ttcctgcaaa aggctcactc agtcccttgc ttgctcagtg gactgggctc cccagggcct 300 aggctgcctt cttttccatg tcc 323 <210> 129 <211> 192 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(192) <223> n = A,T,C or G
<400> 129 acatacatgt gtgtatattt ttaaatatca cttttgtatc actctgactt tttagcatac 60 tgaaaacaca ctaacataat ttntgtgaac catgatcaga tacaacccaa atcattcatc 120 tagcacattc atctgtgata naaagatagg tgagtttcat ttccttcacg ttggccaatg 180 gataaacaaa gt 192 <210> 130 <211> 362 <212> DNA
<213> Homo sapien <220>
<221> misc feature 4~
<222> (1)...(362) <223> n = A,T,C or G
<400> 130 cccttttttatggaatgagtagactgtatgtttgaanatttanccacaacctctttgaca60 tataatgacgcaacaaaaaggtgctgtttagtcctatggttcagtttatgcccctgacaa120 gtttccattgtgttttgccgatcttctggctaatcgtggtatcctccatgttattagtaa180 ttctgtattccattttgttaacgcctggtagatgtaacctgctangaggctaactttata240 cttatttaaaagctcttattttgtggtcattaaaatggcaatttatgtgcagcactttat300 tgcagcaggaagcacgtgtgggttggttgtaaagctctttgctaatcttaaaaagtaatg360 gg 362 <210> 131 <211> 332 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(332) <223> n = A,T,C or G
<400> 131 ctttttgaaa gatcgtgtcc actcctgtgg acatcttgtt ttaatggagt ttcccatgca 60 gtangactgg tatggttgca gctgtccaga taaaaacatt tgaagagctc caaaatgaga 120 gttctcccag gttcgccctg ctgctccaag tctcagcagc agcctctttt aggaggcatc 180 ttctgaacta gattaaggca gcttgtaaat ctgatgtgat ttggtttatt atccaactaa 240 cttccatctg ttatcactgg agaaagccca gactccccan gacnggtacg gattgtgggc 300 atanaaggat tgggtgaagc tggcgttgtg gt 332 <210> 132 <211> 322 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(322) <223> n = A,T,C or G
<400> 132 acttttgcca ttttgtatat ataaacaatc ttgggacatt ctcctgaaaa ctaggtgtcc 60 agtggctaag agaactcgat ttcaagcaat tctgaaagga aaaccagcat gacacagaat 120 ctcaaattcc caaacagggg ctctgtggga aaaatgaggg aggacctttg tatctcgggt 180 tttagcaagt taaaatgaan atgccaggaa aggcttattt atcaacaaag agaagagttg 240 ggatgcttct aaaaaaaact ttggtagaga aaataggaat gctnaatcct agggaagcct 300 gtaacaatct acaattggtc ca 322 <210> 133 <211> 278 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(278) <223> n = A,T,C or G

<400> 133 acaagccttcacaagtttaactaaattgggattaatctttctgtanttatctgcataatt60 cttgtttttctttccatctggctcctgggttgacaatttgtggaaacaactctattgcta120 ctatttaaaaaaaatcacaaatctttccctttaagctatgttnaattcaaactattcctg180 ctattcctgttttgtcaaagaaattatatttttcaaaatatgtntatttgtttgatgggt240 cccacgaaacactaataaaaaccacagagaccagcctg 278 <210> 134 <211> 121 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(121) <223> n = A,T,C or G
<400> 134 gtttanaaaa cttgtttagc tccatagagg aaagaatgtt aaactttgta ttttaaaaca 60 tgattctctg aggttaaact tggttttcaa atgttatttt tacttgtatt ttgcttttgg 120 t 121 <210> 135 <211> 350 <212> DNA
<213> Homo sapien <220>
<221> mis'c_feature <222> (1). .(350) <223> n = A,T,C or G
<400>

acttanaaccatgcctagcacatcagaatccctcaaagaacatcagtataatcctatacc60 atancaagtggtgactggttaagcgtgcgacaaaggtcagctggcacattacttgtgtgc120 aaacttgatacttttgttctaagtaggaactagtatacagtncctaggantggtactcca180 gggtgccccccaactcctgcagccgctcctctgtgccagnccctgnaaggaactttcgct240 ccacctcaatcaagccctgggccatgctacctgcaattggctgaacaaacgtttgctgag300 ttcccaaggatgcaaagcctggtgctcaactcctggggcgtcaactcagt 350 <210> 136 <211> 399 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(399) <223> n = A,T,C or G
<400> 136 tgtaccgtgaagacgacagaagttgcatggcagggacagggcagggccgaggccagggtt60 gctgtgattgtatccgaatantcctcgtgagaaaagataatgagatgacgtgagcagcct120 gcagacttgtgtctgccttcaanaagccagacaggaaggccctgcctgccttggctctga180 cctggcggccagccagccagccacaggtgggcttcttccttttgtggtgacaacnccaag240 aaaactgcagaggcccagggtcaggtgtnagtgggtangtgaccataaaacaccaggtgc300 tcccaggaacccgggcaaaggccatccccacctacagccagcatgcccactggcgtgatg360 ggtgcaganggatgaagcagccagntgttctgctgtggt 399 <210> 137 <211> 165 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(165) <223> n = A,T,C or G
<400> 137 actggtgtgg tngggggtga tgctggtggt anaagttgan gtgaottcan gatggtgtgt 60 ggaggaagtg tgtgaacgta gggatgtaga ngttttggcc gtgctaaatg agcttcggga 120 ttggctggtc ccactggtgg tcactgtcat tggtggggtt cctgt 165 <210> 138 <211> 338 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(338) <223> n = A,T,C or G
<400> 138 actcactggaatgccacattcacaacagaatcagaggtctgtgaaaacattaatggctcc60 ttaacttctccagtaagaatcagggacttgaaatggaaacgttaacagccacatgcccaa120 tgctgggcagtctcccatgccttccacagtgaaagggcttgagaaaaatcacatccaatg180 tcatgtgtttccagccacaccaaaaggtgcttggggtggagggctgggggcatananggt240 cangcctcaggaagcctcaagttccattcagctttgccactgtacattccccatntttaa300 aaaaactgatgccttttttttttttttttgtaaaattc 338 <210> 139 <211> 382 <212> DNA
<213> Homo sapien <400> 139 gggaatcttggtttttggcatctggtttgcctatagccgaggccactttgacagaacaaa60 gaaagggacttcgagtaagaaggtgatttacagccagcctagtgcccgaagtgaaggaga120 attcaaacagacctcgtcattcctggtgtgagcctggtcggctcaccgcctatcatctgc180 atttgccttactcaggtgctaccggactctggcccctgatgtctgtagtttcacaggatg240 ccttatttgtcttctacaccccacagggccccctacttcttcggatgtgtttttaataat300 gtcagctatgtgccccatcctccttcatgccctccctccctttcctaccactgctgagtg' gcctggaacttgtttaaagtgt 382 <210> 140 <211> 200 <212> DNA
<213> Homo sapien <220>
<221> misc_,-mature <222> (1)...(200) <223> n = A,T,C or G

<400> 140 accaaanctt ctttctgttg tgttngattt tactataggg gtttngcttn ttctaaanat 60 acttttcatt taacancttt tgttaagtgt caggctgcac tttgctccat anaattattg 120 ttttcacatt tcaacttgta tgtgtttgtc tcttanagca ttggtgaaat cacatatttt 180 atattcagca taaaggagaa 200 <210> 141 <211> 335 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(335) <223> n = A,T,C or G
<400> 141 actttatttt caaaacactc atatgttgca aaaaacacat agaaaaataa agtttggtgg 60 gggtgctgac taaacttcaa gtcacagact tttatgtgac agattggagc agggtttgtt 120 atgcatgtag agaacccaaa ctaatttatt aaacaggata gaaacaggct gtctgggtga 180 aatggttctg agaaccatcc aattcacctg tcagatgctg atanactagc tcttcagatg 240 tttttctacc agttcagaga tnggttaatg actanttcca atggggaaaa agcaagatgg 300 attcacaaac caagtaattt taaacaaaga cactt 335 <210> 142 <211> 459 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(459) <223> n = A,T,C or G
<400>

accaggttaatattgccacatatatcctttccaattgcgggctaaacagacgtgtattta60 gggttgtttaaagacaacccagcttaatatcaagagaaattgtgacctttcatggagtat120 ctgatggagaaaacactgagttttgacaaatcttattttattcagatagcagtctgatca180 cacatggtccaacaacactcaaataataaatcaaatatnatcagatgttaaagattggtc240 ttcaaacatcatagccaatgatgccccgcttgcctataatctctccgacataaaaccaca300 tcaacacctcagtggccaccaaaccattcagcacagcttccttaactgtgagctgtttga360 agctaccagtctgagcactattgactatntttttcangctctgaatagctctagggatct420 cagcangggtgggaggaaccagctcaaccttggcgtant 459 <210> 143 <211> 140 <212> DNA
<213> Homo sapien <400> 143 acatttcctt ccaccaagtc aggactcctg gcttctgtgg gagttcttat cacctgaggg 60 aaatccaaac agtctctcct agaaaggaat agtgtcacca accccaccca tctccctgag 120 accatccgac ttccctgtgt 140 <210> 144 <211> 164 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(164) <223> n = A,T,C or G
<400> 144 acttcagtaa caacatacaa taacaacatt aagtgtatat tgccatcttt gtcattttct 60 atctatacca ctctcccttc tgaaaacaan aatcactanc caatcactta tacaaatttg 120 aggcaattaa tccatatttg ttttcaataa ggaaaaaaag atgt 164 <210> 145 <211> 303 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(303) <223> n = A,T,C or G
<400>

acgtagaccatccaactttgtatttgtaatggcaaacatccagnagcaattcctaaacaa60 actggagggtatttatacccaattatcccattcattaacatgccctcctcctcaggctat120 gcaggacagctatcataagtcggcccaggcatccagatactaccatttgtataaacttoa180 gtaggggagtccatccaagtgacaggtctaatcaaaggaggaaatggaacataagcccag240 tagtaaaatnttgcttagctgaaacagccacaaaagacttaccgccgtggtgattaccat300 caa 303 <210> 146 <211> 327 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(327) <223> n = A,T,C or G
<400>

actgcagctcaattagaagtggtctctgactttcatcancttctccctgggctccatgac60 actggcctggagtgactcattgctctggttggttgagagagctcctttgccaacaggcct120 ccaagtcagggctgggatttgtttcctttccacattctagcaacaatatgctggccactt180 cctgaacagggagggtgggaggagccagcatggaacaagctgccactttctaaagtagcc240 agacttgcccctgggcctgtcacacctactgatgaccttctgtgcctgcaggatggaatg300 taggggtgagctgtgtgactctatggt 327 <210> 147 <211> 173 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(173) <223> n = A,T,C or G
<400> 147 acattgtttt tttgagataa agcattgana gagctctcct taacgtgaca caatggaagg 60 actggaacac atacccacat ctttgttctg agggataatt ttctgataaa gtcttgctgt 120 atattcaagc acatatgtta tatattattc agttccatgt ttatagccta gtt 173 <210> 148 <211> 477 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(477) <223> n = A,T,C or G
<400> 148 acaaccactttatcteatcgaatttttaacccaaactcactcactgtgcctttctatcct60 atgggatatattatttgatgctccatttcatcacacatatatgaataatacactcatact120 gccctactacctgctgcaataatcacattcccttcctgtcctgaccctgaagccattgggl80 gtggtcctagtggccatcagtccangcctgcaccttgagcccttgagctccattgctcac240 nccancccacctcaccgaccccatcctcttacacagctacctccttgctctctaacccca300 tagattatntccaaattcagtcaattaagttactattaacactctacccgacatgtccag360 caccactggtaagccttctccagccaacacacacacacacacacncacacacacacatat420 ccaggcacaggctacctcatcttcacaatcacccctttaattaccatgctatggtgg 477 <210> 149 <211> 207 <212> DNA
<213> Homo sapien <400> 149 acagttgtat tataatatca agaaataaac ttgcaatgag agcatttaag agggaagaac 60 taacgtattt tagagagcca aggaaggttt ctgtggggag tgggatgtaa ggtggggcct 120 gatgataaat aagagtcagc caggtaagtg ggtggtgtgg tatgggcaca gtgaagaaca 180 tttcaggcag agggaacagc agtgaaa 207 <210> 150 <211> 111 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(111) <223> n = A,T,C or G
<400> 150 accttgattt cattgctgct ctgatggaaa cccaactatc taatttagct aaaacatggg 60 cacttaaatg tggtcagtgt ttggacttgt taactantgg catctttggg t 111 <210> 151 <211> 196 <212> DNA
<213> Homo sapien <400> 151 agcgcggcag gtcatattga acattccaga tacctatcat tactcgatgc tgttgataac 60 agcaagatgg ctttgaactc agggtcacca ccagctattg gaccttacta tgaaaaccat 120 ggataccaac cggaaaaccc ctatcccgca cagcccactg tggtccccac tgtctacgag 180 gtgcatccgg ctcagt 196 <210> 152 <211> 132 <212> DNA
<213> Homo sapien <400> 152 acagcacttt cacatgtaag aagggagaaa ttcctaaatg taggagaaag ataacagaac 60 cttccccttt tcatctagtg gtggaaacct gatgctttat gttgacagga atagaaccag 120 gagggagttt gt 132 <210> 153 <211> 285 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(285) <223> n = A,T,C or G .
<400> 153 acaanaccca nganaggcca ctggccgtgg tgtca'tggcc tccaaacatg aaagtgtcag 60 cttctgctct tatgtcctca tctgacaact ctttaccatt tttatcctcg ctcagcagga 120 gcacatcaat aaagtccaaa gtcttggact tggccttggc ttggaggaag tcatcaacac 180 cctggctagt gagggtgcgg cgccgctcct ggatgacggc atctgtgaag tcgtgcacca 240 gtctgcaggc cctgtggaag cgccgtccac acggagtnag gaatt 285 <210> 154 <211> 333 <212> DNA
<213> Homo sapien <400> 154 accacagtcctgttgggccagggcttcatgaccctttctgtgaaaagccatattatcacc60 accccaaatttttccttaaatatctttaactgaaggggtcagcctcttgactgcaaagac120 cctaagccggttacacagctaactcccactggccctgatttgtgaaattgctgctgcctg180 attggcacaggagtcgaaggtgttcagctcccctcctccgtggaacgagactctgatttg240 agtttcacaaattctcgggccacctcgtcattgctcctctgaaataaaatccggagaatg300 gtcaggcctgtctcatccatatggatcttccgg 333 <210> 155 <211> 308 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(308) <223> n = A,T,C or G
<400> 155 actggaaata ataaaaccca catcacagtg ttgtgtcaaa gatcatcagg gcatggatgg 60 gaaagtgctt tgggaactgt aaagtgccta acacatgatc gatgattttt gttataatat 120 ttgaatcacg gtgcatacaa actctcctgc ctgctcctcc tgggccccag ccccagcccc 180 atcacagctc actgctctgt tcatccaggc ccagcatgta gtggctgatt cttcttggct 240 gcttttagcc tccanaagtt tctctgaagc caaccaaacc tctangtgta aggcatgctg 300 gccctggt <210> 156 <211> 295 <212> DNA
<213> Homo sapien <400> 156 accttgctcg gtgcttggaa catattagga actcaaaata tgagatgata acagtgccta 60 ttattgatta ctgagagaac tgttagacat ttagttgaag attttctaca caggaactga 120 gaataggaga ttatgtttgg ccctcatatt ctctcctatc ctccttgcct cattctatgt 180 ctaatatatt ctcaatcaaa taaggttagc ataatcagga aatcgaccaa ataccaatat 240 aaaaccagat gtctatcctt aagattttca aatagaaaac aaattaacag actat 295 <210> 157 <211> 126 <212> DNA
<213> Homo sapien <400> 157 acaagtttaa atagtgctgt cactgtgcat gtgctgaaat gtgaaatcca ccacatttct 60 gaagagcaaa acaaattctg tcatgtaatc tctatcttgg gtcgtgggta tatctgtccc 120 cttagt 126 <210> 158 <211> 442 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(442) <223> n = A,T,C or G
<400>

acccactggtcttggaaacacccatccttaatacgatgatttttctgtcgtgtgaaaatg60 aanccagcaggctgcccctagtcagtccttccttccagagaaaaagagatttgagaaagt120 gcctgggtaattcaccattaatttcctcccccaaactctctgagtcttcccttaatattt180 ctggtggttctgaccaaagcaggtcatggtttgttgagcatttgggatcccagtgaagta240 natgtttgtagccttgcatacttagcccttcccacgcacaaacggagtggcagagtggtg300 ccaaccctgttttcccagtccacgtagacagattcacagtgcggaattctggaagctgga360 nacagacgggctctttgcagagccgggactctgaganggacatgagggcctctgcctctg420 tgttcattctctgatgtcctgt 442 <210> 159 <211> 498 <212> DNA
<213> Homo sapien <220>
<221> misc_feature, <222> (1)...(498) <223> n = A,T,C or G
<400> 159 acttccaggt aacgttgttg tttccgttga gcctgaactg atgggtgacg ttgtaggttc 60 tccaacaaga actgaggttg cagagcgggt agggaagagt gctgttccag ttgcacctgg 120 gctgctgtgg actgttgttg attcctcact acggcccaag gttgtggaac tggcanaaag 180 gtgtgttgttgganttgagctcgggcggctgtggtaggttgtgggctcttcaacaggggc240 tgctgtggtgccgggangtgaangtgttgtgtcacttgagcttggccagctctggaaagt300 antanattcttcctgaaggccagcgcttgtggagctggcangggtcantgttgtgtgtaa360 cgaaccagtgctgctgtgggtgggtgtanatcctccacaaagcctgaagttatggtgtcn420 tcaggtaanaatgtggtttcagtgtccctgggcngctgtggaaggttgtanattgtcacc480 aagggaataagctgtggt 498 <210> 160 <211> 380 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(380) <223> n = A,T,C or G
<400> 160 acctgcatac agcttccctg ccaaactcac aaggagacat caacctctag acagggaaac 60 agcttcagga tacttccagg agacagagcc accagcagca aaacaaatat tcccatgcct 120 ggagcatggc atagaggaag ctganaaatg tggggtctga ggaagccatt tgagtctggc 180 cactagacat ctcatcagcc acttgtgtga agagatgccc catgacccca gatgcctctc 240 ccacccttac ctccatctca cacacttgag ctttccactc tgtataattc taacatcctg 300 gagaaaaatg gcagtttgac cgaacctgtt cacaacggta gaggctgatt tctaacgaaa 360 cttgtagaat gaagcctgga 380 <210> 161 <211> 114 <212> DNA
<213> Homo sapien <400> 161 actccacatc ccctctgagc aggcggttgt cgttcaaggt gtatttggcc ttgcctgtca 60 cactgtccac tggcccctta tccacttggt gcttaatccc tcgaaagagc atgt 114 <210> 162 <211> 177 <212> DNA
<213> Homo sapien <400> 162 actttctgaa tcgaatcaaa tgatacttag tgtagtttta atatcctcat atatatcaaa 60 gttttactac tctgataatt ttgtaaacca ggtaaccaga acatccagtc atacagcttt 120 tggtgatata taacttggca ataacccagt ctggtgatac ataaaactac tcactgt 177 <210> 163 <211> 137 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(137) <223> n = A,T,C or G
<400> 163 catttataca gacaggcgtg aagacattca cgacaaaaac gcgaaattct atcccgtgac 60 canagaaggc agctacggct actcctacat cctggcgtgg gtggccttcg cctgcacctt 120 catcagcggc atgatgt ~ 137 <210> 164 <211> 469 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(469) <223> n = A,T,C or G
<400>

cttatcacaatgaatgttctcctgggcagcgttgtgatctttgccaccttcgtgacttta60 tgcaatgcatcatgctatttcatacctaatgagggagttccaggagattcaaccaggaaa120 tgcatggatctcaaaggaaacaaacacccaataaactcggagtggcagactgacaactgt180 gagacatgcacttgctacgaaacagaaatttcatgttgcacccttgtttctacacctgtg240 ggttatgacaaagacaactgccaaagaatcttcaagaaggaggactgcaagtatatcgtg300 gtggagaagaaggacccaaaaaagacctgttctgtcagtgaatggataatctaatgtgct360 tctagtaggcacagggctcccaggccaggcctcattctcctctggcctctaatagtcaat420 gattgtgtagccatgcctatcagtaaaaagatntttgagcaaacacttt 469 <210> 165 <211> 195 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(195) <223> n = A,T,C or G
<400> 165 acagtttttt atanatatcg acattgccgg cacttgtgtt cagtttcata aagctggtgg 60 atccgctgtc atccactatt ccttggctag agtaaaaatt attcttatag cccatgtccc 120 tgcaggccgc ccgcccgtag ttctcgttcc agtcgtcttg gcacacaggg tgccaggact 180 tcctctgaga tgagt 195 <210> 166 <211> 383 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(383) <223> n = A,T,C or G
<400> 166 acatcttagtagtgtggcacatcagggggccatcagggtcacagtcactcatagcctcgc60 cgaggtcggagtccacaccaccggtgtaggtgtgctcaatcttgggcttggcgcccacct120 ttggagaagggatatgctgcacacacatgtccacaaagcctgtgaactcgccaaagaatt180 tttgcagaccagcctgagcaaggggcggatgttcagcttcagctcctccttcgtcaggtg240 gatgccaacctcgtctanggtccgtgggaagctggtgtccacntcacctacaacctgggc300 gangatcttataaagaggctccnagataaactccacgaaacttctctgggagctgctagt360 nggggcctttttggtgaactttc 383 <210> 167 <211> 247 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(247) <223> n = A,T,C or G
<400> 167 acagagccagaccttggccataaatgaancagagattaagactaaaccccaagtcganat60 tggagcagaaactggagcaagaagtgggcctggggctgaagtagagaccaaggccactgc120 tatanccatacacagagccaactctcaggccaaggcnatggttggggcaganccagagac180 tcaatctgantccaaagtggtggctggaacactggtcatgacanaggcagtgactctgac240 tgangtc 247 <210> 168 <211> 273 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(273) <223> n = A,T,C or G
<400> 168 acttctaagt tttctagaag tggaaggatt gtantcatcc tgaaaatggg tttacttcaa 60 aatccctcan ccttgttctt cacnactgtc tatactgana gtgtcatgtt tccacaaagg 120 gctgacacct gagcctgnat tttcactcat ccctgagaag ccctttccag tagggtgggc 180 aattcccaac ttccttgcca caagcttccc aggctttctc ccctggaaaa ctccagcttg~ 240 agtcccagat acactcatgg gctgccctgg gca 273 <210> 169 <211> 431 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(431) <223> n = A,T,C or G
<400> 169 acagccttggcttccccaaactccacagtctcagtgcagaaagatcatcttccagcagtc60 agctcagaccagggtcaaaggatgtgacatcaacagtttctggtttcagaacaggttcta120 ctactgtcaaatgaccccccatacttcctcaaaggctgtggtaagttttgcacaggtgag180 ggcagcagaaagggggtanttactgatggacaccatcttctctgtatactccacactgac240 .

cttgccatgggcaaaggcccctaccacaaaaacaataggatcactgctgggcaccagctc300 acgcacatcactgacaaccgggatggaaaaagaantgccaactttcatacatccaactgg360 aaagtgatctgatactggattcttaattaccttcaaaagcttctgggggccatcagctgc420 tcgaacactga 431 <210> 170 <211> 266 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(266) <223> n = A,T,C or G
<400>

acctgtgggctgggctgttatgcctgtgccggctgctgaaagggagttcagaggtggagc60 tcaaggagctctgcaggcattttgccaancctctccanagcanagggagcaacctacact120 ccccgctagaaagacaccagattggagtcctgggagggggagttggggtgggcatttgat180 gtatacttgtcacctgaatgaangagccagagaggaangagacgaanatganattggcct240 tcaaagctaggggtctggcaggtgga 266 <210> 171 <211> 1248 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(1248) <223> n = A,T,C or G
<400>

ggcagccaaatcataaacggcgaggactgcagcccgcactcgcagccctggcaggcggca60 ctggtcatggaaaacgaattgttctgctcgggcgtcctggtgcatccgcagtgggtgctg120 tcagccgcacactgtttccagaagtgagtgcagagctcctacaccatcgggctgggcctg180 cacagtcttgaggccgaccaagagccagggagccagatggtggaggccagcctctccgta240 cggcacccagagtacaacagacccttgctcgctaacgacctcatgctcatcaagttggac300 gaatccgtgtccgagtctgacaccatccggagcatcagcattgcttcgcagtgccctacc360 gcggggaactcttgcctcgtttctggctggggtctgctggcgaacggcagaatgcctacc420 gtgctgcagtgcgtgaacgtgtcggtggtgtctgaggaggtctgcagtaagctctatgac480 ccgctgtaccaccccagcatgttctgcgccggcggagggcaagaccagaaggactcctgc540 aacggtgactctggggggcccctgatctgcaacgggtacttgcagggccttgtgtctttc600 ggaaaagccccgtgtggccaagttggcgtgccaggtgtctacaccaacctctgcaaattc660 actgagtggatagagaaaaccgtccaggccagttaactctggggactgggaacccatgaa720 attgacccccaaatacatcctgcggaaggaattcaggaatatctgttcccagcccctcct780 ccctcaggcccaggagtccaggcccccagcccctcctccctcaaaccaagggtacagatc840 cccagcccctcctccctcagacccaggagtccagaccccccagcccctcctccctcagac900 ccaggagtccagcccctcctccctcagacccaggagtccagaccccccagcccctcctcc960 ctcagacccaggggtccaggcccccaacccctcctccctcagactcagaggtccaagccc1020 ccaacccntcattccccagacccagaggtccaggtcccagcccctcntccctcagaccca1080 gcggtccaatgccacctagactntccctgtacacagtgcccccttgtggcacgttgaccc1140 aaccttaccagttggtttttcatttttngtccctttcccctagatccagaaataaagttt1200 aagagaagngcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1248 <210> 172 <211> 159 <212> PRT
<213> Homo sapien <220>
<221> VARIANT
<222> (2)...(159) <223> Xaa = Any Amino Acid <400> 172 Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Pro Leu Leu A1a Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Gly Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val Ser Glu Glu Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met Phe Cys Ala Gly Gly Gly Gln Xaa Gln Xaa Asp Ser Cys Asn Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser Phe Gly Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val Tyr Thr Asn Leu Cys Lys Phe Thr Glu Trp Tle Glu Lys Thr Val Gln Ala Ser <210> 173 <211> 1265 <212> DNA ' <213> Homo sapien <220>
<221> misc_feature <222> (1)...(1265) <223> n = A,T,C or G
<400>

ggcagcccgcactcgcagccctggcaggcggcactggtcatggaaaacgaattgttctgc60 tcgggcgtcctggtgcatccgcagtgggtgctgtcagccgcacactgtttccagaactcc120 tacaccatcgggctgggcctgcacagtcttgaggccgaccaagagccagggagccagatg180 gtggaggccagcctctccgtacggcacccagagtacaacagacccttgctcgctaacgac240 ctoatgctcatcaagttggacgaatccgtgtccgagtctgacaccatccggagcatcagc300 attgcttcgcagtgccctaccgcggggaactcttgcctcgtttctggctggggtctgctg360 gcgaacggtgagctcacgggtgtgtgtctgccctcttcaaggaggtcctctgcccagtcg420 cgggggctgacccagagctctgcgtcccaggcagaatgcctaccgtgctgcagtgcgtga480 acgtgtcggtggtgtctgaggaggtctgcagtaagctctatgacccgctgtaccacccca540 gcatgttctgcgccggcggagggcaagaccagaaggactcctgcaacggtgactctgggg600 ggcccctgatctgcaacgggtacttgcagggccttgtgtctttcggaaaagccccgtgtg660 gccaagttggcgtgccaggtgtctacaccaacctctgcaaattcactgagtggatagaga720 aaaccgtccaggccagttaactctggggactgggaacccatgaaattgacccccaaatac780 atcctgcggaaggaattcaggaatatctgttcccagcccctcctccctcaggcccaggag840 tccaggcccccagcccctcctccctcaaaccaagggtacagatccccagcccctcctccc900 tcagacccaggagtccagaccccccagcccctcctccctcagacccaggagtccagcccc960 tcctccntcagacccaggagtccagaccccccagcccctcctccctcagacccaggggtt1020 gaggcccccaacccctcctccttcagagtcagaggtccaagcccccaacccctcgttccc1080 cagacccagaggtnnaggtcccagcccctcttccntcagacccagnggtccaatgccacc1140 tagattttccctgnacacagtgcccccttgtggnangttgacccaaccttaccagttggt1200 ttttcatttttngtccctttcccctagatccagaaataaagtttaagagangngcaaaaa1260 aaaaa 1265 <2l0> 174 <211> 1459 <212> DNA .
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(1459) <223> n = A,T,C or G
<400> 174 ggtcagccgcacactgtttccagaagtgagtgcagagctcctacaccatcgggctgggcc60 tgcacagtcttgaggccgaccaagagccagggagccagatggtggaggccagcctctccg120 tacggcacccagagtacaacagacccttgctcgctaacgacctcatgctcatcaagttgg180 acgaatccgtgtccgagtctgacaccatccggagcatcagcattgcttcgcagtgcccta240 ccgcggggaactcttgcctcgtttctggctggggtctgctggcgaacggtgagctcacgg300 gtgtgtgtctgccctcttcaaggaggtcctctgcccagtcgcgggggctgacccagagct360 ctgcgtcccaggcagaatgcctaccgtgctgcagtgcgtgaacgtgtcggtggtgtctga420 ngaggtctgcantaagctctatgacccgctgtaccaccccancatgttctgcgccggcgg480 agggcaagaccagaaggactcctgcaacgtgagagaggggaaaggggagggcaggcgact540 cagggaagggtggagaagggggagacagagacacacagggccgcatggcgagatgcagag600 atggagagacacacagggagacagtgacaactagagagagaaactgagagaaacagagaa660 ataaacacaggaataaagagaagcaaaggaagagagaaacagaaacagacatggggaggc720 .

agaaacacacacacatagaaatgcagttgaccttccaacagcatggggcctgagggcggt780 gacctccacccaatagaaaatcctcttataacttttgactccccaaaaacctgactagaa840 atagcctactgttgacggggagccttaccaataacataaatagtcgatttatgcatacgt900 tttatgcattcatgatatacctttgttggaattttttgatatttctaagctacacagttc960 gtctgtgaatttttttaaattgttgcaactctcctaaaatttttctgatgtgtttattga1020 aaaaatccaagtataagtggacttgtgcattcaaaccagggttgttcaagggtcaactgt1080 gtacccagagggaaacagtgacacagattcatagaggtgaaacacgaagagaaacaggaa1140 aaatcaagactctacaaagaggctgggcagggtggctcatgcctgtaatcccagcacttt1200 gggaggcgaggcaggcagatcacttgaggtaaggagttcaagaccagcctggccaaaatg1260 gtgaaatcctgtctgtactaaaaatacaaaagttagctggatatggtggcaggcgcctgt1320 aatcccagctacttgggaggctgaggcaggagaattgcttgaatatgggaggcagaggtt1380 gaagtgagttgagatcacaccactatactccagctggggcaacagagtaagactctgtct1440 caaaaaaaaaaaaaaaaaa 1459 <210> 175 <211> 1167 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(1167) <223> n = A,T,C or G
<400>

gcgcagccctggcaggcggcactggtcatggaaaacgaattgttctgctcgggcgtcctg60 gtgcatccgcagtgggtgctgtcagccgcacactgtttccagaactcctacaccatcggg120 ctgggcctgcacagtcttgaggccgaccaagagccagggagccagatggtggaggccagc180 ctctccgtacggcacccagagtacaacagactcttgctcgctaacgacctcatgctcatc240 aagtt"ggacgaatccgtgtccgagtctgacaccatccggagcatcagcattgcttcgcag300 tgccctaccgcggggaactcttgcctcgtntctggctggggtctgctggcgaacggcaga360 atgcctaccgtgctgcactgcgtgaacgtgtcggtggtgtctgaggangtctgcagtaag420 ctctatgacccgctgtaccaccccagcatgttctgcgccggcggagggcaagaccagaag480 gactcctgcaacggtgactctggggggcccctgatctgcaacgggtacttgcagggcctt540 gtgtctttcggaaaagccccgtgtggccaacttggcgtgccaggtgtctacaccaacctc600 tgcaaattcactgagtggatagagaaaaccgtccagnccagttaactctggggactggga660 acccatgaaattgacccccaaatacatcctgcggaangaattcaggaatatctgttccca720 gcccctcctccctcaggcccaggagtccaggcccccagcccctcctccctcaaaccaagg780 gtacagatccccagcccctcctccctcagacccaggagtccagaccccccagcccctcnt840 ccntcagacccaggagtccagcccctcctccntcagacgcaggagtccagaccccccagc900 ccntcntccgtcagacccaggggtgcaggcccccaacccctcntccntcagagtcagagg960 tccaagcccccaacccctcgttccccagacccagaggtncaggtcccagcccctcctccc1020 tcagacccagcggtccaatgccacctagantntccctgtacacagtgcccccttgtggca1080 ngttgacccaaccttaccagttggtttttcattttttgtccctttcccctagatccagaa1140 ataaagtntaagagaagcgcaaaaaaa 1167 <210> 176 <211> 205 <212> PRT
<213> Homo sapien <220>
<221> VARIANT
<222> (1)...(205) <223> Xaa = Any Amino Acid <400> 176 Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro Gln Trp Val Leu Ser Ala Ala His Cys Phe Gln Asn Ser Tyr Thr Ile Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser Gln Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Leu Leu Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr Ala Gly 85 90 '' 95 Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Gly Arg Met Pro Thr Val Leu His Cys Val Asn Val Ser Val Val Ser Glu Xaa Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met Phe Cys Ala Gly Gly Gly Gln Asp Gln Lys Asp Ser Cys Asn Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser Phe Gly Lys Ala Pro Cys Gly Gln heu Gly Val Pro Gly Val Tyr Thr Asn Leu Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Gln Xaa Ser <210> 177 <21~1> 1119 <212> DNA
<213> Homo sapien <400> 177 gcgcactcgcagccctggcaggcggcactggtcatggaaaacgaattgttctgctcgggc60 gtcctggtgcatccgcagtgggtgctgtcagccgcacactgtttccagaactcctacacc120 atcgggctgggcctgcacagtcttgaggccgaccaagagccagggagccagatggtggag180 gccagcctctccgtacggcacccagagtacaacagacccttgctcgctaacgacctcatg240 ctcatcaagttggacgaatccgtgtccgagtctgacaccatccggagcatcagcattgct300 tcgcagtgccctaccgcggggaactcttgcctcgtttctggctggggtctgctggcgaac360 gatgctgtgattgccatccagtcccagactgtgggaggctgggagtgtgagaagctttcc420 caaccctggcagggttgtaccatttcggcaacttccagtgcaaggacgtcctgctgcatc480 ctcactgggtgctcactactgctcactgcatcacccggaacactgtgatcaactagccag540 caccatagttctccgaagtcagactatcatgattactgtgttgactgtgctgtctattgt600 actaaccatgccgatgtttaggtgaaattagcgtcacttggcctcaaccatcttggtatc660 cagttatcctcactgaattgagatttcctgcttcagtgtcagccattcccacataatttc720 tgacctacagaggtgagggatcatatagctcttcaaggatgctggtactcccctcacaaa780 ttcatttctcctgttgtagtgaaaggtgcgccctctggagcctcccagggtgggtgtgca840 ggtcacaatgatgaatgtatgatcgtgttcccattacccaaagcctttaaatccctcatg900 ctcagtacaccagggcaggtctagcatttcttcatttagtgtatgctgtccattcatgca960 accacctcaggactcctggattctctgcctagttgagctcctgcatgctgcctccttggg1020 gaggtgagggagagggcccatggttcaatgggatctgtgcagttgtaacacattaggtgc1080 ttaataaacagaagctgtgatgttaaaaaaaaaaaaaaa 1119 <210> 178 <211> 164 <212> PRT
<213> Homo sapien <220>
<221> VARIANT
<222> (1)...(164) <223> Xaa = Any Amino Acid <400> 178 Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro Gln Trp Val Leu Ser Ala Ala His Cys Phe Gln Asn Ser Tyr Thr Ile Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser Gln Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Pro Leu Leu Ala Asn Asp Leu Met Leu Tle Lys Leu Asp Glu Ser Val Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Asp Ala Val Ile Ala Ile Gln Ser Xaa Thr Val Gly Gly Trp Glu Cys Glu Lys Leu Ser Gln Pro Trp Gln Gly Cys.Thr Ile Ser Ala Thr Ser Ser Ala Arg Thr Ser Cys Cys Ile Leu Thr Gly Cys Ser Leu Leu Leu Thr Ala Ser Pro Gly Thr Leu <210> 179 <211> 250 <212> DNA
<213> Homo sapien <400> 179 ctggagtgccttggtgtttcaagcccctgcaggaagcagaatgcaccttctgaggcacct60 ccagctgcccccggccgggggatgcgaggctcggagcacccttgcccggctgtgattgct120 gccaggcactgttcatctcagcttttctgtccctttgctcccggcaagcgcttctgctga180 aagttcatatctggagcctgatgtcttaacgaataaaggtcccatgctccacccgaaaaa240 aaaaaaaaaa 250 <210> 180 <211> 202 <212> DNA
<213> Homo sapien <400> 180 actagtccag tgtggtggaa ttccattgtg ttgggcccaa cacaatggct acctttaaca 60 tcacccagac cccgcccctg cccgtgcccc acgctgctgc taacgacagt atgatgctta 120 ctctgctact cggaaactat ttttatgtaa ttaatgtatg ctttcttgtt tataaatgcc 180 tgatttaaaa aaaaaaaaaa as 202 <210> 181 <211> 558 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(558) <223> n = A,T,C or G
<400>

tccytttgktnaggtttkkgagacamccckagacctwaanctgtgtcacagacttcyngg60 aatgtttaggcagtgctagtaatttcytcgtaatgattctgttattactttcctnattct120 ttattcctctttcttctgaagattaatgaagttgaaaattgaggtggataaatacaaaaa180 ggtagtgtgatagtataagtatctaagtgcagatgaaagtgtgttatatatatccattca240 aaattatgcaagttagtaattactcagggttaactaaattactttaatatgctgttgaac300 ctactctgttccttggctagaaaaaattataaacaggactttgttagtttgggaagccaa360 attgataatattctatgttctaaaagttgggctatacataaattattaagaaatatggaw420 ttttattcccaggaatatggkgttcattttatgaatattacscrggatagawgtwtgagt480 aaaaycagttttggtwaataygtwaatatgtcmtaaataaacaakgctttgacttatttc540 caaaaaaaaaaaaaaaaa 558 <210> 182 <211> 479 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(479) <223> n = A,T,C or G
<400> 182 acagggwttkgrggatgctaagsccccrgarwtygtttgatccaaccctggcttwttttc60 agaggggaaaatggggcctagaagttacagmscatytagytggtgcgmtggcacccctgg120 cstcacacagastcccgagtagctgggactacaggcacacagtcactgaagcaggccctg180 ttwgcaattcacgttgccacctccaacttaaacattcttcatatgtgatgtccttagtca240 ctaaggttaaactttcccacccagaaaaggcaacttagataaaatcttagagtactttca300 tactmttctaagtcctcttccagcctcactkkgagtcctmcytgggggttgataggaant360 ntctcttggctttctcaataaartctctatycatctcatgtttaatttggtacgcatara420 awtgstgaraaaattaaaatgttctggttymactttaaaaaraaaaaaaaaaaaaaaaa 479 <210> 183 <211> 384 <212> DNA
<213> Homo sapien <400> 183 aggcgggagcagaagctaaagccaaagcccaagaagagtggcagtgccagcactggtgcc60 agtaccagtaccaataacagtgccagtgccagtgccagcaccagtggtggcttcagtgct120 ggtgccagcctgaccgccactctcacatttgggctcttcgctggccttggtggagctggt180 gccagcaccagtggcagctctggtgcctgtggtttctcctacaagtgagattttagatat240 tgttaatcctgccagtctttctcttcaagccagggtgcatcctcagaaacctactcaaca300 cagcactctaggcagccactatcaatcaattgaagttgacactctgcattaratctattt360 gccatttcaaaaaaaaaaaaaaaa 384 <210> 184 <211> 496 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(496) <223> n = A,T,C or G
<400> 184 accgaattgggaccgctggcttataagcgatcatgtyyntccrgtatkacctcaacgagc60 agggagatcgagtctatacgctgaagaaatttgacccgatgggacaacagacctgctcag120 cccatcctgctcggttctccccagatgacaaatactctsgacaccgaatcaccatcaaga180 aacgcttcaaggtgctcatgacccagcaaccgcgccctgtcctctgagggtcccttaaac240 tgatgtcttttctgccacctgttacccctcggagactccgtaaccaaactcttcggactg300 tgagccctgatgcctttttgccagccatactctttggcatccagtctctcgtggcgattg360 attatgcttgtgtgaggcaatcatggtggcatcacccataaagggaacacatttgacttt420 tttttctcatattttaaattactacmagawtattwmagawwaaatgawttgaaaaactst480 taaaaaaaaaaaaaaa 496 <210> 185 <211> 384 <212> DNA
<213> Homo sapien <400> 185 gctggtagcctatggcgkggcccacggaggggctcctgaggccacggracagtgacttcc60 caagtatcytgcgcsgcgtcttctaccgtccctacctgcagatcttcgggcagattcccc120 aggaggacatggacgtggccctcatggagcacagcaactgytcgtcggagcccggcttct180 gggcacaccctcctggggcccaggcgggcacctgcgtctcccagtatgcc.aactggctgg240 tggtgctgctcctcgtcatcttcctgctcgtggccaacatcctgctggtcaacttgctca300 ttgccatgttcagttacacattcggcaaagtacagggcaacagcgatctctactgggaag360 gcgcagcgttaccgcctcatccgg 384 <210> 286 <211> 577 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(577) <223> n = A,T,C or G
<400> 186 gagttagctc ctccacaacc ttgatgaggt cgtctgcagt ggcctctcgc ttcataccgc 60 tnccatcgtc atactgtagg tttgccacca cytcctggca tcttggggcg gcntaatatt l20 ccaggaaact ctcaatcaag tcaccgtcga tgaaacctgt gggctggttc tgtcttccgc 180 tcggtgtgaaaggatctcccagaaggagtgctcgatcttccccacacttttgatgacttt240 attgagtcgattctgcatgtccagcaggaggttgtaccagctctctgacagtgaggtcac300 cagccctatcatgccgttgamcgtgccgaagarcaccgagccttgtgtgggggkkgaagt360 ctcacccagattctgcattaccagagagccgtggcaaaagacattgacaaactcgcccag420 gtggaaaaagamcamctcctggargtgctngccgctcctcgtcmgttggtggcagcgctw480 tccttttgacacacaaacaagttaaaggcattttcagcccccagaaanttgtcatcatcc540 aagatntcgcacagcactnatccagttgggattaaat 577 <210> 187 <211> 534 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(534) <223> n = A,T,C or G
<400>

aacatcttcctgtataatgctgtgtaatatcgatccgatnttgtctgstgagaatycatw60 actkggaaaagmaacattaaagcctggacactggtattaaaattcacaatatgcaacact120 ttaaacagtgtgtcaatctgctcccyynactttgtcatcaccagtctgggaakaagggta180 tgccctattcacacctgttaaaagggcgctaagcatttttgattcaacatcttttttttt240 gacacaagtccgaaaaaagcaaaagtaaacagttatyaatttgttagccaattcactttc300 ttcatgggacagagccatytgatttaaaaagcaaattgcataatattgagcttygggagc360 tgatatttgagcggaagagtagcctttctacttcaccagacacaactccctttcatattg420 ggatgttnacnaaagtwatgtctctwacagatgggatgcttttgtggcaattctgttctg480 aggatctcccagtttatttaccacttgcacaagaaggcgttttcttcctcaggc 534 <210> 188 <211> 761 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(761) <223> n = A,T,C or G
<400>

agaaaccagtatctctnaaaacaacctctcataccttgtggacctaattttgtgtgcgtg60 tgtgtgtgcgcgcatattatatagacaggcacatcttttttacttttgtaaaagcttatg120 cctctttggtatctatatctgtgaaagttttaatgatctgccataatgtcttggggacct180 ttgtcttctgtgtaaatggtactagagaaaacacctatnttatgagtcaatctagttngt240 tttattcgacatgaaggaaatttccagatnacaacactnacaaactctccctkgackarg300 ggggacaaagaaaagcaaaactgamcataaraaacaatwacctggtgagaarttgcataa360 acagaaatwrggtagtatattgaarnacagcatcattaaarmgttwtkttwttctccctt420 gcaaaaaacatgtacngacttcccgttgagtaatgccaagttgttttttttatnataaaa480 cttgcccttcattacatgtttnaaagtggtgtggtgggccaaaatattgaaatgatggaa540 ctgactgataaagctgtacaaataagcagtgtgcctaacaagcaacacagtaatgttgac600 atgcttaattcacaaatgctaatttcattataaatgtttgctaaaatacactttgaacta660 tttttctgtnttcccagagctgagatnttagattttatgtagtatnaagtgaaaaantac720 gaaaataataacattgaagaaaaananaaaaaanaaaaaaa 761 <210> 189 <211> 482 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(482) <223> n = A,T,C or G
<400> 189 tttttttttttttgccgatnctactattttattgcaggangtgggggtgtatgcaccgca60 caccggggctatnagaagcaagaaggaaggagggagggcacagccccttgctgagcaaca120 aagccgcctgctgccttctctgtctgtctcctggtgcaggcacatggggagaccttcccc180 aaggcaggggccaccagtccaggggtgggaatacagggggtgggangtgtgcataagaag240 tgataggcacaggccacccggtacagacccctcggctcctgacaggtngatttcgaccag300 gtcattgtgccctgcccaggcacagcgtanatctggaaaagacagaatgctttccttttc360 aaatttggctngtcatngaangggcanttttccaanttnggctnggtcttggtacncttg420 gttcggcccagctccncgtccaaaaantattcacccnnctccnaattgcttgcnggnccc480 <210> 190 <211> 471 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(471) <223> n = A,T,C or G
<400> 190 ttttttttttttttaaaacagtttttcacaacaaaatttattagaagaatagtggttttg60 aaaactctcgcatccagtgagaactaccatacaccacattacagctnggaatgtnctcca120 aatgtctggtcaaatgatacaatggaaccattcaatcttacacatgcacgaaagaacaag180 cgcttttgacatacaatgcacaaaaaaaaaaggggggggggaccacatggattaaaattt240 taagtactcatcacatacattaagacacagttctagtccagtcnaaaatcagaactgcnt300 tgaaaaatttcatgtatgcaatccaaccaaagaacttnattggtgatcatgantnctcta360 ctacatcnaccttgatcattgccaggaacnaaaagttnaaancacncngtacaaaaanaa420 tctgtaattnanttcaacctccgtacngaaaaatnttnnttatacactcCc 471 <210> 191 <211> 402 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(402) <223> n = A,T,C or G
<400>

gagggattgaaggtctgttctastgtcggmctgttcagccaccaactctaacaagttgct60 gtcttccactcactgtctgtaagctttttaacccagacwgtatcttcataaatagaacaa120 attcttcaccagtcacatcttctaggacctttttggattcagttagtataagctcttcca180 cttcctttgttaagacttcatctggtaaagtcttaagttttgtagaaaggaattyaattg240 ctcgttctctaacaatgtcctctccttgaagtatttggctgaacaacccacctaaagtcc300 ctttgtgcatccattttaaatatacttaatagggcattgktncactaggttaaattctgc360 aagagtcatctgtctgcaaaagttgcgttagtatatctgcca 402 <210> 192 <211> 601 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(601) <223> n = A,T,C or G
<400> 192 gagctcggatccaataatctttgtctgagggcagcacacatatncagtgccatggnaact60 ggtctaccccacatgggagcagcatgccgtagntatataaggtcattccctgagtcagac120 atgcytytttgaytaccgtgtgccaagtgctggtgattctyaacacacytccatcccgyt180 cttttgtggaaaaactggcacttktctggaactagcargacatcacttacaaattcaccc240 acgagacacttgaaaggtgtaacaaagcgaytcttgcattgctttttgtccctccggcac300 cagttgtcaatactaacccgctggtttgcctccatcacatttgtgatctgtagctctgga360 tacatctcctgacagtactgaagaacttcttcttttgtttcaaaagcarctcttggtgcc420 tgttggatcaggttcccatttcccagtcyg.aatgttcacatggcatatttwacttcccac480 aaaacattgcgatttgaggctcagcaacagcaaatcctgttccggcattggctgcaagag540 cctcgatgtagccggccagcgccaaggcaggcgccgtgagccccaccagcagcagaagca600 g 601 <210> 193 <211> 608 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(608) <223> n = A,T,C or G
<400>

atacagcccanatcccaccacgaagatgcgcttgttgactgagaacctgatgcggtcact60 ggtcccgctgtagccccagcgactctccacctgctggaagcggttgatgctgcactcytt120 cccaacgcaggcagmagcgggsccggtcaatgaactccaytcgtggcttggggtkgacgg180 tkaagtgcaggaagaggctgaccacctcgcggtccaccaggatgcccgactgtgcgggac240 ctgcagcgaaactcctcgatggtcatgagcgggaagcgaatgaggcccagggccttgccc300 agaaccttccgcctgttctctggcgtcacctgcagctgctgccgctgacactcggcctcg360 gaccagcggacaaacggcrttgaacagccgcacctcacggatgcccagtgtgtcgcgctc420 caggammgscaccagcgtgtccaggtcaatgtcggtgaagccctccgcgggtratggcgt480 ctgcagtgtttttgtcgatgttctccaggcacaggctggccagctgcggttcatcgaaga540 gtcgcgcctgcgtgagcagcatgaaggcgttgtcggctcgcagttcttcttcaggaactc600 cacgcaat 608 <210> 194 <211> 392 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(392) <223> n = A,T,C or G
<400> 194 gaacggctgg accttgcctc gcattgtgct tgctggcagg gaataccttg gcaagcagyt 60 ccagtccgag cagccccaga ccgctgccgc bcgaagctaa gcctgcctct ggccttcccc 120 tccgcctcaa tgcagaacca gtagtgggag cactgtgttt agagttaaga gtgaacactg 180 tttgatttta cttgggaatt tcctctgtta tatagctttt cccaatgcta atttccaaac 240 aacaacaaca aaataacatg tttgcctgtt aagttgtata aaagtaggtg attctgtatt 300 taaagaaaat attactgtta catatactgc ttgcaatttc tgtatttatt gktnctstgg 360 aaataaatat agttattaaa ggttgtcant cc 392 <210> 295 <211> 502 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(502) <223> n = A,T,C or G
<.400> 195 ccsttkgaggggtkaggkyccagttyccgagtggaagaaacaggccaggagaagtgcgtg60 ccgagctgaggcagatgttcccacagtgacccccagagccstgggstatagtytctgacc120 cctcncaaggaaagaccacsttctggggacatgggctggagggcaggacctagaggcacc180 aagggaaggccccattccggggstgttccccgaggaggaagggaaggggctctgtgtgcc240 ccccasgaggaagaggccctgagtcctgggatcagacaccccttcacgtgtatccccaca300 caaatgcaagctcaccaaggtcccctctcagtccccttccstacaccctgamcggccact360 gscscacacccacccagagcacgccacccgccatggggartgtgctcaaggartcgcngg420 gcarcgtggacatctngtcccagaagggggcagaatctccaataganggactgarcmstt480 gctnanaaaaaaaaanaaaaas 502 <210> 196 <211> 665 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(665) <223> n = A,T,C or G
<400>

ggttacttggtttcattgccaccacttagtggatgtcatttagaaccattttgtctgctc60 cctctggaagccttgcgcagagcggactttgtaattgttggagaataactgctgaatttt120 wagctgtttkgagttgattsgcaccactgcacccacaacttcaatatgaaaacyawttga180 actwatttattatcttgtgaaaagtataacaatgaaaattttgttcatactgtattkatc240 aagtatgatgaaaagcaawagatatatattcttttattatgttaaattatgattgccatt300 attaatcggcaaaatgtggagtgtatgttcttttcacagtaatatatgccttttgtaact360 tcacttggttattttattgtaaatgarttacaaaattcttaatttaagaraatggtatgt420 watatttatttcattaatttctttcctkgtttacgtwaattttgaaaagawtgcatgatt480 tcttgacagaaatcgatcttgatgctgtggaagtagtttgacccacatccctatgagttt540 ttcttagaatgtataaaggttgtagcccatcnaacttcaaagaaaaaaatgaccacatac600 tttgcaatcaggctgaaatgtggcatgctnttctaattccaactttataaactagcaaan660 aagtg 665 <210> 197 <211> 492 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(492) <223> n = A,T,C or G
<400>

ttttntttttttttttttgcaggaaggattccatttattgtggatgcattttcacaatat60 atgtttattggagcgatccattatcagtgaaaagtatcaagtgtttataanatttttagg120 aaggcagattcacagaacatgctngtcngcttgcagttttacctcgtanagatnacagag180 aattatagtcnaaccagtaaacnaggaatttacttttcaaaagattaaatccaaactgaa240 caaaattctaccctgaaacttactccatccaaatattggaataanagtcagcagtgatac300 attctcttctgaactttagattttctagaaaaatatgtaatagtgatcaggaagagctct360 tgttcaaaagtacaacnaagcaatgttcccttaccataggccttaattcaaactttgatc420 catttcactcccatcacgggagtcaatgctacctgggacacttgtattttgttcatnctg480 ancntggcttas 492 <210> 198 <211> 478 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(478) <223> n = A,T,C or G
<400>

tttnttttgnatttcantctgtannaantattttcattatgtttattanaaaaatatnaa60 tgtntccacnacaaatcatnttacntnagtaagaggccanctacattgtacaacatacac120 tgagtatattttgaaaaggacaagtttaaagtanacncatattgccgancatancacatt180 tatacatggcttgattgatatttagcacagcanaaactgagtgagttaccagaaanaaat240 natatatgtcaatcngatttaagatacaaaacagatcctatggtacatancatcntgtag300 gagttgtggctttatgtttactgaaagtcaatgcagttcctgtacaaagagatggccgta360 agcattctagtacctctactccatggttaagaatcgtacacttatgtttacatatgtnca420 gggtaagaattgtgttaagtnaanttatggagaggtccangagaaaaatttgatncaa 478 <210> 199 <211> 482 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(482) <223> n = A,T,C or G
<400> ' agtgacttgtcctccaacaaaaccccttgatcaagtttgtggcactgacaatcagaccta60 tgctagttcctgtcatctattcgctactaaatgcagactggaggggaccaaaaaggggca120 tcaactccagctggattattttggagcctgcaaatctattcctacttgtacggactttga180 agtgattcagtttcctctacggatgagagactggctcaagaatatcctcatgcagcttta240 tgaagccnactctgaacacgctggttatctnagatgagaancagagaaataaagtcnaga300 aaatttacctggangaaaagaggctttnggctggggaccatcccattgaaccttctctta360 anggactttaagaanaaactaccacatgtntgtngtatcctggtgccnggccgtttantg420 aacntngacnncacccttntggaatanantcttgacngcntcctgaacttgctcctctgc480 ga 482 <210> 200 <211> 270 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(270) <223> n = A,T,C or G
<400>

cggccgcaagtgcaactccagctggggccgtgcggacgaagattctgccagcagttggtc60 cgactgcgacgacggcggcggcgacagtcgcaggtgcagcgcgggcgcctggggtcttgc120 aaggctgagctgacgccgcagaggtcgtgtcacgtcccacgaccttgacgccgtcgggga180 cagccggaacagagcccggtgaangcgggaggcctcggggagcccctcgggaagggcggc240 ccgagagatacgcaggtgcaggtggccgcc 270 <210> 201 <211> 419 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(419) <223> n = A,T,C or G
<400>

ttttttttttttttggaatctactgcgagcacagcaggtcagcaacaagtttattttgca60 gctagcaaggtaacagggtagggcatggttacatgttcaggtcaacttcctttgtcgtgg120 ttgattggtttgtctttatgggggcggggtggggtaggggaaancgaagcanaantaaca180 tggagtgggtgcaccctccctgtagaacctggttacnaaagcttggggcagttcacctgg240 tctgtgaccgtcattttcttgacatcaatgttattagaagtcaggatatcttttagagag300 tccactgtntctggagggagattagggtttcttgccaanatccaancaaaatccacntga360 aaaagttggatgatncangtacngaataccganggcatanttctcatantcggtggcca 419 <210> 202 <211> 509 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (509) <223> n = A,T,C or G
<400>

tttntttttttttttttttttttttttttttttttttttttttttttttttttttttttt60 tggcacttaatccatttttatttcaaaatgtctacaaantttnaatncnccattatacng120 gtnattttncaaaatctaaannttattcaaatntnagccaaantccttacncaaatnnaa180 tacncncaaaaatcaaaaatatacntntctttcagcaaacttngttacataaattaaaaa240 aatatatacggctggtgttttcaaagtacaattatcttaacactgcaaacatntttnnaa300 ggaactaaaataaaaaaaaacactnccgcaaaggttaaagggaacaacaaattcntttta360 caacancnncnattataaaaatcatatctcaaatcttaggggaatatatacttcacacng420 ggatcttaacttttactncactttgtttatttttttanaaccattgtnttgggcccaaca480 caatggnaatnccnccncnctggactagt 509 <210> 203 <211> 583 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1).,.(583) <223> n = A,T,C or G
<400> 203 ttttttttttttttttttgacecccctcttataaaaaacaagttaccattttattttact60 tacacatatttattttataattggtattagatattcaaaaggcagcttttaaaatcaaac120 taaatggaaactgccttagatacataattcttaggaattagcttaaaatctgcctaaagt180 gaaaatcttctctagctcttttgactgtaaatttttgactcttgtaaaacatccaaattc240 atttttcttgtctttaaaattatctaatctttccattttttccctattccaagtcaattt300 gcttctctagcctcatttcctagctcttatctactattagtaagtggcttttttcctaaa360 agggaaaacaggaagaganaatggcacacaaaacaaacattttatattcatatttctacc420 tacgttaataaaatagcattttgtgaagccagctcaaaagaaggcttagatccttttatg480 tccattttagtcactaaacgatatcnaaagtgccagaatgcaaaaggtttgtgaacattt540 attcaaaagctaatataagatatttcacatactcatctttctg 583 <210> 204 <211> 589 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(589) <223> n = A,T,C or G
<400>

ttttttttntttttttttttttttttnctcttctttttttttganaatgaggatcgagtt60 tttcactctctagatagggcatgaagaaaactcatctttccagctttaaaataacaatca120 aatctcttatgctatatcatattttaagttaaactaatgagtcactggcttatcttctcc180 tgaaggaaatctgttcattcttctcattcatatagttatatcaagtactaccttgcatat240 tgagaggtttttcttctctatttacacatatatttccatgtgaatttgtatcaaaccttt300 attttcatgcaaactagaaaataatgtnttcttttgcataagagaagagaacaatatnag360 cattacaaaactgctcaaattgtttgttaagnttatccattataattagttnggcaggag420 ctaatacaaatcacatttacngacnagcaataataaaactgaagtaccagttaaatatcc480 aaaataattaaaggaacatttttagcctgggtataattagctaattcactttacaagcat540 ttattnagaatgaattcacatgttattattccntagcccaacacaatgg 589 <210> 205 <211> 545 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(545) <223> n = A,T,C or G
<400> 205 tttttnttttttttttcagtaataatcagaacaatatttatttttatatttaaaattcat60 agaaaagtgccttacatttaataaaagtttgtttctcaaagtgatcagaggaattagata120 tngtcttgaacaccaatattaatttgaggaaaatacaccaaaatacattaagtaaattat180 ttaagatcatagagcttgtaagtgaaaagataaaatttgacctcagaaactctgagcatt240 aaaaatccactattagcaaataaattactatggacttcttgctttaattttgtgatgaat300 atggggtgtcactggtaaaccaacacattctgaaggatacattacttagtgatagattct360 tatgtactttgctanatnacgtggatatgagttgacaagtttctctttcttcaatctttt420 aaggggcngangaaatgaggaagaaaagaaaaggattacgcatactgttctttctatngg480 aaggattaga tatgtttcct ttgccaatat taaaaaaata ataatgttta ctactagtga 540 aaccc 545 <210> 206 <211> 487 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(487) <223> n = A,T,C or G
<400>

ttttttttttttttttagtcaagtttctnatttttattataattaaagtcttggtcattt60 catttattagctctgcaacttacatatttaaattaaagaaacgttnttagacaactgtna120 caatttataaatgtaaggtgccattattgagtanatatattcctccaagagtggatgtgt180 cccttctcccaccaactaatgaancagcaacattagtttaattttattagtagatnatac240 actgctgcaaacgctaattctcttctccatccccatgtngatattgtgtatatgtgtgag300 ttggtnagaatgcatcancaatctnacaatcaacagcaagatgaagctaggcntgggctt360 tcggtgaaaatagactgtgtctgtctgaatcaaatgatctgacctatcctcggtggcaag420 aactcttcgaaccgcttcctcaaaggcngctgccacatttgtggcntctnttgcacttgt480 ttcaaaa 487 <210> 207 <211> 332 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(332) <223> n = A,T,C or G
<400> 207 tgaattggctaaaagactgcatttttanaactagcaactcttatttctttcctttaaaaa60 tacatagcattaaatcccaaatcctatttaaagacctgacagcttgagaaggtcactact120 gcatttataggaccttctggtggttctgctgttacntttgaantctgacaatccttgana180 atctttgcatgcagaggaggtaaaaggtattggattttcacagaggaanaacacagcgca240 gaaatgaaggggccaggcttactgagcttgtccactggagggctcatgggtgggacatgg300 aaaagaaggcagcctaggccctggggagccca 332 <210> 208 <211> 524 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(524) <223> n = A,T,C or G
<400>

agggcgtggtgcggagggcgttactgttttgtctcagtaacaataaatacaaaaagactg60 gttgtgttccggccccatccaaccacgaagttgatttctcttgtgtgcagagtgactgat120 tttaaaggacatggagcttgtcacaatgtcacaatgtcacagtgtgaagggcacactcac180 tcccgcgtgattcacatttagcaaccaacaatagctcatgagtccatacttgtaaatact240 tttggcagaatacttnttgaaacttgcagatgataactaagatccaagatatttcccaaa300 gtaaatagaa gtgggtcata atattaatta cctgttcaca tcagcttcca tttacaagtc 360 atgagcccag acactgacat caaactaagc ccacttagac tcctcaccac cagtctgtcc 420 tgtcatcaga caggaggctg tcaccttgac caaattctca ccagtcaatc atctatccaa 480 aaaccattac ctgatccact tccggtaatg caccaccttg gtga 524 <210> 209 <211> 159 <212> DNA
<213> Homo sapien <400> 209 gggtgaggaa atccagagtt gccatggaga aaattccagt gtcagcattc ttgctccttg 60 tggccctctc ctacactctg gccagagata ccacagtcaa acctggagcc aaaaaggaca 120 caaaggactc tcgacccaaa ctgccccaga ccctctcca 159 <210> 210 <211> 256 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(256) <223> n = A,T,C or G
<400> 210 actccctggcagacaaaggcagaggagagagctctgttagttctgtgttgttgaactgcc60 actgaatttctttccacttggactattacatgccanttgagggactaatggaaaaacgta120 tggggagattttanccaatttangtntgtaaatggggagac~ggggcaggcgggagagat180 ttgcagggtgnaaatggganggctggtttgttanatgaacagggacataggaggtaggca240 ccaggatgctaaatca 256 <210> 211 <211> 264 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(264) <223> n = A,T,C or G
<400> 211 acattgtttttttgagataaagcattgagagagctctccttaacgtgacacaatggaagg60 actggaacacatacccacatctttgttctgagggataattttctgataaagtcttgctgt7.20 atattcaagcacatatgttatatattattcagttccatgtttatagcctagttaaggagal80 ggggagatacattcngaaagaggactgaaagaaatactcaagtnggaaaacagaaaaaga240 aaaaaaggagcaaatgagaagcct , 264 <210> 212 <211> 328 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(328) <223> n = A,T,C or G

cattacaaaactgctcaaattgtttgttaagnttatccattataattagttnggcaggag420 ctaata <400> 212 acccaaaaatccaatgctgaatatttggcttcattattcccanattctttgattgtcaaa60 ggatttaatgttgtctcagcttgggca~ttcagttaggacctaaggatgccagccggcag120 gtttatatatgcagcaacaatattcaagcgcgacaacaggttattgaacttgcccgccag180 ttnaatttcattcccattgacttgggatccttatcatcagccagagagattgaaaattta240 cccctacnactctttactctctgganagggccagtggtggtagctataagcttggccaca300 tttttttttcctttattcctttgtcaga 328 <210> 213 .<211> 250 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(250) <223> n = A,T,C or G
<400> 213 acttatgagcagagcgacatatccnagtgtagactgaataaaactgaattctctccagtt60 taaagcattgctcactgaagggatagaagtgactgccaggagggaaagtaagccaaggct120 cattatgccaaagganatatacatttcaattctccaaacttcttcctcattccaagagtt180 ttcaatatttgcatgaacctgctgataanccatgttaanaaacaaatatctctctnacct240 tctcatcggt 250 <210> 214 <211> 444 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(444) <223> n = A,T,C or G
<400>

acccagaatccaatgctgaatatttggcttcattattcccagattctttgattgtcaaag60 gatttaatgttgtctcagcttgggcacttcagttaggacctaaggatgccagccggcagg120 tttatatatgcagcaacaatattcaagcgcgacaacaggttattgaacttgcccgccagt180 tgaatttcattcccattgacttgggatccttatcatcagccanagagattgaaaatttac240 ccctacgactctttactctctggagagggccagtggtggtagctataagcttggccacat300 ttttttttcctttattcctttgtcagagatgcgattcatccatatgctanaaaccaacag360 agtgacttttacaaaattcctataganattgtgaataaaaccttacctatagttgccatt420 actttgctctccctaatatacctc , 444 <210> 215 <211> 366 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(366) <223> n = A,T,C or G
<400> 215 acttatgagc agagcgacat atccaagtgt anactgaata aaactgaatt ctctccagtt 60 taaagcattgctcactgaagggatagaagtgactgccaggagggaaagtaagccaaggct120 cattatgccaaagganatatacatttcaattctccaaacttcttcctcattccaagagtt180 ttcaatatttgcatgaacctgctgataagccatgttgagaaacaaatatctctctgacct240 tctcatcggtaagcagaggctgtaggcaacatggaccatagcgaanaaaaaacttagtaa300 tccaagctgttttctacactgtaaccaggtttccaaccaaggtggaaatctcctatactt360 ggtgcc 366 <210> 216 <211> 260 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(260) <223> n = A,T,C or G
<400> 216 ctgtataaac agaactccac tgcangaggg agggccgggc caggagaatc tccgcttgtc 60 caagacaggg gcctaaggag ggtctccaca ctgctnntaa gggctnttnc atttttttat 120 taataaaaag tnnaaaaggc ctcttctcaa cttttttccc ttnggctgga aaatttaaaa 180 atcaaaaatt tcctnaagtt ntcaagctat catatatact ntatcctgaa aaagcaacat 240 aattcttcct tccctccttt 260 <210> 217 <211> 262 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(262) <223> n = A,T,C or G
<400>

acctacgtgggtaagtttanaaatgttataatttcaggaanaggaacgcatataattgta60 tcttgcctataattttctattttaataaggaaatagcaaattggggtggggggaatgtag120 ggcattctacagtttgagcaaaatgcaattaaatgtggaaggacagcactgaaaaatttt180 atgaataatctgtatgattatatgtctctagagtagatttataattagccacttacccta240 atatccttcatgcttgtaaagt 262 <210> 218 <211> 205 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(205) <223> n = A,T,C or G
<400> 218 accaaggtgg tgcattaccg gaantggatc aangacacca tcgtggccaa cccctgagca 60 cccctatcaa ctcccttttg tagtaaactt ggaaccttgg aaatgaccag gccaagactc 120 aggcctcccc agttctactg acctttgtcc ttangtntna ngtccagggt tgctaggaaa 180 anaaatcagc agacacaggt gtaaa 205 <210> 219 <211> 114 <212> DNA
<213> Homo sapien <400> 219 tactgttttg tctcagtaac aataaataca aaaagactgg ttgtgttccg gccccatcca 60 accacgaagt tgatttctct tgtgtgcaga gtgactgatt ttaaaggaca tgga 114 <210> 220 <211> 93 <212> DNA' <213> Homo sapien <400> 220 actagccagc acaaaaggca gggtagcctg aattgcttt~ tgctctttac atttctttta 60 aaataagcat ttagtgctca gtccctactg agt 93 <210> 221 <211> 167 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(167) <223> n = A,T,C or G
<400> 221 actangtgca ggtgcgcaca aatatttgtc gatattccct tcatcttgga ttccatgagg 60 tcttttgccc agcctgtggc tctactgtag taagtttctg ctgatgagga gccagnatgc 120 cccccactac cttccctgac gctccccana aatcacccaa cctctgt 167 <210> 222 <211> 351 <212> DNA
<213> Homo sapien <400> 222 agggcgtggtgcggagggcggtactgacctcattagtaggaggatgcattctggcacccc60 gttcttcacctgtcccccaatccttaaaaggccatactgcataaagtcaacaacagataa120 atgtttgctgaattaaaggatggatgaaaaaaattaataatgaatttttgcataatccaa180 ttttctcttttatatttctagaagaagtttctttgagcctattagatcccgggaatcttt240 taggtgagcatgattagagagcttgtaggttgcttttacatatatctggcatatttgagt300 ctcgtatcaaaacaatagattggtaaaggtggtattattgtattgataagt 351 <210> 223 <211> 383 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(383) <223> n = A,T,C or G
<400> 223 aaaacaaaca aacaaaaaaa acaattcttc attcagaaaa attatcttag ggactgatat 60 tggtaattat ggtcaattta atwrtrttkt ggggcatttc cttacattgt cttgacaaga 120 ttaaaatgtctgtgccaaaattttgtattttatttggagacttcttatcaaaagtaatgc180 tgccaaaggaagtctaaggaattagtagtgttcccmtcacttgtttggagtgtgctattc240 taaaagattttgatttcctggaatgacaattatattttaactttggtgggggaaanagtt300 ataggaccacagtcttcacttctgatacttgtaaattaatcttttattgcacttgttttg360 accattaagctatatgtttaaaa 383 <210> 224 <211> 320 <212> DNA
<213> Homo sapien <400> 224 cccctgaagg cttcttgtta gaaaatagta cagttacaac caataggaac aacaaaaaga 60 aaaagtttgt gacattgtag tagggagtgt gtacccctta ctccccatca aaaaaaaaat 120 ggatacatgg ttaaaggata raagggcaat attttatcat atgttctaaa agagaaggaa 180 gagaaaatac tactttctcr aaatggaagc ccttaaaggt gctttgatac tgaaggacac 240 aaatgtggcc gtccatcctc ctttaragtt gcatgacttg gacacggtaa ctgttgcagt 300 tttaractcm gcattgtgac 320 <210> 225 <211> 1214 <212> DNA
<213> Homo sapien <400> 225 gaggactgcagcccgcactcgcagccctggcaggcggcactggtcatggaaaacgaattg60 ttctgctcgggcgtcctggtgcatccgcagtgggtgctgtcagccgcacactgtttccag120 aactcctacaccatcgggctgggcctgcacagtcttgaggccgaccaagagccagggagc180 cagatggtggaggccagcctctccgtacggcacccagagtacaacagacccttgctcgct240 aacgacctcatgctcatcaagttggacgaatccgtgtccgagtctgacaccatccggagc300 atcagcattgcttcgcagtgccctaccgcggggaactcttgcctcgtttctggctggggt360 ctgctggcgaacggcagaatgcctaccgtgctgcagtgcgtgaacgtgtcggtggtgtct420 gaggaggtctgcagtaagctctatgacccgctgtaccaccccagcatgttctgcgccggc480 ggagggcaagaccagaaggactcctgcaacggtgactctggggggcccctgatctgcaac540 gggtacttgcagggccttgtgtctttcggaaaagccccgtgtggccaagttggcgtgcca600 ggtgtctacaccaacctctgcaaattcactgagtggatagagaaaaccgtccaggccagt660 taactctggggactgggaacccatgaaattgacccccaaatacatcctgcggaaggaatt720 caggaatatctgttcccagcccctcctccctcaggcccaggagtccaggcccccagcccc780 tcctccctcaaaccaagggtacagatccccagcccctcctccctcagacc'caggagtcca840 gaccccccagcccctcctccctcagacccaggagtccagcccctcctccctcagacccag900 gagtccagaccccccagcccctcctccctcagacccaggggtccaggcccccaacccctc960 ctccctcagactcagaggtccaagcccccaacccctccttccccagacccagaggtccag1020 gtcccagcccctcctccctcagacccagcggtccaatgccacctagactctccctgtaca1080 cagtgcccccttgtggcacgttgacccaaccttaccagttggtttttcattttttgtccc1140 tttcccctagatccagaaataaagtctaagagaagcgcaaaaaaaaaaaaaaaaaaaaaa1200 aaaaaaaaaaaaaa 1214 <210> 226 <211> 119 <212> DNA
<213> Homo sapien <400> 226 acccagtatg tgcagggaga cggaacccca tgtgacagcc cactccacca gggttcccaa 60 agaacctggc ccagtcataa tcattcatcc tgacagtggc aataatcacg ataaccagt 119 <210> 227 <211> 818 <212> DNA
<213> Homo sapien <400>

acaattcatagggacgaccaatgaggacagggaatgaacccggctctcccccagccctga60 tttttgctacatatggggtcccttttcattctttgcaaaaacactgggttttctgagaac120 acggacggttcttagcacaatttgtgaaatctgtgtaraaccgggctttgcaggggagat180 aattttcctcctctggaggaaaggtggtgattgacaggcagggagacagtgacaaggcta240 gagaaagccacgctcggccttctctgaaccaggatggaacggcagacccctgaaaacgaa300 gcttgtcccc,ttccaatcagccacttctgagaacccccatctaacttcctactggaaaag360 agggcctcctcaggagcagtccaagagttttcaaagataacgtgacaactaccatctaga420 ggaaagggtgcaccctcagcagagaagccgagagcttaactctggtcgtttccagagaca480 acctgctggctgtcttgggatgcgcccagcctttgagaggccactaccccatgaacttct540 gccatccactggacatgaagctgaggacactgggcttcaacactgagttgtcatgagagg600 gacaggctctgccctcaagccggctgagggcagcaaccactctcctcccctttctcacgc660 aaagccattcccacaaatccagaccataccatgaagcaacgagacccaaacagtttggct720 caagaggatatgaggactgtctcagcctggctttgggctgacaccatgcacacacacaag780 gtccacttctaggttttcagcctagatgggagtcgtgt 818 <210> 228 <211> 744 <212> DNA
<213> Homo sapien <400>

actggagacactgttgaacttgatcaagacccagaccaccccaggtctccttcgtgggat60 gtcatgacgtttgacatacctttggaacgagcctcctccttggaagatggaagaccgtgt120 tcgtggccgacctggcctctcctggcctgtttcttaagatgcggagtcacatttcaatgg180 taggaaaagtggct~cgtaaaatagaagagcagtcactgtggaactaccaaatggcgaga240 tgctcggtgcacattggggtgctttgggataaaagatttatgagccaactattctctggc300 accagattctaggccagtttgttccactgaagcttttcccacagcagtccacctctgcag360 gctggcagctgaatggcttgccggtggctctgtggcaagatcacactgagatcgatgggt420 gagaaggctaggatgcttgtctagtgttcttagctgtcacgttggctccttccaggttgg480 ccagacggtgttggccactcccttctaaaacacaggcgccctcctggtgacagtgacccg540 ccgtggtatgccttggcccattccagcagtcccagttatgcatttcaagtttggggtttg600 ttcttttcgttaatgttcctctgtgttgtcagctgtcttcatttcctgggctaagcagca660 ttgggagatgtggaccagagatccactccttaagaaccagtggcgaaagacactttcttt720 cttcactctgaagtagctggtggt 744 <2l0> 229 <211> 300 <212> DNA
<213> Homo sapien <400> 229 cgagtctggg ttttgtctat aaagtttgat ccctcctttt ctcatccaaa tcatgtgaac 60 cattacacat cgaaataaaa gaaaggtggc agacttgccc aacgccaggc tgacatgtgc 120 tgcagggttg ttgtttttta attattattg ttagaaacgt cacccacagt ccctgttaat 180 ttgtatgtga cagccaactc tgagaaggtc ctatttttcc acctgcagag gatccagtct 240 cactaggctc ctccttgccc tcacactgga gtctccgcca gtgtgggtgc ccactgacat 300 <210> 230 <211> 301 <212> DNA
<213> Homo sapien <400> 230 cagcagaaca aatacaaata tgaagagtgc aaagatctca taaaatctat gctgaggaat 60 gagcgacagttcaaggaggagaagcttgcagagcagctcaagcaagctgaggagctcagg120 caatataaagtcctggttcacactcaggaacgagagctgacccagttaagggagaagttg180 cgggaagggagagatgcctccctctcattgaatgagcatctccaggccctcctcactccg240 gatgaaccggacaagtcccaggggcaggacctccaagaaacagacctcggccgcgaccac300 g 301 <210> 231 <211> 301 <212> DNA
<213> Homo sapien <400> 231 gcaagcacgc tggcaaatct ctgtcaggtc agctccagag aagccattag tcattttagc 60 caggaactcc aagtccacat ccttggcaac tggggacttg cgcaggttag ccttgaggat 120 ggcaacacgg gacttctcat caggaagtgg gatgtagatg agctgatcaa gacggccagg 180 tctgaggatg gcaggatcaa tgatgtcagg ccggttggta ccgccaatga tgaacacatt 240 tttttttgtg gacatgccat ccatttctgt caggatctgg ttgatgactc ggtcagcagc 300 c 301 <210> 232 <211> 301 <212> DNA
<213> Homo sapien <400> 232 agtaggtatttcgtgagaagttcaacaccaaaactggaacatagttctccttcaagtgtt60 ggcgacagcggggcttcctgattctggaatataactttgtgtaaattaacagccacctat120 agaagagtccatctgctgtgaaggagagacagagaactctgggttccgtcgtcctgtcca180 cgtgctgtaccaagtgctggtgccagcctgttacctgttctcactgaaaatctggctaat240 gctcttgtgtatcacttctgattctgacaatcaatcaatcaatggcctagagcactgact300 g 301 <210> 233 <211> 301 <212> DNA
<213> Homo sapien <400> 233 atgactgacttcccagtaaggctctctaaggggtaagtaggaggatccacaggatttgag60 atgctaaggccccagagatcgtttgatccaaccctcttattttcagaggggaaaatgggg120 cctagaagttacagagcatctagctggtgcgctggcacccctggcctcacacagactccc180 gagtagctgggactacaggcacacagtcactgaagcaggccctgttagcaattctatgcg240 tacaaattaacatgagatgagtagagactttattgagaaagcaagagaaaatcctatcaa300 c 301 <210> 234 <21l> 301 <212> DNA
<213> Homo sapien <400> 234 aggtcctacacatcgagactcatccatgattgatatgaatttaaaaattacaagcaaaga60 cattttattcatcatgatgctttcttttgtttcttcttttcgttttcttctttttctttt120 tcaatttcagcaacatacttctcaatttcttcaggatttaaaatcttgagggattgatct180 cgcctcatgacagcaagttcaatgtttttgccacctgactgaaccacttccaggagtgcc240 ttgatcaccagcttaatggtcagatcatctgcttcaatggcttcgtcagtatagttcttc300 t 301 <210> 235 <211> 283 <212> DNA
<213> Homo sapien <400> 235 tggggctgtg catcaggcgg gtttgagaaa tattcaattc tcagcagaag ccagaatttg 60 aattccctca tcttttaggg aatcatttac caggtttgga gaggattcag acagctcagg 120 tgctttcact aatgtctctg aacttctgtc cctctttgtt catggatagt ccaataaata 180 atgttatctt tgaactgatg ctcataggag agaatataag aactctgagt gatatcaaca 240 ttagggattc aaagaaatat tagatttaag ctcacactgg tca ~ 283 <210> 236 <211> 301 <212> DNA
<213> Homo sapien <400>'236 aggtcctccaccaactgcctgaagcacggttaaaattgggaagaagtatagtgcagcata60 aatacttttaaatcgatcagatttccctaacccacatgcaatcttcttcaccagaagagg120 tcggagcagcatcattaataccaagcagaatgcgtaatagataaatacaatggtatatag180 tgggtagacggcttcatgagtacagtgtactgtggtatcgtaatctggacttgggttgta240 aagcatcgtgtaccagtcagaaagcatcaatactcgacatgaacgaatataaagaacacc300 a 301 <210> 237 <211> 301 <212> DNA
<213> Homo sapien <400> 237 cagtggtagt ggtggtggac gtggcgttgg tcgtggtgcc ttttttggtg cccgtcacaa 60 actcaatttt tgttcgctcc tttttggcct tttccaattt gtccatctca attttctggg 120 ccttggctaa tgcctcatag taggagtcct cagaccagcc atggggatca aacatatcct 180 ttgggtagtt ggtgccaagc tcgtcaatgg cacagaatgg atcagcttct cgtaaatcta 240 gggttccgaa attctttctt cctttggata atgtagttca tatccattcc ctcctttatc 300 t 301 <210> 238 <211> 301 <212> DNA
<213> Homo sapien <400> 238 gggcaggttt tttttttttt ttttttgatg gtgcagaccc ttgctttatt tgtctgactt 60 gttcacagtt cagccccctg ctcagaaaac caacgggcca gctaaggaga ggaggaggca 120 ccttgagact tccggagtcg aggctctcca gggttcccca gcccatcaat cattttctgc 180 accccctgcc tgggaagcag ctccctgggg ggtgggaatg ggtgactaga agggatttca 240 gtgtgggacc cagggtctgt tcttcacagt aggaggtgga agggatgact aatttcttta 300 t 301 <210> 239 <211> 239 <212> DNA
<213> Homo sapien <400> 239 ataagcagct agggaattct ttatttagta atgtcctaac ataaaagttc acataactgc 60 ttctgtcaaa ccatgatact gagctttgtg acaacccaga aataactaag agaaggcaaa 120 cataatacct tagagatcaa gaaacattta cacagttcaa ctgtttaaaa atagctcaac 180 attcagccag tgagtagagt gtgaatgcca gcatacacag tatacaggtc cttcaggga 239 <210> 240 <211> 300 <212> DNA
<213> Homo sapien <400>

ggtcctaatgaagcagcagcttccacattttaacgcaggtttacggtgatactgtccttt60 gggatctgccctccagtggaaccttttaaggaagaagtgggcccaagctaagttccacat120 gctgggtgagccagatgacttctgttccctggtcactttcttcaatggggcgaatggggg180 ctgccaggtttttaaaatcatgcttcatcttgaagcacacggtcacttcaccctcctcac240 gctgtgggtgtactttgatgaaaatacccactttgttggcctttctgaagctataatgtc300 <210> 241 <211> 301 <212> DNA
<213> Homo sapien <400>

gaggtctggtgctgaggtctctgggctaggaagaggagttctgtggagctggaagccaga60 cctctttggaggaaactccagcagctatgttggtgtctctgagggaatgcaacaaggctg120 ctcctccatgtattggaaaactgcaaactggactcaactggaaggaagtgctgctgccag180 tgtgaagaaccagcctgaggtgacagaaacggaagcaaacaggaacagccagtcttttct240 tcctcctcctgtcatacggtctctctcaagcatcctttgttgtcaggggcctaaaaggga300 g 301 <210> 242 <211> 301 <212> DNA
<213> Homo sapien <400>

ccgaggtcctgggatgcaaccaatcactctgtttcacgtgacttttatcaccatacaatt60 tgtggcatttcctcattttctacattgtagaatcaagagtgtaaataaatgtatatcgat120 gtcttcaagaatatatcattcctttttcactagaacccattcaaaatataagtcaagaat180 cttaatatcaacaaatatatcaagcaaactggaaggcagaataactaccataatttagta240 taagtacccaaagttttataaatcaaaagccctaatgataaccatttttagaattcaatc300 a 301 <210> 243 <211> 301 <212> DNA
<213> Homo sapien <400> 243 aggtaagtcccagtttgaagctcaaaagatctggtatgagcataggctcatcgacgacat60 ggtggcccaagctatgaaatcagagggaggcttcatctgggcctgtaaaaactatgatggl20 tgacgtgcagtcggactctgtggcccaagggtatggctctctcggcatgatgaccagcgt180 gctggtttgtccagatggcaagacagtagaagcagaggctgcccacgggactgtaacccg240 tcactaccgcatgttccagaaaggacaggagacgtccaccaatcccattgcttccatttt300 t 301 <210> 244 <211> 300 <212> DNA

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

~~ TTENANT LES PAGES 212 A 288 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
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NOTE POUR LE TOME / VOLUME NOTE:

Claims

What is Claimed:

1. An isolated polynucleotide comprising a sequence selected from the group consisting of:
(a) sequences provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(b) complements of the sequences provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, '79-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and,384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;
(d) sequences that hybridize to a sequence provided in SEQ ID NO:
1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942 under moderately stringent conditions;
(e) sequences having at least 75% identity to a sequence of SEQ ID
NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942;

(f) sequences having at least 90% identity to a sequence of SEQ ID
NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942; and (g) degenerate variants of a sequence provided in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939 and 942.

2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 866-877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943;
(b) sequences having at least 70% identity to a sequence of SEQ ID
NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, S73-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943;
(c) sequences having at least 90% identity to a sequence of SEQ ID
NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 877, 879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941 and 943;
(d) sequences encoded by a polynucleotide of claim 1;

(e) sequences having at least 70% identity to a sequence encoded by a polynucleotide of claim 1; and (f) sequences having at least 90% identity to a sequence encoded by a polynucleotide of claim 1.

3. An expression vector comprising a polynucleotide of claim 1 operably linked to an expression control sequence.

4. A host cell transformed or transfected with an expression vector according to claim 3.

5. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide of claim 2.

6. A method for detecting the presence of a cancer in a patient, comprising the steps of:
(a) obtaining a biological sample from the patient;
(b) contacting the biological sample with a binding agent that binds to a polypeptide of claim 2;
(c) detecting in the sample an amount of polypeptide that binds to the binding agent; and (d) comparing the amount of polypeptide to a predetermined cut-off value and therefrom determining the presence of a cancer in the patient.

7. A fusion protein comprising at least one polypeptide according to claim 2.

8. An oligonucleotide that hybridizes to a sequence recited in SEQ
ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-93I, 938, 939 and 942 under moderately stringent conditions.

9. A method for stimulating and/or expanding T cells specific for a tumor protein, comprising contacting T cells with at least one component selected from the group consisting of:
(a) polypeptides according to claim 2;
(b) polynucleotides according to claim 1; and (c) antigen-presenting cells that express a polypeptide according to claim 2, under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.

10. An isolated T cell population, comprising T cells prepared according to the method of claim 9.

11. A composition comprising a first component selected from the group consisting of physiologically acceptable carriers and immunostimulants, and a second component selected from the group consisting of:
(a) polypeptides according to claim 2;
(b) polynucleotides according to claim 1;
(c) antibodies according to claim 5;
(d) fusion proteins according to claim 7;
(e) T cell populations according to claim 10; and (f) antigen presenting cells that express a polypeptide according to claim 2.

12. A method for stimulating an immune response in a patient, comprising administering to the patient a composition of claim 11.

13. A method for the treatment of a cancer in a patient, comprising administering to the patient a composition of claim 11.

14. A method for determining the presence of a cancer in a patient, comprising the steps of:
(a) obtaining a biological sample from the patient;
(b) contacting the biological sample with an oligonucleotide according to claim 8;
(c) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; and (d) compare the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence of the cancer in the patient.

15. A diagnostic kit comprising at least one oligonucleotide according to claim 8.

16. A diagnostic kit comprising at least one antibody according to claim 5 and a detection reagent, wherein the detection reagent comprises a reporter group.

17. A method for inhibiting the development of a cancer in a patient, comprising the steps of:
(a) incubating CD4+ and/or CD8+ T cells isolated from a patient with at least one component selected from the group consisting of: (i) polypeptides according to claim 2; (ii) polynucleotides according to claim 1; and (iii) antigen presenting cells that express a polypeptide of claim 2, such that T cell proliferate;
(b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient.