JP2002534056A - Preparation and use of excellent vaccines - Google Patents

Abstract

  (57) [Summary] The present invention provides an isolated population of polynucleotides comprising or corresponding to at least one of the polynucleotides shown in Table 1 and their respective complements. Further, a polynucleotide encoding a ligand or an antibody that binds to a cell surface portion of an antigen presenting cell, or a polynucleotide encoding an engineering protein, wherein the polynucleotide contains or corresponds to the polynucleotide shown in Table 1 or a complement thereof. provide. The invention further provides a polynucleotide encoding a transcription factor, wherein the polynucleotide comprises or corresponds to a polynucleotide set forth in Table 1 or a complement thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] TECHNICAL FIELD The present invention relates to the use of human malignancies, such as cancer (malignancy), relates to the immunotherapy was enhanced. Background The complex relationship between the immune system and tumor cells during the course of disease development has not been fully understood. However, simply the fact that the host immune system has the potential to recognize and eventually eradicate tumor cells guarantees that immunotherapy is one of the most promising approaches for treating cancer. . Most tumors express altered or abnormal gene products as a result of unregulated cell growth and malignant transformation. These aberrant gene products are often immunogenic to the host immune system and can render tumor cells susceptible to immune cell lysis. Gilboa et al. (1998) Cancer Imm. Immuno
ther. 46: 82-87.

[0002] Cytotoxic T lymphocyte (CTL) -mediated cellular immunity is considered an important weapon in the host defense system against many tumors. A variety of molecular factors determine whether a tumor cell can be recognized by the host immune system and eventually lysed into CTL. Lindauer et al. Mol. Med. 76:32
-47. Tumor-associated antigens are proteolytically degraded to small peptide epitopes that compete for binding to and presentation by a limited number of major histocompatibility complex (MHC) molecules. The formed MHC-peptide complex can be recognized by native CTL via its T cell receptor. Further activation of untreated CTL requires the function of a costimulator, most of which is associated with professional antigen presenting cells (APCs). Activating antigen-specific CTLs then differentiate into cytolytic effector cells capable of lysing tumor cells bearing a particular tumor antigen.

[0003] A variety of tumor-associated antigens have been identified to date, most of which are melanoma-associated. Lindauer et al., Supra. Tumor-associated antigens can be divided into four classes: differentiation antigens, such as gp100, which are normal proteins that are overexpressed by tumor cells; viral antigens, such as HPV16 E6 and E7; MAGE
And a mutated protein such as ras or p53. All of these tumor antigens, when properly processed and conveniently presented by MHC molecules, can be targeted for recognition and binding by T cell receptors.

[0004] However, merely presenting tumor antigens via the MHC and subsequently being recognized by the T cell receptor activates a strong cytotoxic immune response capable of lysing tumor cells. Is not enough. Effective CTL activation requires many co-factors with immunostimulatory functions. Indeed, the binding and stimulation of T cell receptors in the absence of these cofactors can render T cells unresponsive to additional antigenic stimuli, a condition that has led to many tumor self- Is an anergic state that may be responsible for tolerance to

[0005] Auxiliary stimulatory functions have been primarily associated with specialized antigen presenting cells (APCs). For example, upon exposure to certain signals, such as inflammatory agents, APCs have the ability to upregulate T cell proliferation and IL-2 production, which are processes required for CTL activation. APCs also secrete T cell growth factors and produce antigen-specific C
It is also known to amplify the TL reaction. In addition, activated APCs secrete chemokines capable of inducing the migration of immune effector cells to the site of antigen presentation; or, such as draining lymphocytes, where favorable APC: T cell interactions may occur. By migrating to sites rich in T cells, antigen presentation and CT
It is possible to provide a favorable lymphatic environment for L activation.

[0006] For antigen presentation, a transgene encoding a tumor antigen in vivo was transformed by APC
Although a variety of gene-based vaccines have been used to deliver E. coli, few have proven to be effective in eliciting anti-tumor immune activity. Vaccines based on these genes, including genetically modified APCs, may be effective at presenting antigens, but have a functional status of endogenous APCs, such as stimulation / enhancement of T cell activation. Does not provide any adjustment. The present invention relates to this limitation,
And there is provided an enhanced vaccine composition for eliciting an effective anti-tumor immune response. DISCLOSURE OF THE INVENTION The present invention provides an isolated population of polynucleotides comprising or corresponding to at least one of the polynucleotides set forth in Table 1 and their respective complements. The invention also provides a polynucleotide encoding a ligand or antibody or a recombinant protein that binds to a cell surface protein of an antigen presenting cell, wherein the polynucleotide is a polynucleotide set forth in Table 1 or a complement thereof. Or corresponding to the polynucleotide or complement. The invention further provides a polynucleotide encoding a transcription factor, wherein the polynucleotide comprises or corresponds to a polynucleotide set forth in Table 1 or a complement thereof.

[0007] Further provided herein is comprising encoding an immunostimulatory factor that is differentially expressed in antigen presenting cells, and comprising or comprising the tag shown in Table 1 or a complement thereof. Or corresponding to the complement, including the first polynucleotide,
It is a polynucleotide. In one embodiment, the first polynucleotide is P
Encodes a factor selected from the group consisting of ARC, TARC, monocyte chemoattractant protein-4 (MDP-4), MDC, escalectin, MCP-2 or biologically active fragments thereof. . The polynucleotide may further include first and second promoters, wherein the first and second polynucleotides are under the transcriptional control of the first and second promoters, respectively.

[0008] Also provided in the present invention includes encoding an immunostimulatory factor that is differentially expressed in antigen presenting cells, and comprises or comprises a tag set forth in Table 1 or a complement thereof. A polynucleotide comprising a first polynucleotide corresponding to a complement, as well as a second polynucleotide that regulates expression of the first polynucleotide. Also provided are a first polynucleotide that encodes an antigen and a second polynucleotide that regulates expression of a third polynucleotide that encodes an immunostimulatory factor that is differentially expressed in antigen presenting cells , Wherein the third polynucleotide comprises or corresponds to a tag set forth in Table 1. The first polynucleotide may encode PARC, monocyte chemoattractant protein-4 (MDP-4), MDC, escalectin, MCP-2 or a biologically active fragment thereof. Also provided herein is a first polynucleotide that encodes a designed protein or polypeptide that binds to a cell surface protein of an antigen presenting cell, and thereby regulates directly or indirectly through a signaling pathway, and A polynucleotide comprising a second polynucleotide comprising a tag set forth in Table 1 or encoding an immunostimulatory factor corresponding to said tag. A promoter may be operably linked to the polynucleotide to direct its expression.

[0009] The polynucleotide may be inserted into a gene delivery vehicle or host cell. Alternatively, it may be linked to a chip or a database for computational analysis.

[0010] The compositions of the present invention are useful for inducing an immune response in a subject. They are also useful for screening for candidate therapeutic agents that modulate the genotype of antigen presentation and that modulate the expression of polynucleotides that are differentially expressed in antigen presenting cells. BRIEF DESCRIPTION OF THE TABLES The table shows a series of mRNA sequences identified by SAGE analysis. Tags isolated from diverse populations were isolated and analyzed: tags expressed on monocytes; tags expressed on monocyte-derived immature dendritic cells; and stimulated and matured using TNFα. These are tags expressed in monocyte-derived mature dendritic cells. The vertical columns of the table are as follows: "Deposit" is the deposit number of the EST in the public database; "Tag" is the 10-mer SAGE tag; "SEQ ID NO" is SEQ ID NO: of the corresponding tag found in the claim. The description identifies the known gene or EST corresponding to the tag. If the description is blank or "N
If "M" is included, no match was found and the tag is identified as a new tag. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Unless otherwise indicated, the practice of the present invention will involve the use of conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. use. Such techniques are explained fully in the literature. These methods are
It is described in the following publications. For example, Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd edition (1
989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (edited by FM Ausubel et al., (1987)); a series of METHODS IN ENZY MOLOGY (Academic Press)
, Inc. ); "PCR: A PRACTICAL APPROACH" (
M. MacPherson et al., IRL Press at Oxford.
University Press (1991)); PCR 2: APR
ACTICAL APPROACH (MJ MacPherson, B.S.
D. Hames and G.W. R. Supervised by Taylor (1995)); ANT
IBODIES: A LABORATORY MANUAL (supervised by Harlow and Lane (1988)); and ANIMAL CELL CULTU
See RE (edited by RI Freshney (1987)).

As used herein, certain terms may have the meanings defined below. The singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells and includes a mixture thereof.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of," as used in defining compositions and methods, means excluding other elements of essential significance to the combination. Thus, a composition consisting essentially of elements as defined herein may contain trace amounts of contaminants from isolation and purification methods, as well as phosphate buffered saline, preservatives and the like. Will not exclude pharmaceutically acceptable carriers. "Consisting of"
Means eliminating more than trace elements of other components and substantial method steps for administering the compositions of the present invention. These transition terms (transititi
Embodiments defined by each of the (on term) are within the scope of the present invention.

The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably to refer to a polymeric form of nucleotides of any length. A polynucleotide may include deoxyribonucleotides, ribonucleotides, and / or analogs thereof. Nucleotides may have any three-dimensional structure and may perform any function, known or unknown. The term "polynucleotide" includes, for example, single, double-stranded and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids , Vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may also include modified nucleic acid molecules.

“Gene” refers to a polynucleotide comprising at least one open reading frame capable of encoding a particular polypeptide or protein after being transcribed and translated.

“Gene product” refers to an amino acid (eg, a peptide or polypeptide) produced when a gene is transcribed and translated. As used herein, a second polynucleotide “corresponds” to a first polynucleotide if it is related to another (first) polynucleotide by any of the following relationships: 1) The second polynucleotide comprises the first polynucleotide, and the second polynucleotide encodes the gene product. 2) The second polynucleotide is 5 'or 3' of the first polynucleotide in cDNA, RNA, genomic DNA, or a fragment of any of these polynucleotides. For example, the second polynucleotide may be a fragment of a gene containing the first and second polynucleotides. The first and second polynucleotides are related in that they are components of a gene that encodes a gene product, eg, a protein or antibody. However, it is not necessary herein that the second polynucleotide comprises or overlaps with the first polynucleotide to be included within the definition of "corresponding". For example, the first polynucleotide may be a fragment of the 3 'untranslated region of the second polynucleotide, and may include, for example, the promoter sequence of the gene containing the tag. The first and second polynucleotides may be fragments of a gene encoding a gene product. The second polynucleotide may be an exon of the gene, while the first polynucleotide may be an intron of the gene. 3) The second polynucleotide is the complement of the first polynucleotide.

“Foreign polynucleotide” is a DNA sequence that is foreign to the cell, vector or position therein in which it is located. A “sequence tag” or “tag” or “SAGE tag” is a short oligonucleotide containing a defined nucleotide sequence that occurs at a particular position in a gene transcript. The length of the tag is generally less than about 20 nucleotides, preferably
It is between 9 and 15 nucleotides, and most preferably 10 nucleotides. Tags may be used to identify the corresponding transcript and the gene to which the transcript is transcribed. The tag may further include an exogenous nucleotide sequence that facilitates the identification and utility of the tag. Such auxiliary sequences include, but are not limited to, restriction endonuclease cleavage sites, and well-known primer sequences for sequencing and cloning.

The term “peptide”, in its broadest sense, refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics (pept
idomimetics). The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as ester, ether bonds, and the like. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids and includes both glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A peptide of three or more amino acids is
If the peptide chain is short, it is commonly called an oligopeptide. If the peptide chain is long, the peptide is commonly called a polypeptide or protein.

The term “cDNA” refers to complementary DNA, which is an enzyme such as reverse transcriptase,
An mRNA molecule present in a cell or organism that has been converted to DNA. A "cDNA library" is a collection in which all mRNA molecules present in a cell or organism are converted into cDNA molecules by reverse transcriptase and then inserted into a "vector".

“Probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that hybridizes to a target and thereby serves as a reagent for detecting a target that may be present in a sample of interest. Usually, the probe will include a label or a means to which the label can bind, before or after the hybridization reaction. Suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes.

A “primer” binds to a target or “template” that may be present in a sample of interest by hybridizing to the target, and then facilitates the polymerization of a polynucleotide complementary to the target. , Generally unbound (free) 3
'Short polynucleotide with -OH groups. "Polymerase chain reaction" (
“PCR”) is a “primer pair” consisting of “upstream” and “downstream” primers.
Or a "primer set", as well as a catalyst for polymerization, such as a DNA polymerase,
And typically using a thermostable polymerase enzyme to make a duplicate copy of the target polynucleotide. Methods of PCR are well known in the art,
For example, “PCR: A PRACTICAL APPROACH” (M. Ma
cPherson et al., IRL Press at Oxford Unive.
rsity Press (1991)). All methods of producing duplicate copies of a polynucleotide, such as PCR or gene cloning,
In this specification, these are collectively referred to as "duplicates." Primers can also be used as probes in hybridization reactions such as Southern or Northern blot analysis. Sambrook et al., Supra.

A “promoter” is an RNA polymerase that binds and initiates transcription.
A region on a DNA molecule. In an operon, the promoter is usually located at the end of the operator and is close to, but external to, the operator. The nucleotide sequence of the promoter determines both the nature of the enzyme bound to it and the rate of RNA synthesis.

The term “genetically modified” means including and / or expressing a foreign gene or nucleic acid sequence that subsequently modifies the genotype or phenotype of the cell or its progeny.

As used herein, “expression” or “expressed” means that the polynucleotide is
Refers to the process by which RNA is transcribed or transcription is enhanced. In another aspect,
RNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA if a suitable eukaryotic host is chosen.

“Differentially expressed” when applied to a gene refers to the differential production of mRNA transcribed from the gene or the protein product encoded by the gene. Differentially expressed genes are compared to normal or control cell expression levels.
It may be overexpressed or underexpressed. In one aspect, the term refers to a difference that is 3-fold, preferably 5-fold, or preferably 10-fold higher or lower than the expression level detected in the control sample. The term "differentially expressed" also refers to a nucleotide sequence in a cell or tissue that is expressed when silent in control cells, or not expressed when expressed in control cells. Point to.

A “native” or “native” antigen is a polypeptide, protein or fragment containing an epitope, which has been isolated from a natural biological source and which, in a subject, has an antigen receptor, in particular a T Said polypeptide, protein or fragment capable of specifically binding to a cell antigen receptor (TCR). The antigen also
Along with a substance that is immunogenic, ie, an immunogen, also refers to a substance that is immunologically unresponsive or induces anergy, ie, anergen.

As used herein, “self-antigen” also refers to a natural or wild-type antigen that is an antigenic peptide that induces little or no immune response in a subject due to self-tolerance to the antigen. An example of an autoantigen is the human melanoma antigen gp 10
0.

The term “tumor-associated antigen” or “TAA” refers to an antigen associated with or specific for a tumor. Examples of known TAAs include gp100, MART and MA
GE.

The term “lysis” refers to the action of rupture of the cell wall and / or cell membrane of cells through cytotoxic T cell lymphocyte (CTL) -mediated cellular immunity. In a preferred embodiment, lysis of the cells is performed to release cell components from the lysed cells. For the purposes of the present invention, "cellular component" means any component found within a cell. Such components include, but are not limited to, proteins, lipoproteins, glycoproteins, lipids, carbohydrates, nucleic acids, steroids, prostaglandins, and combinations and complexes thereof. Components are also referred to as "endogenous antigens."

“Gene delivery vehicle” is defined as a molecule that can be any molecule capable of delivering an inserted polynucleotide into a host cell. Examples of gene delivery vehicles are liposomes, viruses such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and the art, which has been described for expression in a variety of eukaryotic and prokaryotic hosts. Other recombinant vehicles typically used in the field, and may be used for simple protein expression with gene therapy.

“Viral vectors” are in vivo, ex vivo or in vivo.
It is defined as a recombinantly produced virus or virus particle containing a polynucleotide to be delivered to a host cell in vitro. Examples of viral vectors include:
Retrovirus vectors, adenovirus vectors, adeno-associated virus vectors and their equivalents are included. In aspects where gene transfer is mediated by a retroviral vector, vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof, and a therapeutic gene. As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" refers to the entry of a virus into a cell and the integration of the viral genome into the genome of the host cell, thereby stabilizing the gene or nucleic acid sequence in the host cell. Has the same meaning as the process introduced and refers to the process. The virus may enter the host cell via the normal mechanism of infection, or may be modified to bind to a different host cell surface receptor or ligand for entry into the cell. As used herein, a retroviral vector refers to a viral particle capable of introducing an exogenous nucleic acid into a cell through a virus or virus-like entry mechanism.

The term “isolated” refers to the separation of a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, from cellular or other components normally associated with nature. Means that it is. Further, a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof that is "enriched,""separated," or "diluted" may have a concentration or number of molecules per volume that is More "enriched" or less "separated" than the naturally occurring counterpart distinguishes it from the naturally occurring counterpart. Although not explicitly mentioned for each of the inventions disclosed herein, for each of the compositions disclosed below, all of the above aspects, and under appropriate conditions, are provided herein. It should be understood that Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the naturally occurring polynucleotide that is being isolated. Proteins produced in bacterial cells are provided as a separate embodiment from naturally occurring proteins isolated from naturally produced eukaryotic cells.

[0033] A "host cell" or "recipient cell" is any individual that could be, or was, a recipient of a vector or uptake of an exogenous nucleic acid molecule, polynucleotide and / or protein. It is intended to also include cells or cell cultures of The term is intended to include the progeny of a single cell, and the progeny is not necessarily completely identical (in morphology or in genomic or total DNA complementation) to the original parent cell by natural, accidental or deliberate mutation. In the body). The cells can be prokaryotic or eukaryotic and include, but are not limited to, bacterial cells, yeast cells, animal cells, and mammalian cells, eg, murine, rat, monkey or human.

A “subject” is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in experiments for comparison purposes. Controls may be "positive" or "negative."
For example, if the purpose of the experiment is to determine the correlation between a particular type of cancer and the altered expression level of the proto-oncogene, generally a positive control (a syndrome with such alterations and characteristic of the disease) It is preferred to use a subject or a sample derived from the subject, and a negative control (a subject or a sample derived from the subject lacking altered expression and the clinical syndrome of the disease).

The term “major histocompatibility complex” or “MHC” refers to a complex of genes encoding cell surface molecules required for antigen presentation to T cells and rapid graft rejection. The proteins encoded by the MHC complex are known as "MHC molecules" and are classified into Class I and Class II MHC molecules. Class I
MHC molecules include membrane heterodimer proteins composed of chains encoded by MHC, non-covalently associated with b2-microglobulin. Class I M
HC molecules are expressed by almost all nucleated cells and have been shown to function in antigen presentation to CD8 ⁺ T cells. In humans, class I molecules include HLA-A, -B, and -C. Class II MHC molecules also include membrane heterodimeric proteins consisting of non-covalently linked a and b chains. Class II MHCs are known to be involved in antigen presentation to CD4 ⁺ T cells and include HLA-DP, -DQ, and DR in humans. The term "MHC restriction" allows an antigen to be recognized only after the antigen has been processed and the resulting antigenic peptide has been shown bound to either a self-class I or class II MHC molecule Refers to the characteristics of T cells. Methods for identifying and comparing MHC are well known in the art and
llen, M .; (1994) Human Immunol. 40: 25-
32; Santamaria, P.M. (1993) Human Immun.
ol. 37: 39-50; and Hurley, C.E. K. Et al. (1997) T
issue Antigens 50: 401-415.

The term “antigen presenting cell (APC)” refers to the induction of an effective cellular immune response against one or more antigens recognized by and presented by specific effector cells of the immune system. In the form of an antigen-MHC complex,
Refers to a type of cell capable of presenting one or more antigens. Many types of cells may be able to present antigen on their cell surface for T cell recognition, but only specialized APCs present antigen in effective amounts, and It has the ability to activate T cells for a cytotoxic T lymphocyte (CTL) response. APCs can be intact whole cells, such as macrophages, B cells and dendritic cells; or purified complexed to other naturally occurring or synthetic molecules, such as beta2-microglobulin. It may be an MHC class I molecule.

The term “dendritic cells (DC)” refers to a diverse population of morphologically similar cell types found in various lymphoid and non-lymphoid tissues (Stcinman (199
1) Ann. Rev .. Immunol. 9: 271-296). Dendritic cells constitute the most potent and preferred APC in organisms. At least a subset, if not all, of dendritic cells are derived from bone marrow progenitor cells, circulate in small numbers in peripheral blood, and appear as immature Langerhans cells or terminally differentiated mature cells. Dendritic cells may differentiate from monocytes, but have distinct phenotypes. For example, a particular differentiation marker, the CD14 antigen,
Absent or present at low levels, but carried by monocytes. Also, dendritic cells have no phagocytosis, whereas monocytes are strong phagocytic cells. It has been shown that DCs provide all the signals necessary for T cell activation and proliferation.

“Co-stimulatory molecules” are involved in interactions between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Studies accumulated over the past several years have shown that resting T cells are likely to require at least two signals for induction of cytokine gene expression and proliferation (Schw).
artz R.S. H. (1990) Science 248: 1349-1356.
And Jenkins M. K. (1992) Immunol. Today
13: 69-73). One signal that confers specificity can be generated by the interaction of the TCR / CD3 complex with the appropriate MHC / peptide complex. The second signal is not antigen-specific and is referred to as a "co-stimulation" signal. This signal was originally defined as the activity provided by bone marrow-derived accessory cells such as macrophages and dendritic cells, so-called "professional" APCs. Some molecules have been shown to enhance costimulatory activity. These are thermostable antigens (HSA) (Liu Y. et al. (1992) J. Exp. Med.
175: 437-445); Chondroitin sulfate-modified MHC invariant chain (ICS)
(Naujokas MF et al. (1993) Cell 74: 257-268.
); Cell Adhesion Molecule 1 (ICAM-1) (Van Eventer G.A.
1990) J. Immunol. 144: 4579-4586); and B
7-1 and B7-2 / B70 (Schwartz RH (1992) Ce)
111: 1065-1068). Co-stimulatory molecules include, for example, Beck
It is commercially available from a variety of sources, including man Coulter. Although not always explicitly mentioned, molecules having a biological activity similar to wild-type or purified co-stimulatory molecules (eg, recombinantly produced or muteins thereof) are: It is intended to be used within the spirit and scope of the present invention.

As used herein, the term “cytokine” refers to any one of a number of factors that affect a cell in a variety of ways, such as inducing growth or proliferation. Non-limiting examples of cytokines that may be used alone or in combination in the practice of the present invention include interleukin-2 (IL-2), stem cell factor (S
CF), interleukin 3 (IL-3), interleukin 6 (IL-6),
Interleukin 12 (IL-12), G-CSF, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1α)
), Interleukin-11 (IL-11), MIP-1, leukemia inhibitory factor (L
IF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The invention also includes culturing conditions in which one or more cytokines are specifically excluded from the medium. Cytokines are commercially available from a number of sources, such as Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R & D.
Systems (Minneapolis, MN) and Immunex (Seattle, WA). Although not always explicitly mentioned, molecules having biological activity similar to wild-type or purified cytokines (eg, recombinantly produced or muteins thereof) are within the spirit and scope of the present invention. It is intended to be used within.

The term “culturing” refers to the in vitro growth of cells or organisms on or in various types of media. It is understood that the progeny of the proliferating cell in culture may not be completely identical (ie, morphologically, genetically, or phenotypically) to the parent cell. By "expanded" is meant any growth or division of a cell.

“Composition” means a combination of an active agent and another compound or composition, either inert (eg, a detectable agent or label) or active, eg, an adjuvant.

A “pharmaceutical composition” refers to an active agent, which is inactive or active, and which is administered in vitro.
It is intended to include carrier combinations that make them suitable for ro, in vivo or ex vivo diagnostic or therapeutic use.

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount may be administered in one or more administrations, applications or dosages. The terms "cancer,""neoplasm," and "tumor" are used interchangeably and, whether singular or plural, undergo a malignant transformation that causes them to be pathogenic to the host organism. Refers to a cell that is Primary cancer cells (ie, cells obtained near the site of malignant transformation) can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cell includes not only primary cancer cells, but also any cells derived from cancer cell ancestry. This includes metastatic cancer cells, and in v
It includes in vitro cultures and cell lines. When referring to a type of cancer that usually appears as a solid tumor, a "clinically detectable" tumor is; It can be detected on the basis of this. Biochemical or immunological findings alone may not be sufficient to meet this definition. Tumor cells often express tumor-specific antigens. The term "tumor-associated antigen" or "TAA" refers to an antigen associated with or specific for a tumor.

As used herein, “solid support” is not limited to a particular type of support. Rather, a number of supports are available and known to those of ordinary skill in the art. For solid support, silica gel, resin, derivatized plastic film, glass beads,
Includes cotton, plastic beads, and alumina gel. Suitable solid supports can be selected based on the desired end use and suitability for a variety of synthesis protocols. For example, for peptide synthesis, the solid support may be a resin such as polystyrene (eg, Bachem Inc., Peninsula Laborat).
PAM resin obtained from E. ories, etc.), POLYHIPE ⁶ resin (Amin
otech, Canada), polyamide resins (Peninsula L)
obtained from Aboratories), polystyrene resin polyethylene glycol is grafted (TentaGel ^a, Rapp Polymere, Germany Tubingen) or polydimethylacrylamide resin (Millig
en / Biosearch, obtained from California). In a preferred embodiment of the peptide synthesis, the solid support refers to a polydimethylacrylamide resin.

“Transgenic animal” refers to a genetically engineered animal or the progeny of a genetically engineered animal. The transgenic animal may contain a genetic component from at least one unrelated organism (eg, from a bacterium, virus, plant, or other animal), or may contain a mutation that interferes with the expression of the gene product. .

The present invention provides an enhanced anti-tumor vaccine, wherein one or more polypeptides encoding a tumor antigen is linked to one or more polypeptides encoding an immunostimulatory factor associated with APC function. I do. Co-administration of tumor antigens and APC-related stimulators not only allows for appropriate antigen presentation to endogenous APCs,
APC functions with respect to 1) presentation of co-stimulatory signals; 2) migration to sites rich in T cells; 3) secretion of T cell growth factors; or 4) secretion of chemokines for recruitment of immune effector cells. Will increase.

The immunostimulatory factor of the present invention may include any polypeptide factor that modulates the immune response mediated by APCs and corresponding T cells. For example, co-stimulatory factors that are differentially expressed in APC may be used to directly enhance APC function in vivo. Co-stimulators have been described above and include, but are not limited to, thermostable antigen (HSA); chondroitin sulfate modified MHC invariant chain (Ii-CS); cell adhesion molecule 1; 1 and B7-2 / B70
including. Further, the immunostimulatory factor of the present invention may be a gene regulator that regulates the expression and activity of the above-described APC-related auxiliary stimulatory factor. In addition, autocrine using a ligand of an APC-related co-stimulator, the genetically modified APC then secretes a soluble ligand that binds to cell surface receptors and is available to activate the APC A loop may be generated.

For the purposes of the present invention, polypeptides and polynucleotides encoding cell-specific antigens are, in one embodiment, tumor-associated antigens that have been previously characterized, such as the melanoma-associated antigen gp 100 (Kawakami et al.) (1997) Inter
n. Rev .. Immunol. 14: 173-192); MUC-1 (H
enderson et al. (1996) Cancer Res. 56: 3763);
MART-1 (Kawakami et al. (1994) Proc. Natl. Ac
ad. Sci. 91: 3515; Ribas et al. (1997) Cancer.
Res. 57: 2865); HER-2 / neu (U.S. Pat.
No. 214); MAGE (PCT / US92 / 04354); HPV16, 18E
6 and E7 (Ressing et al. (1996) Cancer Res. 56 (
1): 582; Restifo (1996) Current Opinion
in Immunol. 8: 658; Stern (1996) Adv.
Cancer Res. 69: 175; Tindle et al. (1995) Cli.
n. Exp. Immunol. 101: 265; van Driel et al. (1996) Annals of Medicine 28: 471); CEA
(U.S. Pat. No. 5,274,087); PSA (Lundwall, A. (1).
989) Biochem. Biophys. Research Commu
161: 1151); prostate-specific membrane antigen (PSMA) (
Israeli et al. (1993) Cancer Research 53: 227.
Tyrosinase (U.S. Pat. Nos. 5,530,096 and 4,898,81);
No. 4; Brichard et al. (1993) J. Mol. Exp. Med. 178:
489); Tyrosinase-related protein 1 or 2 (TRP-1 and TRP-
2), NYESO-1 (Chen et al. (1997) Proc. Natl. Ac)
ad. Sci. U. S. A. 94: 1914); alternatively, it may be the GA733 antigen (US Pat. No. 5,185,254).

Selection of Immunostimulators of the Invention Embodiments of the present invention include immunostimulators that are preferentially or differentially expressed on monocyte-derived dendritic cells. Many comparative gene expression analyzes may be used to identify genes and mRNAs that are preferentially or differentially expressed in monocyte-derived dendritic cells as compared to other cells, such as monocyte progenitor cells. One preferred method is the SAGE analysis-serial analysis of gene expression (Serial Analysis of Gene Expr).
ession) (Velculescu et al. (1995) Science
270: 484-487 and U.S. Patent No. 5,695,937).

SAGE provides tools to isolate, sequence and classify expressed genes and gene expression levels in cells at any time in the cell cycle and under various environmental stimuli. SAGE provides quantitative gene expression data without the prior need for a hybridization probe for each transcript. SAGE is based on two principles. First, short sequence tags (9-11 base pairs) contain sufficient information to uniquely identify a transcript, if derived from a defined location in the transcript. Second, many transcript tags can be spliced into a single molecule and then sequenced to reveal the identity of multiple tags simultaneously. The expression pattern of any population of transcripts can be assessed quantitatively by determining the abundance of individual tags and identifying the gene corresponding to each tag. Velculescu et al. (1995) 484, supra.

Isolation and Characterization of the Macromolecules of the Invention In one embodiment, the invention provides for the isolation of costimulators that are preferentially or differentially expressed on APCs, such as monocyte-derived dendritic cells. And provide characterization. SAGE analysis as described above may be used to identify a population of sequence tags that correspond to gene transcripts that are preferentially or differentially expressed in dendritic cells but not in their monocyte precursor cells. . In one aspect, the transcript or gene has been previously identified but is not previously known to be preferentially or differentially expressed in monocyte-derived dendritic cells. In another embodiment, the transcript or gene disclosed in the present invention is "novel", wherein the tag or its respective complement is the previously identified expressed sequence tag (EST) or characterized gene. Does not include or does not correspond to the sequence of

As one non-limiting example, SAGE analysis shows that chemokines PARC and TARC (Pulmonary and TARC) are known for their ability to recruit activated T cells.
Activation-Regulated Chemokine, Hie
Shima et al. (1997) J. Am. Imm. 159 (3): 1140-1149
And Thymus and Activation-Regulated C
Hemokine, Imai et al. (1996) J. Am. Biol. Chem.
271 (35): 21514-21521) has been shown to be differentially expressed by monocyte-derived immature dendritic cells, a fact which was not recognized prior to the present invention.

In a separate aspect of the invention, the immunostimulatory factor as claimed is monocyte-derived D
It may be a costimulator that is differentially expressed in C. As used herein, a co-stimulator will include at least a portion of a protein sufficient to allow binding to a co-stimulatory ligand expressed on the corresponding T cell surface.

According to an alternative embodiment of the present invention, a gene is used that encodes a transcription factor capable of up-regulating the expression and activity of a costimulator discussed above. The encoded transcription factor may be a naturally occurring protein involved in the gene regulatory pathway of a costimulator that is differentially expressed in dendritic cells. Examples of transcription factors: Nuclear factor kappa B (NFκB) (Grohmann et al. (1998), Imm
unity 9 (3) pp. 315-323), X61498; relB-familiar rel family (Wu et al. (1998) Immunity 9 (6) 839-
847); CCAAT / enhancer binding protein (Yamanaka
(1998) Bioorganic and Medicinal Chem.
issue Letters 1 (1), pages 213-221), Y1 152
5; interferon-stimulated gene factor 3 (ISGF-3) (Schindler
Et al., 1992 p. N. A. S. USA 89 (16) 7836-7839
STAT5 (Welte et al. (1997) Europe).
an Journal of Immunology 27 (10) 2737-
2740), U43185; and NFAT-X (Masuda et al., 1).
995 Molecular and Cellular Biology 1
5 (5), pages 2697-2706) and U14510. Alternatively, the nucleotides encoding the transcription factors may be designed via recombinant DNA technology. When included in a vaccine of the invention, these nucleotides are capable of producing a transcription factor that will transactivate expression of an endogenous gene.

Thus, according to one aspect of the present invention, the immunostimulatory factor as claimed is a transcription factor that regulates the gene expression of a costimulatory factor that is differentially expressed in monocyte-derived DCs. Is also good.

Differential gene expression analysis is also expected to reveal genes encoding cell surface proteins that are preferentially expressed on immature or mature dendritic cell surfaces compared to monocyte precursors Will. Examples of such dendritic cell surface proteins that may be targeted by activating ligands or designed binding molecules (eg, antibodies) include: IFN
Alpha / beta receptor, X89814; IL-13 receptor, Y09328; C
D27 ligand, L08096; CD1b, M28826; CD151, D29
963; CD53, M60871; LFA-1, M15395; and WSX1
And a cytokine receptor, AF053004. These cell surface molecules
By acting as accessory stimulation signals or as regulators of DC function or migration, they can play a pivotal role in the function of dendritic cells. Naturally occurring ligands specific for these cell surface molecules or recombinant proteins (eg, antibodies) generated to have specificity for these cell surface molecules interact with the cell surface proteins and cause dendritic cell It may be expected to stimulate function or promote maintenance of an activated state or to stimulate the migration of dendritic cells to T cell rich sites. Accordingly, the present invention provides that one or more genes encoding one or more tumor antigens may be used for comparative gene expression analysis (eg, SAGE).
A) one or more genes encoding secreted proteins that bind to cell surface proteins identified as being differentially expressed in mature or immature dendritic cells and have the ability to modulate the activity of said proteins A vaccine linked to the vaccine. In this manner, a novel autocrine loop is established, whereby the genetically modified APC produces a ligand, which is secreted from the APC, where it binds to a cell surface receptor on the cell. And it is possible to stimulate the genetically modified dendritic cells.

In accordance with the present invention, the polynucleotide sequence encoding the tumor antigen and the polynucleotide sequence encoding the immunostimulator may be constructed as separate molecules in the vaccine composition of the present invention. Alternatively, two nucleotide sequences may be covalently linked using standard recombinant DNA techniques or chemical synthesis to form a single polynucleotide construct. Recombinant DNA constructs may be designed in a vaccine composition to have a linking sequence under the control of one transcription regulatory region capable of mediating the expression of both a tumor antigen and an immunostimulator.

The present invention also includes polynucleotides that differ from those of the above-described polynucleotides, but that encode substantially the same amino acid sequence. These modified, but phenotypically equivalent, polynucleotides are referred to as "functionally equivalent nucleic acids." As used herein, a “functionally equivalent nucleic acid” will function in substantially the same manner to produce the same protein product as the nucleic acids disclosed herein (eg, the degeneracy of the genetic code). Or nucleic acids characterized by slight and insignificant sequence variations having conservative amino acid variations. For example, conservative fluctuations include
Substitution of a non-polar residue with another non-polar residue, or substitution of a charged residue with a similarly charged residue, is included. These sequence variations include those recognized by those skilled in the art as not substantially altering the tertiary structure of the encoded protein.

The polynucleotides of the invention may comprise additional sequences, for example additional coding sequences within the same transcription unit, regulatory elements such as promoters, ribosome binding sites, and polyadenyl sites, additional transcription units under the control of the same or different promoters,
Sequences that enable cloning, expression, and transformation of host cells, as well as any such sequences that may be desirable to provide aspects of the invention, may be included.

Indeed, the present invention also provides a promoter sequence derived from the cell genome, wherein the promoter sequence corresponds to a regulatory region of a gene that is differentially transcribed in control cells compared to control cells. The promoter is identified by: 1) probing the cDNA library with a probe corresponding to the SAGE tag sequence or performing an anchor PCR using primers based on the SAGE tag sequence to produce a portion of the desired cDNA and Determined. Examples of cell types in which differential expression of the gene is associated with promoter function include the partial cDN obtained in step 1 above.
Using the A product as a probe, cloning the 5 'end of the cDNA, and also using the 5' end of the cDNA as a probe, cloning the promoter of the gene encoding the cDNA from a genomic library It depends. These promoters are identified using the methods described below in combination with standard molecular techniques. Functionally equivalent sequences, as defined above, are further provided by the present invention.

[0062] In one aspect, the promoter is a sequence from the APC genome, wherein the promoter region corresponds to a regulatory region of a gene that is differentially transcribed by APC. In a further aspect, the APC is a TNF-α treated dendritic cell. In a further aspect, the APC is an immature dendritic cell. In a further aspect, the expression of genes from these two cell sources is compared to the genes expressed on monocytes from which the dendritic cell population is derived.

The promoter identified above may be operably linked to a foreign polynucleotide, forcing differential transcription of the foreign polynucleotide in the cell from which the promoter is derived. A foreign polynucleotide is intended to include any sequence that completely or partially encodes a polypeptide or protein.
Heterologous polynucleotides also include sequences encoding ribozymes and antisense molecules.

Heterologous polynucleotides also include genes encoding regulatory proteins and oligonucleotides, as well as therapeutic genes encoding dominant negative oligonucleotides and peptides. Generally, gene therapy will involve the introduction of a single therapeutic gene, but treatment of a particular disease may require one or more genes. In one embodiment, the therapeutic gene is a dominant negative mutant of a wild-type immunosuppressant. Alternatively, the therapeutic gene may be a wild-type copy of a defective gene or a functional homolog.

In one aspect, the tag identified in the table corresponds to a polynucleotide that encodes a polypeptide or protein that is biologically active as an antigen, eg, a natural, modified, autologous, or tumor-associated antigen Or comprises the polynucleotide. An antigen is identified by exhibiting overexpression or cell-specific expression of a tag identified herein. Using the methods described below, the gene containing or corresponding to the tag is identified, cloned, and inserted into the APC.
A tag corresponds to an antigen if the CTL response increases under appropriate experimental conditions. A peptide is identified as immunogenic if an appropriate immune response is elicited.

The present invention also includes the co-administration of immunostimulators and foreign polynucleotides, both under the control of a promoter. In one embodiment, the promoter is an APC-specific promoter. In an alternative embodiment, the promoter is specific for the tissue identified in the table. The immunostimulatory factors of the present invention include any polypeptide factor that modulates the immune response mediated by APCs and corresponding T cells. For example, using an auxiliary stimulator differentially expressed in APC,
The function may be directly enhanced. Co-stimulators have been described above.

The polynucleotides of the present invention may be introduced into and expressed in a host cell suitable for producing a cell-based vaccine. These methods are described in further detail below.

The polynucleotides and sequences identified above may be conjugated to a detectable marker, such as an enzymatic label or a radioisotope, to detect expression of the nucleic acid and / or gene in the cell. A wide variety of suitable detectable markers are known in the art, including fluorescent, radioactive, enzymatic or other ligands that provide a detectable signal, such as avidin / biotin. In a preferred embodiment, it may be desirable to use a fluorescent or enzymatic tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of an enzyme tag, a colorimetric indicator substrate is known that can be used to provide a means that is visible to the human eye or spectrometer and that can identify specific hybridization with a complementary nucleic acid-containing sample. Can be

The polynucleotides and sequences included in the present invention may be obtained using chemical synthesis, recombinant cloning techniques, PCR, or any combination thereof. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. One of skill in the art may obtain the desired polynucleotide using the sequence data provided herein, by using a DNA synthesizer, or by ordering a commercial service.

The polynucleotides and sequences of the present invention may be inserted into a suitable vector, and the vector may be subsequently introduced into a suitable host cell for replication and / or amplification. A polynucleotide may be introduced into a host cell by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide may be maintained within the cell as a non-integrated vector (eg, a plasmid) or may be integrated into the host cell genome. Amplified DNA can be isolated from host cells by standard methods. For example, Sambr
See OK et al. (1989) supra. RNA may also be obtained from transformed host cells, or by using DNA-dependent RNA polymerase,
It may be obtained directly from NA.

The present invention further includes a variety of gene delivery vehicles comprising a polynucleotide of the present invention. Gene delivery vehicles include both viral and non-viral vectors, for example, naked plasmid DNA or DNA / liposome complexes. Vectors are generally classified as cloning and expression vectors. Cloning vectors are useful for obtaining replica copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a depository for further recovery. Using expression vectors (and host cells containing these expression vectors), it is possible to obtain polypeptides produced from the polynucleotides they contain. Suitable cloning and expression vectors include any known in the art, for example, for use in bacterial, mammalian, yeast and insect expression systems. Polypeptides produced in a variety of expression systems are also within the scope of the invention and are described above.

When the vector is used for gene therapy in vivo or ex vivo,
Pharmaceutically acceptable vectors, such as replication defective retrovirus or adenovirus vectors, are preferred. Pharmaceutically acceptable vectors containing the nucleic acids of the present invention may be further modified for transient or stable expression of the inserted polynucleotide. As used herein, the term "pharmaceutically acceptable vector" includes, but is not limited to, a vector capable of selectively targeting and introducing nucleic acids into dividing cells. Or a transport vehicle is included. An example of such a vector is a "replication-defective" vector, which is defined as incapable of producing viral proteins and preventing the vector from spreading in infected host cells. An example of a replication defective retroviral vector is LNL6 (Miller AD et al. (1989).
) BioTechniques 7: 980-990). The methodology of using replication defective retroviruses for retrovirus-mediated gene transfer of genetic markers is well established (Correll et al. (1989) Proc. Nat.
l. Acad. Sci. USA 86: 8912; Bordignon
(1989) Proc. Natl. Acad. Sci. USA 891
2-52; Culver K .; (1991) Proc. Natl. Aca
d. Sci. USA 88: 3155; and Rill, D.A. R. (19
91) Blood 79 (10): 2694). Clinical studies have shown that there are few or no side effects associated with viral vectors.
See Anderson (1992) Science 256: 808-13.

Compositions comprising the polynucleotides and sequences of the present invention, either in isolated form or contained in a vector or host cell, are further provided herein. When these compositions are used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.

The vectors of the present invention may comprise one or more polynucleotides comprising the sequences shown in the table or the complement thereof. The vectors of the present invention also encode other polypeptides that enhance, facilitate, or regulate the desired result, such as fusion components that facilitate protein purification, and sequences that increase the immunogenicity of the resulting protein or polypeptide. May also be included.

[0075] Also included in the invention are host cells that have been transformed with the vectors as described above. Both prokaryotic and eukaryotic host cells may be used. Prokaryotic hosts include bacterial cells such as E. coli and Mycobacteria (Myco).
bacteria). Among eukaryotic hosts are yeast, insect, bird, plant and mammalian cells. Host systems are known in the art and need not be described in detail herein. Examples of mammalian cells include, but are not limited to, C
OS, HeLa, and CHO cells, and APCs, such as dendritic cells, are included.

The host cells of the present invention may also be used, inter alia, as a repository of polynucleotides that are differentially expressed in the cell or as a vehicle for the production of polynucleotides and encoded polypeptides. Good.

Polypeptides of the Invention The present invention relates to a population of proteins or polypeptides expressed from a population of polynucleotides of the invention, which are produced recombinantly from wild-type and prokaryotic and eukaryotic host cells. Muteins, along with polypeptides and proteins
Provided is said population intended to comprise analogs, fusions and fragments thereof. In some embodiments, the term also includes antibodies and anti-idiotype antibodies.

[0078] It is understood that equivalents or variants of the wild-type polypeptide or protein are also within the scope of the invention. An "equivalent" is at least a portion of the fragment that differs from the wild-type sequence encoded by the polynucleotide of the present invention by any combination of additions, deletions, or substitutions, but that is appropriate for the context in which it is used. Retain one functional characteristic. For example, an equivalent of a polypeptide of the invention may be capable of eliciting an immune response with similar antigenic specificity as that elicited by a wild-type polypeptide. As will be apparent to those skilled in the art,
Equivalents may also be associated with or associated with other substances or agents that promote, enhance or regulate their function.

The present invention includes modified polypeptides containing conservative or non-conservative substitutions that do not significantly affect their properties, such as the immunogenicity of the peptide or its tertiary structure. Modification of a polypeptide is a routine practice in the art. Amino acid residues that may be conservatively substituted for one another include, but are not limited to: glycine / alanine;
Valine / isoleucine / leucine; asparagine / glutamine; aspartic / glutamic acid; serine / threonine; lysine / arginine; and phenylalanine / tyrosine. These polypeptides may also be used with other glycosylated and non-glycosylated polypeptides, as well as other post-translational modifications, such as glycosylation with different sugars.
Acetylation and phosphorylation are included.

The polypeptides of the invention may also be linked to a chemically functional moiety.
Typically, the moiety is a label capable of producing a detectable signal. These binding polypeptides are useful in detection systems, such as for imaging breast tumors. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds,
Substrates, cofactors and inhibitors are included. Examples of patents illustrating the use of such markers include U.S. Patent Nos. 3,817,837; 3,850,752;
No. 39,350; No. 3,996,345; No. 4,277,437; No. 4,2
75,149; and 4,366,241. The moiety may be covalently attached to the polypeptide, may be recombinantly attached, or may be through a second reagent, such as a secondary antibody, protein A, or a biotin-avidin complex. It may be conjugated to a polypeptide.

Other functional moieties include agents that enhance immunological reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioreactivity modifiers, paramagnetic labels and drugs. . Agents that enhance immunological reactivity include, but are not limited to, bacterial superantigens. Agents that facilitate coupling to a solid support include, but are not limited to, biotin or avidin. Immunogenic carriers include, but are not limited to, any physiologically acceptable buffer.

[0082] The invention also includes fusion proteins comprising a polypeptide encoded by a polynucleotide disclosed herein and fragments thereof. Such a fusion may be between two or more polypeptides of the present invention and related or unrelated polypeptides. Useful fusion partners include sequences that facilitate subcellular localization of the polypeptide or enhance immunological reactivity or coupling of the polypeptide to an immunoassay support or vaccine carrier.
For example, the polypeptide may be fused to a bioreactive modifier. Examples of bioreactive modifiers include, but are not limited to, green fluorescent protein (GFP)
And cytokines or lymphokines such as interleukin-2 (IL-2), interleukin-4 (IL-4), GM-CSF, and α-interferon. Another useful fusion sequence is one that facilitates purification. Examples of such sequences are known in the art and include those encoding epitopes such as Myc, HA (from influenza virus hemagglutinin), His-6, or FLAG. Other fusion sequences that facilitate purification include glutathione S-transferase (GST), maltose binding protein (MBP)
Or from a protein such as the Fc portion of an immunoglobulin. For immunological purposes, tandem repeat polypeptide moieties may be used as antigens, thereby producing highly immunogenic proteins.

[0083] The proteins of the present invention may also be combined with a variety of liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions. Examples of non-aqueous solvents include propylethylene glycol, propylene glycol, and vegetable oil. When used to prepare antibodies, the carrier may also non-specifically include an adjuvant, which is useful for increasing a specific immune response. One skilled in the art can readily determine whether an adjuvant is needed and select an adjuvant. However, for purposes of illustration only, suitable adjuvants include, but are not limited to, complete and incomplete Freund's, mineral salts and polynucleotides.

[0084] The proteins and polypeptides of the present invention can be obtained by several methods well known to those skilled in the art, including purification, chemical synthesis and recombinant methods. Full-length protein can be obtained from cells or tissue lysates from non-metastatic or metastatic breast tumor tissue by methods such as immunoprecipitation with antibodies, as well as gel filtration, ion exchange, reverse phase, and as described herein. Purification can be achieved by standard techniques such as affinity chromatography using the fusion protein. Such methodologies include, for example, Deutscher et al. (1999) GUIDE T.
O PROTEIN PURIFICATION: METHODS IN E
NZYMOLOGY (see Vol. 182, Academic Press). Thus, the present invention also provides methods obtainable by these methods, as well as methods for obtaining these proteins and polypeptides, and provides the obtained products.

[0085] Proteins and polypeptides can also be obtained from commercially available automated peptide synthesizers, such as the Perkin Elmer / Applied Biosystem.
ms, Inc. Can be obtained by chemical synthesis using Model 430A or 431A, manufactured in Foster City, California, USA.
The synthetic protein or polypeptide may be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the present invention also provides protein sequences and reagents, such as amino acids and enzymes, and chemically synthesizes the proteins of the present invention by linking the amino acids together in a suitable orientation and in a linear sequence. A method is also provided.

Alternatively, proteins and polypeptides can be obtained as described, for example, in Sambrook et al.
989), using well-known recombinant methods, as described above, using the host cells and vector systems described above.

Antibodies Also provided herein are populations of antibodies capable of specifically binding to a protein or polypeptide as described above. The antibodies of the present invention include polyclonal and monoclonal antibodies. These include, but are not limited to, mouse, rat, and rabbit or human antibodies. The invention also includes functionally equivalent antibodies and fragments thereof. As used herein, with respect to the exemplified antibodies, the phrase "functionally equivalent" refers to an antibody, or a fragment thereof, or an antibody that cross-blocks the exemplified antibody when used in an immunoassay, such as immunoblotting or immunoprecipitation. Means any molecule having an antigen binding site (or epitope).

[0088] Antibody fragments include Fab, Fab ', F (ab') ₂ , and Fv regions, or derivatives or combinations thereof. Fab, Fa of immunoglobulin
The b 'and F (ab') ₂ regions may be generated by enzymatic digestion of a monoclonal antibody, using techniques well known to those skilled in the art. Fab fragments may be produced by digesting the monoclonal antibody with papain and contacting the digest with a reducing agent that reductively cleaves disulfide bonds. Fab ′ fragments may be obtained by digesting the antibody with pepsin and reductively cleaving such produced fragments with a reducing agent. In the absence of a reducing agent, enzymatic digestion of the monoclonal antibody with pepsin produces F (ab ') ₂ fragments.

Included within the definition of antibody fragment are chimeric and humanized antibodies (Oi et al.), With single-chain antibodies that can be generated as described in US Pat. No. 4,704,692. (1986) BioTechniques 4 (3): 214). Chimeric antibodies are those in which the various domains of the heavy and light chains of the antibody are encoded by DNA derived from one or more species.

As used herein, with respect to monoclonal antibodies, “hybridoma cell line” refers to any derivative of the parent hybridoma that produces a monoclonal antibody specific for a polypeptide of the invention, regardless of karyotypic generation. Intended to include progeny cells.

Laboratory methods for producing polyclonal and monoclonal antibodies and estimating their corresponding nucleic acid sequences are known in the art.
Harlow and Lane (1988) supra and Sambrook et al.
89) See above. For production of polyclonal antibodies, a suitable host animal,
Typically, a mouse or rabbit is selected. An antigen that is substantially purified is
A polypeptide that corresponds to part or all of a specific loop region within a transmembrane domain, coupled or fused to the entire transmembrane domain, a fragment thereof, or another polypeptide, is suitable for the host. By the method, it is presented to the host immune system. Antigen is generally introduced by intramuscular, intraperitoneal, or transdermal routes by injection into the foot pads of the host. Peptide fragments suitable for generating antibodies may be prepared by chemical synthesis, and are generally coupled to a carrier molecule (eg, keyhole limpet hemocyanin),
The host is then injected for a period suitable for antibody production. Alternatively, the antigen
It may be produced recombinantly as a fusion protein. Examples of components of these fusion proteins include, but are not limited to, myc, HA, FLAG, His
-6, glutathione S-transferase, maltose binding protein or the Fc portion of an immunoglobulin.

A monoclonal antibody of the invention refers to an antibody composition having a homogeneous antibody population.
It is not intended to be limited as to the source of the antibody or the manner in which the antibody is made. Generally, monoclonal antibodies are produced biologically by introducing the protein or fragment thereof into a suitable host, such as a mouse. After an appropriate period of time, the spleen of such animals is excised and individual spleen cells are fused to typically immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatant of each clone is tested for the production of appropriate antibodies specific for the desired region of the antigen using methods well known in the art.

The isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can also be achieved by producing anti-idiotypic antibodies.
It can be achieved by one of ordinary skill in the art (Herlyn et al. (1986) S
science 232: 100). Anti-idiotype antibodies are antibodies that recognize unique determinants on monoclonal antibodies produced by the hybridoma of interest.

The idiotypic identity between the monoclonal antibodies of the two hybridomas indicates that the two monoclonal antibodies are identical with respect to recognition of the same epitope determinant. Thus, by using an antibody to an epitope determinant on a monoclonal antibody, it is possible to identify other hybridomas that express a monoclonal antibody of the same epitope specificity.

It is also possible to produce monoclonal antibodies that mimic an epitope using anti-idiotypic technology. For example, an anti-idiotype monoclonal antibody raised against a first monoclonal antibody will have, within the hypervariable region, a binding domain that is a mirror image of the epitope bound by the first monoclonal antibody. Thus, in this case, anti-idiotype monoclonal antibodies can be used for immunization for the production of these antibodies.

[0096] Other suitable techniques for producing antibodies include, but are not limited to, in vitro exposure of lymphocytes to an antigenic polypeptide or selection of a library of antibodies in phage or similar vectors. . Huse et al. (1989) Sc
issue 246: 1275-1281. Genetically engineered variants of the antibody can be obtained by obtaining a polynucleotide encoding the antibody and applying common molecular biology methods, introducing mutations and translating the variant. , Can be produced. The antibody "derivatives" described above are further provided herein.

[0097] Serum collected from the immunized animal provides a source of polyclonal antibodies. Detailed methods for purifying specific antibody activities from source materials are known in the art. If there is an undesirable activity that cross-reacts with other antigens, e.g., by passing the preparation over an adsorbent made with these antigens attached to a solid phase and eluting or releasing the desired antibodies from the antigens Can be removed. If desired, the specific antibody activity can be determined by protein A chromatography, ammonium sulfate precipitation, ion exchange chromatography, high performance liquid chromatography and immunoaffinity chromatography on a column of immune polypeptide coupled to a solid support, such as. Depending on the technique, it may be further purified.

[0098] Antibody specificity refers to the ability of an antibody to distinguish a polypeptide containing an immune epitope from other polypeptides. One of ordinary skill in the art will determine whether the antibodies share the same specificity as the antibodies of the invention, and whether the antibodies being tested prevent the antibodies of the invention from binding to normally reactive polypeptides. By doing so, it is possible to easily determine without undue experimentation. If the antibody being tested competes with the antibody of the invention, as indicated by reduced binding by the antibody of the invention, the two antibodies may bind to the same or very related epitopes . Alternatively, the antibodies of the invention may be pre-incubated with a polypeptide that is normally reactive and determine whether the antibody being tested is inhibited in its ability to bind to the antigen. If the antibody being tested is inhibited, then it probably has the same or a very relevant epitope specificity as the antibodies of the invention.

[0099] The antibodies of the present invention may be conjugated to a number of different carriers. Therefore,
The invention also provides for a composition comprising the antibody and a carrier. The carrier may be active and / or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural or modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier may be soluble or insoluble for the purposes of the present invention. One of skill in the art would know other suitable carriers for the conjugated antibody, or would be able to ascertain such using routine experimentation.

The antibodies of the present invention may also be conjugated to a detectable agent or hapten.
The complex can be prepared using standard immunochemical techniques, such as those described in Harlow and Lane (198).
8) Useful for detecting a polypeptide (or polypeptide fragment) to which an antibody specifically binds in a sample using immunohistochemistry as described above. There are many different labels and labeling methods known to those of ordinary skill in the art. In the present invention, examples of labeling species that may be used include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds. One of ordinary skill in the art would know other labels suitable for binding to the antibody, or would be able to ascertain such using routine experimentation. Furthermore, the binding of these labels to the antibodies of the present invention can be performed using standard techniques common to those skilled in the art.

[0101] Yet another technique that may result in higher sensitivity consists of coupling the antibody to a low molecular weight hapten. These haptens may then be specifically detected by a second reaction. For example, biotin that reacts with avidin,
Or it is common to use haptens, such as dinitrophenyl, pyridoxal, and fluorescein, that can react with specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

Compositions comprising the antibodies, fragments thereof, or cell lines producing the antibodies are included in the present invention. If these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.

Uses of the Polynucleotides, Polypeptides and Antibodies of the Present Invention The polynucleotides, polypeptides and antibodies encompassed by the present invention provide specific reagents that can be used in standard diagnostic methods. Accordingly, one embodiment of the present invention is to detect differential expression of a polypeptide comprising any one of the sequences shown in the tables, or any one of the disclosed populations or encoded polypeptides. , A method of characterizing monocyte lineage cells.

When assaying for changes at the mRNA level, nucleic acids contained in said sample suspected of containing dendritic cells are first extracted according to standard methods in the art. For example, mRNA can be isolated using various lytic enzymes or chemical solutions as described in Sambrook et al. (1989) supra, or can be extracted with a nucleic acid binding resin according to the accompanying instructions provided by the manufacturer. Subsequently, the mRNA contained in the extracted nucleic acid sample is subjected to hybridization (for example, Northern blot analysis).
And / or by amplification methods based on methods well known in the art or based on the methods exemplified herein.

[0105] Nucleic acid molecules having at least 10 nucleotides and having complementary sequences or homology to the polynucleotides described herein can be used as hybridization probes. It is known in the art that "perfectly matched" probes are not required for specific hybridization. Minor changes in the probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the specificity of hybridization. In general, up to 20% base mismatch (when optimally aligned) is acceptable. The mRN differentially expressed in cells to be probed
Probes useful for the detection of A are preferred. These tags are specified in Table 1 below. More preferably, the probes are at least 80% or 85%, and more preferably 90%, identical to the corresponding gene sequence when the homologous regions are aligned.

The probes can be used in hybridization reactions (eg, Southern and Northern blot analysis) to detect, diagnose or monitor physiological conditions associated with differential expression of these genes. The size of the entire fragment, as well as the size of the complementary portion, will depend on the particular use of the nucleic acid fragment or application. In general, small fragments derived from a known sequence can vary in length from those where the length of the complementary region is, for example, between about 10 to about 100 nucleotides, or up to a length completely identical to the complementary sequence desired to be detected. It may be used in hybridization embodiments.

In general, to increase the stability and selectivity of a hybrid, a nucleotide probe having a complementary sequence covering a length of 10 nucleotides or more is preferable, and this improves the selectivity of a specific hybrid molecule obtained. More preferably, it is possible to design nucleic acid molecules having 50 or more nucleotides or, if desired, longer gene complementarity. Such fragments may be prepared, for example, by directly synthesizing the fragments by chemical methods, or by nucleic acid production techniques such as the PCR ^™ technique using two types of priming oligonucleotides as described in US Pat. No. 4,603,102. Or by introducing a selection sequence into a recombinant vector suitable for recombinant production. Preferred probes are about 50-70, or more preferably 50-100.
Nucleotide length.

In certain embodiments, the nucleic acid sequences of the present invention may be advantageously used in combination with appropriate means, such as labels for hybridization, ie, detection of complementary sequences. A variety of suitable indicating means are known in the art, including fluorescence, radioactivity, enzymes or other ligands, such as avidin / biotin, which can provide a detectable signal. In a preferred embodiment, the use of fluorescent labels other than radioactive or other environmentally unfriendly reagents or enzymes such as urease, alkaline phosphatase or peroxidase may be desired. Examples of enzyme tags are known chromogenic indicator substrates that can be used to provide a human eye or spectroscopically visible means of identifying specific hybridization with a complementary nucleic acid-containing sample.

The nucleotide probes of the present invention can also be used to detect probes or genes or gene transcripts that are differentially expressed in specific body tissues. Preferred primers are those that comprise the sequence shown in the table, or the complement thereof. In addition, primers useful for the detection of the above genes or transcripts should have at least about 8 homologous regions of the gene or transcript to be detected contained in the previously identified sequence.
0% identical. For the purposes of this invention, amplification refers to any method that uses a primer-dependent polymerase that can replicate the target sequence with reasonable accuracy. Amplification is performed using natural or recombinant DNA-polymerases such as T7 DNA polymerase, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.

[0110] A preferred amplification method is PCR. The general procedure for PCR is MacPherso.
n, et al., PCR; A PRACTICAL APPROACH, (IRL Press, Oxford University Press (1991)). However, the PCR conditions used for each amplification reaction are empirically determined. Many parameters affect the success of the reaction. They include annealing temperature and time, extension time, Mg ²⁺ ATP concentration, pH and relative concentrations of primers, template and deoxyribonucleotides.

The DNA fragment obtained after the amplification can be detected by agarose gel electrophoresis, followed by ethidium bromide staining and visualization by ultraviolet irradiation. Specific amplification of the gene or transcript of interest is that the amplified DNA fragment has the expected size,
It can be verified by showing the expected restriction enzyme digestion pattern and / or showing that it hybridizes with the correctly cloned DNA sequence.

The probes and tags of the present invention can also be attached to a solid support for use in high-throughput screening assays using methods known to those of skill in the art. For example, PCT WO 97/10365 and U.S. Patent Nos. 5,405,783, 5
Nos. 4,412,087 and 5,445,934 describe one of the sequences disclosed herein.
Disclosed is the construction of high density oligonucleotide chips that can include or more. Based on the methods disclosed in U.S. Patent Nos. 5,405,783, 5,412,087 and 5,445,934, the probes of the present invention are synthesized on derivatized glass surfaces. The photoprotected nucleoside phosphoramidite is bound to the glass surface, selectively deprotected by photolysis through a photolithographic mask, and reacts with the second protected nucleotide phosphoramidite. The binding / deprotection process is repeated until the desired probe is completed.

[0113] The expression level of the desired gene is determined through exposure of the nucleic acid sample to the probe-modified chip. The extracted nucleic acids are labeled, for example, by a fluorescent tag, preferably during the amplification step. Hybridization of the labeled sample is performed at an appropriate stringent level. The degree of probe-nucleic acid hybridization is quantitatively measured using a detection device such as a confocal microscope. See U.S. Patent Nos. 5,578,832 and 5,631,734. The measurements obtained correlate directly with gene expression levels.

More specifically, the probe and the high-density oligonucleotide probe array
It provides an efficient means of monitoring the expression of multiple genes. The methods of the present invention for monitoring expression can be used in a wide range of situations, including detecting disease, identifying differential gene expression between samples, or screening for substances that up-regulate or down-regulate expression of a particular gene. Would.

In another preferred embodiment, the methods of the invention are used to monitor the expression of genes that specifically hybridize to a probe of the invention in response to a specific stimulus, such as a drug.

[0116] In one embodiment, the hybridized nucleic acid is detected by detecting one or more labels attached to the sample nucleic acid. The label may be incorporated by any of a number of methods known to those skilled in the art. However, in one aspect, the label is simultaneously incorporated during the amplification step during the preparation of the sample nucleic acid. Thus, for example, polymerase chain reaction (PCR) using labeled primers or labeled nucleotides will provide a labeled amplification product. In another embodiment, a labeled nucleotide (eg, fluorescein-labeled UTP and / or CTP)
The labeling product is incorporated into the transcribed nucleic acid by the above-described transcription amplification using)).

Alternatively, the label may be an original nucleic acid sample (eg, mRNA, polyA, mRNA,
cDNA, etc.) or directly after amplification is completed. Methods for attaching a label to a nucleic acid are known to those skilled in the art, for example, nick translation or kinase reaction of the nucleic acid, followed by binding (ligation) of a nucleic acid linker that binds the sample nucleic acid to the label (eg, a fluorescent substance). Labeling (eg, with labeled RNA).

Nucleic acid samples may be modified prior to hybridization to a high density probe array using the methods disclosed in WO 97/10365 to reduce sample complexity, thereby reducing background signal and improving measurement sensitivity. You could do it.

The results of a chip assay are typically analyzed using a computer software program. For example, EP 0717 113A2 and WO 95/206
See 81. The hybridization data is read into a program to calculate the expression level of the target gene. The results are compared to existing datasets of gene expression levels for various types of cells.

The expression of genes characteristic of dendritic cells as compared to monocytes can also be determined by examining the protein products of the polynucleotides of the invention. To determine protein levels,
a) providing a biological sample containing the polypeptide; and (b) combining an antibody reactive with the desired protein product with a component in the sample, wherein the amount of immunospecific binding indicates the level of the protein product. Measuring the amount of immunospecific binding occurring in between.

For protein analysis, various techniques are known in the art. They include radioimmunoassays, ELISAs (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (eg using colloidal gold, enzymes or radioisotope labels), Western blot analysis, These include, but are not limited to, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE. Further, cell sorting analysis can be used to detect cell surface antigens. Such assays include labeling the target cells with an antibody conjugated to a detectable agent, and subsequently separating the labeled cells from unlabeled cells in a cell sorter. A sophisticated cell separation method is Fluorescent Cell Sorting (FACS). In the laser beam, cells moving in a single column in a micro flow are passed, and the fluorescence of each cell to which the fluorescently labeled cells are bound is measured.

An antibody that specifically recognizes and binds to a desired protein product is required to perform the protein analysis described above. These antibodies may be purchased from commercial vendors or generated and screened using methods known in the art. Harlow and Lane (1988)
See supra, and Sambrook et al., (1989) supra.

There are a variety of methods in the art suitable for quantifying the level of mRNA or protein obtained from a cell, and any method capable of quantifying these levels is encompassed by the present invention. For example, the determination of the mRNA level of a gene may, in some respects, be based on mRNA isolated from cells.
Measuring the amount of mRNA in a sample by hybridization or quantitative amplification using at least one oligonucleotide probe that is complementary to the mRNA. Determining the protein product requires measuring the amount of immunospecific binding that occurs with the antibody that reacts with the desired product. A digital image analysis system for detecting the radioactivity or chemiluminescence of the probe can be used to detect a signal generated by the immunospecific binding or hybridization or amplification method, but is not limited thereto.

[0124] The promoter sequences of the present invention are useful for targeted expression of foreign polynucleotides. A promoter is operably linked to an exogenous polynucleotide and can be administered to a patient alone or after transduction into a host cell. The promoter is cell-
Because they are selected for specific expression, targeted expression of the inserted polypeptide can be obtained after incorporation into host cells in vivo or ex vivo.

In some embodiments, the promoter preferentially expresses a polynucleotide, eg, a tumor-associated antigen, operably linked in the APC. Genes encoding immunostimulatory molecules, such as cytokines, or co-stimulatory molecules, can be linked to promoter sequences and genes encoding antigens. Transduction and administration of these polynucleotides to a subject alone or to a host APC are useful for inducing an immune response by educating immune effector cells in vivo.

Screening Assays The present invention provides that a suitable cell is first contacted with an effective amount of a potential agent, followed by a polynucleotide selected from the group consisting of any of SEQ ID NOs: 1-27 or a population derived therefrom. Screening for various agents that modulate the expression of a polynucleotide associated with the phenotype of normal or diseased cells by assaying for changes in the expression level of the same is also provided. For example, changes in the expression levels of any of the polynucleotides in the monocytes or dendritic cells, including the sequences shown in the table, or corresponding, can be assayed. A change in expression level is indicative of a candidate modulator substance. In a particular aspect of the invention, the agent results in a phenotypic change in the recipient cell manifested by the agent-induced decrease in cell apoptosis, cell growth rate or motility. Altered gene expression can be detected by assaying altered mRNA or protein expression using probes, primers and antibodies as described herein.

To carry out the in vitro method, a cell or tissue culture is first provided which has previously been found to express one or more tags or polypeptides corresponding to the tags. The cell may be a cultured cell or a genetically modified cell in which a transcript or its complement, or a tag or its corresponding complement, containing or matching the transcript is expressed. Cells are cultured for a sufficient period of time under conditions (temperature, growth or culture medium, and gas (CO ₂ )) that allow logarithmic growth without density-dependent constraints. It is also desirable to maintain a cell culture that receives no agent, which is tested separately as a control.

As will be apparent to those skilled in the art, suitable cells are cultured in microtiter plates,
While recording the genotypic and / or phenotypic changes, several agents could be assayed simultaneously.

When the agent is a substance other than naked DNA or RNA, the agent is added directly to the cell culture or to the culture medium for addition. As will be apparent to those skilled in the art, an "effective" amount must be added which can be determined empirically. If the agent is a polynucleotide, it could be introduced directly into the cell by transfection or electroporation. Alternatively, it will be inserted into cells using a gene delivery vehicle or other methods described above.

For the purposes of the present invention, “agents” include single or complex forms, biological or chemical, such as organic or inorganic molecules, peptides, proteins (eg, antibodies) or polynucleotides (eg, antisense). But not limited thereto.
A vast number of compounds, for example, polymers such as polypeptides and polynucleotides,
And arrays of synthetic organic compounds based on various core structures can be synthesized, and are also included in the term "agent." Furthermore, various natural sources can provide compounds suitable for screening, such as plant or animal extracts. Although not always explicitly described, it is to be understood that the agent is used alone or in combination with another agent having the same or different biological activity as the agent identified by the inventive screen. It is.

The assays can also be performed in a subject. Where the subject is an animal, such as a rat, mouse or monkey, the method provides a convenient animal model system for the use of the agent prior to clinical trials. In this system, candidate agents are up-regulated (such as restoring tumor suppressor function), down-regulated or eliminated, such as when a transcript expression is altered, ie, as a drug resistance gene or oncogene, or untreated. A candidate agent is a potential drug if symptoms are ameliorated with or in the presence of cells containing transcript expression, as compared to an animal with no diseased cells. It is also beneficial to have another negative control group that is a healthy or untreated cell or animal that provides a basis for comparison. After administering the agent to the subject, a suitable cell or tissue sample is collected and assayed for altered gene expression.

The agents of the invention and the above compounds and derivatives thereof can be combined with a pharmaceutically acceptable carrier for the preparation of a medicament for use in the methods described herein.

The agents of the present invention can be administered to cells or subjects by various delivery systems known in the art. Non-limiting examples include encapsulation in liposomes), microparticles,
Microcapsules, expression by recombinant cells, receptor-mediated endocytosis (Wu and Wu (1987) J. Biol. Chem. 262: 4429-).
4432) and constructs of therapeutic nucleic acids as part of a retrovirus or other vector. Delivery methods include, but are not limited to, transdermal, gene therapy, intraarterial, intramuscular, intravenous, intranasal, and oral routes, and also include continuous delivery systems. In certain embodiments, it will be desirable to administer the pharmaceutical compositions of the invention to the area in need of treatment; this includes, but is not limited to, intraoperative local perfusion, injection, or catheterization, or treatment. A targeted gene supply of the sequence encoding the component may be achieved.

In vivo administration can be performed once, continuously or intermittently throughout the treatment period. The most effective methods and methods for determining the dosage will be known to those skilled in the art and will vary with the composition employed, the purpose of the treatment, the cell to be treated and the subject being treated. Single or multiple administrations can be carried out according to the level and pattern of administration selected by the treating physician. Suitable formulation dosages and active agent administration methods can be found below.

The agents and compositions of the present invention can be used for the manufacture of medicaments and for the treatment of humans and other animals by administering as active ingredients in pharmaceutical compositions according to conventional methods. The pharmaceutical composition can be administered orally, nasally, parenterally, transdermally, or by inhalation therapy,
And may take the form of tablets, troches, granules, capsules, pills, ampules, suppositories or sprays. They may also take the form of active ingredient emulsions in gene therapy, suspensions or solutions in aqueous or non-aqueous diluents, syrups, granules or powders.
In addition to the agents of the present invention, the pharmaceutical compositions may also contain other pharmaceutically active compounds or compounds of the invention.

The formulations of the present invention are suitable for oral administration in addition to the specific ingredients described above, as well as other active ingredients that are common in the art for the type of formulation in question, eg, sweetening, bulking and flavoring agents. Things can also be included. It further aims at combining the agents, compositions and methods of the present invention with other suitable compositions and therapeutic agents.

Genomic Application The present invention further provides a process for preparing a database suitable for analysis of genes expressed in cells by storing information about the sequence of the transcriptome in a digital storage medium. Using this method, a data processing system for standardizing and displaying the expressed genes of a cell is compiled. Data processing systems are useful for analyzing gene expression between two cells by first selecting the cells and then identifying and sequencing the transcriptome of the cells. This information is stored as a transcriptome in a computer-readable recording medium. The transcriptome is then compared to at least one transcript fragment of a reference cell. Subsequently, the compared sequences are analyzed. By this method, a sequence that is specifically expressed and a sequence that is differentially expressed between a reference cell and a selected cell can be specified.

In other words, the present invention first provides a complete set of expressed genes in computer readable form, ie, the transcriptome, homology screens DNA or mRNA of unknown sequence to the transcriptome, and Unknown DN
By determining whether the A-sequence has similarity to any part of the transcriptome contained in the computer-readable form, the unknown sequence for one or more of the preselected cell transcripts or expressed genes is determined. A computer-based method for screening DNA or mRNA homology is provided. In one embodiment, the SEQ ID NOs or polypeptides corresponding to these sequences are transcriptomes compared to test cells.

That is, the information provided herein is based on the high-throughput sequence specificity of individual RNAs or their corresponding cDNAs using an improvement of the system described in WO 95/2068, 96/23078 and 5,618,672. It also provides a means to compare the relative amounts of gene transcripts in various biological materials using statistical analysis.

[0140] Tags or transcripts can also be attached to solid supports suitable for use in high-throughput screening assays. For example, PCT WO 97/10365 discloses the construction of high density oligonucleotide chips. U.S. Pat. No. 5,405,783;
See also 412,087 and 5,445,934. Using this method,
Probes are synthesized on the activated glass surface. The photoprotected nucleoside phosphoramidite is bound to the glass surface that has been selectively deprotected by photolysis with a photolithographic mask and reacted with a second protected nucleoside phosphoramidite. The binding / deprotection step is repeated until the desired probe is completed.

[0141] Gene expression levels are determined by exposing a nucleic acid sample to a probe-modified chip. The extracted nucleic acids are labeled, for example, by a fluorescent tag, preferably during the amplification step. Hybridization of the labeled sample is performed at an appropriate stringent level. The strength of probe-nucleic acid hybridization is
It is measured quantitatively using a detection device such as a confocal microscope. US Patent 5,5
See 78,832 and 5,631,734. The measurements obtained correlate directly with gene expression levels.

The results of the chip assay are typically analyzed using a computer software program. For example, EP 0717 113A2 and WO 95/20681
See Hybridization data is read into the program, which calculates the expression level of the target gene. The results are compared to an existing dataset of gene expression levels for that cell type.

Further usefulness of the database include analysis of the developmental status of test cells, effects of viral or bacterial infection, control of the cell cycle, effects of tumor suppressor genes or lack thereof, polymorphisms within cell types, apoptosis and Includes, but is not limited to, analysis of the effects of regulatory genes.

Non-Human Transgenic Animals From a different perspective, transgenic animal models can be created using the novel polynucleotide sequences associated with the pathological state of cells. In recent years, geneticists have successfully manipulated the genes of developing embryos and introduced foreign genes into these embryos to create transgenic animals, for example, for mice. Once these genes have been integrated into the genome of the recipient embryo, the resulting embryo or adult animal can be analyzed to determine the function of the gene. Mutant animals are created to understand the function of known genes in vivo and to create animal models of human disease (PO
STIMPLANTATION DEVELOPMENT IN THE MO
USE (Edited by Chadwick and Marsh, John Wiley & Sons
(UK)) Chisaka et al. (1992) 355: 516-520;
er et al., pp 277-297 (1992); and Dorin et al. (1992).
Nature 359: 211-215).

The following examples are intended to illustrate, but not to limit, the invention. The following are some techniques available to those of skill in the art suitable for identifying and cloning polynucleotides corresponding to tags having the sequences set forth in the Table.

1) RACE-PCR Technique One of the methods for isolating a gene or cDNA encoding a polypeptide or protein is the 5′-RACE-PCR technique. In this technique, first a poly-A mRNA containing a particularly desired coding sequence is identified by hybridization to the sequences disclosed herein, and then a 3'- containing a sequence disclosed herein.
Reverse transcribed using primers. The newly synthesized cDNA strand is then ligated to an anchor primer of known sequence containing a normal cloning restriction site, preferably linked to the 5 'end. The tagged cDNA is then amplified using a 3'-primer (or a nested primer sharing a sequence homologous to the internal sequence of the coding region) and a 5'-anchor primer. Amplification will be performed under conditions of varying stringency levels, to maximize the specificity of the amplification. 5 '
RACE-PCR can be easily carried out by using a commercially available kit (available from BRL5 Life Technologies, Inc., Clonetech) according to the manufacturer's instructions. 2) Separation of partial cDNA (3 'fragment) by 3' direction PCR reaction This method is an improvement of the method described by Polyak et al. (1997) Nature 389: 300. Briefly, the method involves creating a SAGE tag in the PCR reaction that includes the desired SAGE tag as well as other cDNAs and whose length is defined by the position of the tag relative to the 3 'end of the cDNA. Use. The cDNA product resulting from such a transcript-derived PCR reaction can be used for many amplifications.

RNA from sources believed to express cDNA corresponding to a particular tag
First, it is converted to double-stranded cDNA using standard cDNA protocols. Except that the first-strand synthesis is performed using the modified oligo-dT primer, it can be carried out under the same conditions as those used for the preparation of cDNA suitable for constructing a SAGE library.
For example, the composition 5'-biotin-TCC GGC GCG CCG TTT TC
The oligonucleotide of C CAG TCA CGA ₍₃₀₎ -3 ′ (SEQ ID NO: 208) was used for hybridization and priming from the poly-A tail at the 3 ′ end of a poly-T stretch, followed by a PCR step. Includes an M13 priming site, a 5 'biotin label (B) for capture on avidin-coated magnetic particles, and an AscI restriction endonuclease site for releasing cDNA from streptavidin-coated magnetic particles. In theory, PC
Any DNA region having a size sufficient to hybridize to the R primer and having recognition of an 8-base pair endonuclease can be used.

Subsequently, cDNAs constructed using this or similarly modified oligo-dT primers are described in US Pat. No. 5,695, to adapter ligation in which only one adapter is ligated to the cDNA pool. 937 is performed. After ligation of the adapter, the cDNA is released from the streptavidin-coated magnetic particles and subsequently used as a template for cDNA amplification.

[0149] Various PCR methods were performed using the 3 'modified oligo-dT primer and the PC in the SAGE tag.
It can be performed using an R priming site. The SAGE tag-derived PCR primer used is defined by the 5 'extension of the tag into the adapter sequence and can be of various lengths. Thus, cDNA products suitable for various applications are obtained.

The technique further comprises: (1) changing the length and / or content of the modified oligo-dT primer; (2) ligating adapters other than those already used for the SAGE method;
) PCR from template retained on streptavidin-coated magnetic beads
And (4) first strand cD using non-oligo-dT based primers
It can be further improved by priming NA synthesis.

3) cDNA using gene trapper or improved gene trapper technology
Separation of reagents and manufacturer's instructions for this technology are available from Life Technology (L
if Technologies Inc) Gaithersburg
rsburg), commercially available from Maryland. Briefly, a complex population of single-stranded phagemid DNA containing directional cDNA inserts is hybridized in solution to a biotinylated oligonucleotide probe complementary to the target sequence to enrich for the target sequence. . The target sequence is based on the tag sequence of the present invention. Hybrids are captured on streptavidin-coated magnetic particles. When the magnet collects the magnetic particles from the solution, non-hybridized single-stranded DNA remains in the solution. Subsequently, the captured single-stranded DNA target is released from the biotinylated oligonucleotide. After release, the cDNA clones are further enriched using non-biotinylated target oligonucleotides to specifically convert single-stranded targets to double-stranded DNA. After transduction and plating, typically between 20% and 100% of clones represent the desired cDNA clone. Desired c
To identify DNA clones, colonies are screened by colony hybridization using ³² P-labeled oligonucleotides as described for solution hybridization above, or by DNA sequencing and the entire sequence obtained from multiple clones. A consensus sequence will be determined from the alignment.

4) Separation of cDNAs from the library by probing with SAGE transcripts or tags. Classical methods for constructing cDNA libraries are taught in Sambrook et al., supra. Velculescu et al. (1997) Science 270: 484).
The recent method described in Example 1 describes an expression cDNA cloned into a Zap expression vector.
Can be used to build libraries. A ZAP expression cDNA synthesis kit is available from Stratagene and is used according to the manufacturer's instructions. Plates containing 250 to 2000 plaques are described in Rupert et al. (1988) Mol. Cell. Bio. As described in 8: 3104, hybridization is carried out with the oligonucleotide probe under the same conditions as the conventional description for the standard probe except that the hybridization temperature is lowered to room temperature.
Washing is performed in 6 × standard-saline-citric acid 0.1% SDS for 30 minutes at room temperature. The probe utilizes T4 polynucleotide kinase, ³² P-ATP
Marked with.

5) Identification of Known Genes or ESTs In addition, there are databases that reduce the complexity of ESTs by assembling contiguous EST sequences as temporary genes. For example, TIGR has assembled a human EST sequence as a temporary human consensus sequence in a database called THC. The THC database allows for more specific assignments than ESTs alone. Software programs (TIGR Assembler and TIGEM EST) that can assemble ESTs of any organism into a continuous sequence
Assembly equipment and contiguous assembly program (Huang, X (1
996) Genomics 33: 21-23) also exists.

Isolation, Culture and Expansion of APCs Containing Dendritic Cells Various methods are known in the art for isolating and characterizing APCs containing DCs.
The generation of human dendritic cells from expanded progenitor cells in peripheral blood or bone marrow requires at least 2
Different methods are used. One method is as follows: GM-CSF and IL
-4 in which monocytes are cultured, the immature part is used for SAGE, and the remaining part is treated with TNF-α for 36 hours to promote its maturation. Following this method, the cell line identified herein as mature TNFα dendritic cells was isolated and cultured.

Another method utilizes a larger population of CD34 precursors, such as adherent peripheral blood monocytes, and stimulates them with GM-CSF + IL-4 (eg, Sall
usto et al. (1994) see above).

In another aspect of the invention, Romani et al. (1996) supra, or Bender.
(1996) The above described method is utilized to generate immature or mature dendritic cells from peripheral blood mononuclear cells (PBMC) of a mammal such as a mouse, monkey, or human. Briefly, T- and B-cells are removed from the isolated PBMCs by immunomagnetic techniques. Next, lymph cell-deleted PBMC was 1% for 7 days.
Dendritic cells are produced by culturing in RPMI medium supplemented with autologous human serum and GM-CSF / IL-4. On day 7, non-adherent cells are collected for further processing.

Dendritic cells derived from PBMC in the presence of GM-CSF and IL-4 are immature, and when cytokine stimulation is removed from the medium, they lose the properties of dendritic cells therein, You can go back to fate. A population of dendritic cells having such characteristics was used to separate transcripts of immature dendritic cells. Immature dendritic cells are extremely effective at processing natural protein antigens for the MHC class II restricted pathway (Romani et al. (1989) J. Exp. Med 169: 1).
169).

Further maturation is achieved by culturing in a conditioned medium or treating with LPS or TNF-α, or CD40 ligand for 3 days in a macrophage conditioned medium (CM) containing the necessary maturation factors. Mature dendritic cells are inferior to supplement the ability of the new protein for the purpose of presentation but, resting T cells are better in be stimulated to proliferate and differentiate (CD4 ^- - and ^CD8).

Mature dendritic cells may have a morphological change, such as the formation of motile cytoplasmic processes, the presence of at least one of the following markers: CD83, CD68, HLA-DR or CD86; or CD115. It can be identified from the disappearance of the Fc receptor (Steinman (1991) Ann. Rev. Immunol.
9: 271).

Computer Analysis The tags and populations thereof identified and claimed herein are further available for computer analysis. The sequences and expression profiles of the present invention can be obtained from functionally related programs, for example, Compare R using SAGE software (available from Johns Hopkins University Dr. Ken Kinzler).
saved in the report. The Copper Report provides a table of polynucleotide sequences and their abundance for samples normalized to a fixed number of polynucleotides per library (eg, 25,000). This information can be captured directly in MS-ACCESS for further processing, or by copying the data first to an Excel spreadsheet and then from there to MS-ACCESS. Other programs capable of comparing polynucleotide numbers, such as SYBASE or Oraclc, can also be used as alternatives to MS-ACCESS. These additional functions can be incorporated by expanding the software. Such features include standard Boolean algebra and text search applied in various combinations to reduce large input sets of polynucleotides to a limited subset of polynucleotides of interest that can be processed. Consists of functions.

[0161] Sequence information and abundance from test samples and cells are also entered into functionally useful programs. Researchers will combine the number of specific polynucleotides in a group to create a group containing one or more projects (eg, normal group =
Normal 1 + Normal 2, tumor group = primary tumor + tumor cell line). Other property values (eg, average number, minimum number, maximum number) are calculated for each tag in the group. The researcher will calculate the ratio of the number of individual tags between groups, eg, the ratio of the average number of normal groups to the average number of tumor groups for each polynucleotide. Researchers will calculate statistics of the significance of the observed differences in tag numbers between groups.

To identify a polynucleotide in MS-ACCESS, a query is performed that classifies the polynucleotide tag based on its amount in the sample cells. The output report from the query lists the specific polynucleotides (by sequence) and their amounts that meet the classification criteria in the various sample cells.

The classification is based on the principle that the sample with the selected phenotype is more abundant in the desired gene product (and corresponding nucleotides) than in the sample without the phenotype.

For example, for one or more test samples, if the total number of tags per library is normalized to a fixed number in the reference sample, the polypeptide present at the level of 10 tags is identified. You can ask questions. The results of the search are
For example, if five polynucleotides meet the classification criteria, and thus transfer them into a sample without the phenotype, there are five candidate genes to test to determine if they confer the phenotype. Will show you.

With more strict classification criteria, the classification will be more efficient. That is, if the total number of tags per library is normalized, requesting a polynucleotide that has 5 tags in it and not more than 5 in a test sample would yield a large number of candidates. However, if the difference is increased such that the sample is strongly phenotypic (eg,> 10 in the test sample and <1 in one or more reference samples), the number of candidates identified is limited. You.

[0166] From provisionally selecting a set of classification parameters, performing data analysis, and identifying and testing candidates, the amount of gene product (ie, the amount of polynucleotide) required to confer a given phenotype is determined in advance. What is known is not essential. If the desired candidate is not found, the strictness of the classification criteria can be reduced (ie, the difference is reduced) and the newly found candidate can be tested. By bi-directionally repeating the classification and testing of candidates, the desired candidate may be successfully retrieved.

Determining the amount of gene product (ie, the amount of polynucleotide) required for phenotyping depends on the particular phenotype of interest and the sensitivity of the assay that measures that phenotype.

Thus, the individual polynucleotide sequences of the question report are entered into the program to determine if they are compatible with the known genes or if they have new potential (non-match = NM).

The cDNAs corresponding to the specific sequences are then recovered from the question report and tested individually in a suitable biological assay to determine if they confer phenotype.
Obtaining complementary DNAs for these candidates in candidates corresponding to known genes, and furthermore, whether they confer the specific phenotype of interest when individually tested and transferred into cells without the phenotype Determining is a relatively simple task.
If none of the known genes confer a phenotype, search for cDNAs corresponding to unmatched (No Match) sequences in the query report by PCR cloning,
The novel cDNAs are individually tested for their ability to confer phenotype. If the hypothesis developed up to this point is correct (ie, a single gene product can confer a phenotype, the classification criteria are not strict enough to exclude the desired candidate), one of the candidates in the question report The corresponding cDNA is considered to confer the phenotype and the search ends. However, if none of the candidates gave the phenotype, it would be necessary to reduce the strictness of the classification parameters, to "broader" and capture more candidates to be tested as above.

In one embodiment, the polynucleotide or gene sequence can be compared to a sequence database using, for example, computer methods to match a sample sequence to a known sequence. Sequence identity is determined by CURRENT PROT, ie,
OCOLS IN MOLECULAR BIOLOGY (FM Ausub
el., et al., 1987) Extra number 30, Section 7.7.18, and can be determined by comparing the sequences using a sequence alignment program as described in Table 7.7.1. A preferred alignment program preferably uses default parameters such as: mismatch = 2; open gap = 0; and extended gap = 2
LIGHT Plus (Scientific and Educational)
Software, Pennsylvania). Another preferred program is the BLAST program for alignment of two nucleotide sequences utilizing the following default parameters: open gap = 50; extended gap−2 penalty; gap × dropoff = 0; expect = 1.
0; word size = 11. The BLAST program is available from the following Internet address: http: // www. ncbi. nlm. nih. gov
. Alternatively, hybridization under high, medium, and low stringency conditions can also indicate a degree of sequence identity.

In one embodiment of a vaccine for treating and preventing cancer , the present invention includes a vaccine for treating cancer. Recent advances in vaccine adjuvants have provided effective means of peptide administration such that they have a significant effect on the immune system. Del-Guidice (1994) Expe
rentia 50: 1061-1066. The polynucleotide of the present invention can be administered alone as a cancer vaccine or in combination with a polynucleotide encoding an antigenic peptide. Polynucleotides can be administered in the form of naked DNA or in an expression vector. Although treatment can be facilitated by co-administration of cytokines and / or costimulatory molecules, they can also be administered as proteins or polynucleotides encoding the proteins.

Host Cells Containing the Antigenic Peptide of the Invention The present invention further provides an isolated host cell comprising a polynucleotide of the present invention. In some embodiments, these host cells contain one or more of the inventive peptides in H
The antigenic peptide of the present invention is bound to a cell surface MHC molecule so that it is presented on the cell surface with the MC molecule, ie, the peptide can be recognized by immune effector cells. Isolated host cells that display a polypeptide of the invention in association with an HMC molecule are further useful for expanding and isolating a population of educated antigen-specific immune effector cells. Immune effector cells, such as cytotoxic T lymphocytes, are produced by culturing naive immune effector cells with antigen presenting cells that present polypeptides in association with MHC molecules on the surface of APCs. Populations can be generated using methods known in the art, such as FACS analysis or FICOL ^™ gradients. Methods for generating and culturing immune effector cells and populations produced thereby are also contributions and inventions of the present inventors. Pharmaceutical compositions containing these cells and a pharmaceutically acceptable carrier are useful for adoptive immunotherapy. Prior to in vivo administration, immune effector cells are screened for their ability to lyse melanoma tumor cells in vitro.

Vectors Useful for Gene Transfer Gene Modification In one embodiment, the present invention provides for the introduction of a recombinant polynucleotide encoding an antigenic peptide alone or in combination with an immunostimulator into a subject. Provided is a method for inducing an effective antigen-specific reaction on a sample. Suitable methods and materials for gene transfer, including, for example, virus-mediated gene transfer, lipofection, transduction, transfection, and transduction, are known in the art. A polynucleotide encoding an immunostimulatory factor and a target antigenic peptide can be introduced ex vivo into a host cell, eg, a dendritic cell. Genetically modified host cells can be introduced into target subjects as cell-based vaccines. Or,
Polynucleotides encoding an immunostimulatory factor and a target antigenic peptide can be introduced directly into a subject in the form of a gene-based vaccine.

A variety of viral infection techniques utilizing recombinant viral vectors suitable for gene donation have been developed and will construct a preferred approach of the present invention. Viral vectors utilized for gene transfer include, but are not limited to, viral sequences derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV) and retrovirus.

Viral transduction of cells, such as APCs APCs can be transduced by viral vectors encoding the relevant polypeptide. The most common viral vectors include recombinant poxviruses such as vaccinia and fowlpox (Bronte et al., (1997) Proc.
. Natl. Acad. Sci. USA 94: 3183-3188; Kim et al. (
1997) J. Amer. Immunother. 20: 276-286) and, preferably, adenovirus (Arthur et al., (1997) J. Immunol. 159).
: 1393-1403; Wan et al. (1997) Human Gene Ther.
apy 8: 1355-1363; Huang et al., (1995) J. Am. Virol
. 69: 2257-2263). Retroviruses could also be used for transduction of human APCs (Marin et al., (1996) J. Virol. 7).
0: 2957-2962).

In vitro or ex vivo exposure of human DCs to an adenovirus (Ad) vector at a multiplicity of infection of 500 in a minimum volume of serum-free medium for 16-24 hours, expression of exogenous polynucleotide in 90-100% of DCs Is definitely caused. The transduction rate of DC or other APC can be evaluated by immunofluorescence using a fluorescent antibody specific to the expressed tumor antigen (Kim et al. (1997) J. Imm.
unother. 20: 276-286). Alternatively, the antibody is an enzyme (eg, HR
P) and can produce a colored product upon reaction with the substrate. The actual amount of antigenic polypeptide expressed by APC can be assessed by ELISA.

In vivo transduction of DCs or other APCs can be performed by the administration of Ad (or other viral vectors) via various routes including intravenous, intramuscular, intranasal, intraperitoneal, or intradermal administration. A preferred method is about 1 × 10 ¹⁰ -1 × 10 ¹² i. Multi-dermal intradermal delivery of Ad vector using u total dose. The level of in vivo transduction can be roughly assessed by double staining with an antibody against the APC marker and an antibody against the expressed antigen. Staining methods can be performed on biopsy samples from the site of administration, or on cells from draining lymph nodes or other organs into which APCs (especially DCs) will migrate (Condon et al., (1996)
) Nature Med. 2: 1122-1128; Wan et al., (1997) H.
human Gene Therapy 8: 1355-1363). The amount of antigen expressed at the injection site, or other organ where transduced APC will migrate, can be assessed by ELISA on tissue homogenates.

Although viral gene delivery is more efficient, DCs are transduced in vitro / ex vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation, or the cationic lipid / plasmid DNA complex method. (Arthur et al., (1997) Cancer G).
ene Therapy 4: 17-25). The transduced APC can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular, or intraperitoneal supply route.

In vivo transduction of DC or other APCs could also be achieved by administration of a cationic lipid / plasmid DNA complex supplied via intravenous, intramuscular, intranasal, intraperitoneal or intradermal administration. Intradermal gene gun delivery or injection of naked plasmid DNA can also induce DC transduction (Condon et al.,
(1996) Nature Med. 2: 1122-1128 and Raz et al. (1
994) Proc. Natl. Acad. Sci. USA 91: 9519-95
23). Intramuscular supply of plasmid DNA could also be used for immunization (R
Osato et al. (1997) Human Gene Therapy 8: 1451.
-1458.

The transduction efficiency and level of exogenous polynucleotide expression can be assessed as described above for viral vectors. Administration of a Cell-Based Vaccine to a Subject Genetically modified cells can subsequently be administered to a host subject via various routes including, for example, intravenous infusion, subcutaneous injection, intranasal, intramuscular or intraperitoneal delivery. Cells containing the recombinant polynucleotide can be used to immunize an individual. In vivo administration can be performed once, continuously or intermittently throughout the treatment period. Methods for determining the most effective method of administration and amount are known to those of skill in the art, and include compositions used for treatment, therapeutic purposes,
It will depend on the target cell to be treated and the subject to be treated. Single or multiple administrations can be carried out depending upon the level and pattern of administration selected by the treating physician.

Adoptive Immunotherapy The expanded population of antigen-specific immune effector cells and antigen presenting APCs can be used for adoptive immunotherapy.

In one aspect, adoptive immunotherapy employs an essentially pure population of educated antigen-specific immune effector cells created by co-culturing APCs and naive immune effector cells as described above. Administration. In some embodiments, the APC is a dendritic cell.

In certain embodiments, the adoptive immunotherapy methods described herein are autologous. In this case, APC is made using parent cells separated from a single subject. The expanded population also uses T cells separated from that subject. Finally, an expanded population of antigen-specific cells is administered to the same patient.

In another embodiment, APCs or immune effector cells are administered with an effective amount of a stimulatory cytokine or costimulatory molecule, such as IL-2. Immune Effector Cells The present invention utilizes the antigen presentation matrix described above, including APCs, to stimulate the production of an enriched population of antigen-specific immune effector cells. Thus, the present invention provides a cell population enriched in educated antigen-specific immune effector cells that is specific for an antigenic peptide of the invention. These cells are responsible for the antigenic determinants (
Cross-reaction (specific binding). In some embodiments, the native antigen is on the surface of a tumor cell, and the educated, antigen-specific immune effector cells of the invention inhibit tumor cell growth. When using APC, antigen-
Specific immune effector cells are expanded at the expense of APCs that die in culture. The process by which naive immune effector cells are educated by other cells is described in Coulie (1997) Molec. Med. Today 3: 261-268
Is essentially described.

Effector cell populations suitable for use in the methods of the invention may be autologous or allogeneic, but are preferably autologous. If the effector cells are of the same species,
Preferably, the cells are depleted of alloreactive cells prior to use. This may include, for example, mixing the allogeneic effector cells with the recipient cell population, incubating for a suitable period of time, and then depleting the CD69 + cells, or inactivating the autoreactive cells, or anergy in the autoreactive cell population. Can be achieved by known methods, including inducing

[0186] Hybrid immune effector cells can also be used. Immune effector cell hybrids are known in the art and have been described in various publications. See, for example, International Patent Applications WO 98/46785; and WO 95/16775.

While the foregoing invention will be described in greater detail with reference to the drawings and examples for clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the description and examples should not be construed as limiting the scope of the invention, which is made by the appended claims.

[0188]

【Example】

Example 1 The table illustrates a series of both known and unknown mRNAs that were found by SAGE analysis to be differentially expressed in monocyte-derived dendritic cells compared to monocytes.
For example, SAGE analysis shows that the chemokine PARC can collect activated T cells.
And TARC were used to convert monocyte-derived immature dendritic cells (PBMC-derived monocytes into GM-CSF).
And prepared in culture with IL-4). Other immunostimulators to be described include: monocyte chemoattractant protein-4 (MCP-4
) [Berkhout et al., (1997) Journal of Biologic.
cal Chemistry 272 (26): 16404-16413] U4
6767; macrophage-derived chemokines (MDC) [Godiska et al., (1)
997) Journal of Experimental Medicine
185 (9): 1595-1604] U83171; ecarectin [Matsu
Moto et al., (1998) Journal of Biological Ch.
273 (27): 16976-16984], AB00589.
4; and monocyte chemoattractant protein-2 (MCP-2) [Proost et al., 1996 J.
own of Leukocyte Biology 59 (1): 67
-74] Y10802. The fact that dendritic cells produce these chemokines in large quantities has not been previously reported. The genes encoding these chemokines are:
Within a DNA-based vaccine, it can be linked to single or multiple genes encoding tumor antigens, so that APCs transduced by the vaccine can produce and process not only tumor antigens but also stimulatory chemokines. As shown in Table I, cDNA encoding either mRNA or a combination of mRNAs identified as differentially expressed in immature dendritic cells by differential gene expression analysis (such as SAGE) Alternatively, the gene can be linked to single or multiple tumor antigen genes to prepare an excellent vaccine. Similarly, mRNA identified as being differentially expressed in mature dendritic cells by differential gene expression analysis (such as SAGE)
Alternatively, a cDNA or gene encoding any of the mRNA combinations can be linked to single or multiple tumor antigen genes to prepare an excellent vaccine.

Example 2 Genes encoding mRNA that are differentially expressed in immature or mature dendritic cells are likely to be controlled by specific transcription factors. That is, comparative analysis of gene expression (SA
Another method of providing genes, including mRNAs, identified as differentially expressed in mature or immature dendritic cells (such as GE) by differentially expressed mRN
To provide one or more genes encoding transcription factors (naturally occurring or processed by recombinant DNA technology) capable of trans-acting the expression of the endogenous gene encoding A. That is, the present invention provides for the identification of mRs identified as differentially expressed in mature or immature dendritic cells by comparative analysis of gene expression.
It also relates to a vaccine comprising one or more genes encoding a tumor antigen linked to a gene encoding a transcription factor or transactivator capable of up-regulating the expression of NA.

Example 3 Differential gene expression analysis is also expected to reveal genes encoding cell surface proteins that are preferentially expressed by immature or mature dendritic cells as compared to monocyte progenitor cells You. These cell surface molecules are costimulatory signals of DC function or migration,
Alternatively, by acting as a modulator factor, it may play an important role in the function of dendritic cells. Natural ligands specific for these cell surface molecules, or recombinant proteins (eg, antibodies) made to have specificity for these cell surface molecules, interact with cell surface proteins to form dendritic cells. It would be expected to promote function or act to maintain an activated state, or to promote migration of dendritic cells to T-cell rich sites. That is, the present invention provides that single or multiple genes encoding single or multiple tumor antigens are differentially expressed in mature or immature dendritic cells by comparative analysis of gene expression (such as SAGE). Vaccines linked to one or more genes encoding secreted proteins capable of binding to and modulating the activity of a cell surface protein identified as such. In this manner, the genetically modified APC produces a ligand that is secreted by the APC and
An autocrine loop is established that binds to cell surface receptors on C cells and stimulates genetically altered dendritic cells.

While the foregoing invention has been described in detail by way of drawings and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is made by the appended claims.

[0192]

[Table 1]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0219]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 1/21 G01N 33/15 Z 5/10 33/50 Z G01N 33/15 33/53 M 33/50 33/566 33/53 37/00 102 33/566 C12N 15/00 ZNAA 37/00 102 5/00 A 15/00 F (31) Priority claim number 60 / 090,041 ( 32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 089,853 (32) Priority date 1998 March 19, 1998 (June 19, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 089,997 (32) Priority date June 19, 1998 (1998. 19) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,079 (32) Priority Japan June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,035 (32) Priority date June 19, 1998 ( (19 Jun. 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 089,993 (32) Priority date June 19, 1998 (June 19, 1998) (33) ) Priority claim country United States (US) (31) Priority claim number 60 / 089,992 (32) Priority date June 19, 1998 (1998.19.1998) (33) Priority claim country United States (US (31) Priority claim number 60 / 090,072 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 089,878 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 089,991 (32) Priority Date June 19, 1998 (June 19, 1998) (33) Priority Country United States (US) (31) Priority Owner No. 60 / 090,000 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,048 (32 ) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 089,999 (32) Priority date June 1998 (19) June 19, 1998 (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,043 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,042 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,036 (32) Priority date June 19, 1998 (June 19, 1998) (33) Country claiming priority United States (US) (31) Priority Claim number 60 / 090,044 (32) Priority date June 19, 1998 (June 19, 1998) (3 3) Priority claim country United States (US) (31) Priority claim number 60 / 089,844 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States ( (US) (31) Priority claim number 60 / 090,080 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim No. 60 / 089,833 (32) Priority date June 19, 1998 (June 19, 1998) (33) Country of priority claim United States (US) (31) Priority claim number 60 / 089,994 (32 ) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,077 (32) Priority date June 1998 (19) June 19, 1998 (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,078 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 090,04 (32) Priority date June 19, 1998 (June 19, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 090,076 (32) Priority date 1998 June 19, 1998 (June 19, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 090,045 (32) Priority date June 19, 1998 (June 1998) .19) (33) Priority claim country United States (US) (31) Priority claim number 60 / 111,715 (32) Priority date December 8, 1998 (1998.2.8) (33) Priority Claimed country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL) , SZ, UG, ZW), EA (AM, A Z, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE , DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) BB23 CC03 DD62 EE01

Claims (24)

[Claims] 1. An isolated population of polynucleotides comprising or corresponding to at least one of the polynucleotides set forth in Table 1 and their respective complements. 2. A polynucleotide encoding a ligand or an antibody or a designed protein that binds to a cell surface protein of an antigen presenting cell, and comprises or comprises the polynucleotide shown in Table 1 or a complement thereof. The polynucleotide corresponding to the body. 3. A polynucleotide encoding a transcription factor and comprising or corresponding to a polynucleotide set forth in Table 1 or a complement thereof. 4. A first antibody comprising an immunostimulatory factor that is differentially expressed on an antigen presenting cell and comprising or corresponding to the tag shown in Table 1 or a complement thereof. A polynucleotide comprising the polynucleotide of 5. The method according to claim 1, wherein the first polynucleotide is PARC, TARC, monocyte chemoattractant protein-4 (MDP-4), MDC, or escalectin.
5. The polynucleotide of claim 4, which encodes an agent selected from the group consisting of ctin), MCP-2, or biologically active fragments thereof. 6. The polynucleotide of claim 4, further comprising a first and second promoter, wherein the first and second polynucleotides are under the transcriptional control of the first and second promoters, respectively. . 7. The method of claim 4, further comprising a single promoter, wherein the first and second polynucleotides are under the transcriptional control of the single promoter.
Polynucleotide. 8. A novel polynucleotide comprising or corresponding to a tag shown in Table 1. 9. A gene delivery vehicle comprising the polynucleotide of claim 1-8. 10. A host cell comprising the polynucleotide of claim 1-8. 11. An array of probes comprising the polynucleotide of claim 1-8 bound to a chip. 12. A first polynucleotide comprising an immunostimulatory factor that is differentially expressed in antigen presenting cells, and comprising or corresponding to the tag shown in Table 1, and a first polynucleotide. A polynucleotide, comprising a second polynucleotide that regulates expression of the polynucleotide. 13. A method according to claim 12, wherein the first polynucleotide encodes an antigen and the second polynucleotide regulates the expression of a third polynucleotide encoding an immunostimulatory factor that is differentially expressed in antigen-presenting cells. Wherein the third polynucleotide comprises or corresponds to a tag set forth in Table 1. 14. The first polynucleotide encodes PARC, monocyte chemoattractant protein-4 (MDP-4), MDC, escalectin, MCP-2 or a biologically active fragment thereof. 14. The polynucleotide of claim 12 or 13. 15. A gene delivery vehicle comprising the polynucleotide of claim 12-14. 16. A host cell comprising the polynucleotide of claim 12-14. 17. A first polynucleotide encoding a recombinant protein or polypeptide that binds to a cell surface protein of an antigen presenting cell and thereby regulates, directly or indirectly, through a signaling pathway, and as shown in Table 1. Or a second polynucleotide encoding an immunostimulatory agent corresponding to or corresponding to the tag. 18. The polynucleotide of claim 17, further comprising first and second promoters, wherein the first and second polynucleotides are under the transcriptional control of the first and second promoters, respectively. . 19. The polynucleotide of claim 17, further comprising a single promoter, wherein said first and second polynucleotides are under the transcriptional control of said single promoter. 20. A gene delivery vehicle comprising the polynucleotide of claim 17-19. 21. A host cell comprising the polynucleotide of claims 17-19. 22. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the polynucleotide of claim 1, 12 or 17. 23. A method for regulating the genotype of an antigen-presenting cell, comprising introducing the polynucleotide of claim 1, 12 or 17 into a cell. 24. A method for screening for a candidate therapeutic agent that modulates the expression of a polynucleotide that is differentially expressed on an antigen presenting cell, comprising contacting the cell with an effective amount of a potential agent. Assaying for a change in the expression level of a polynucleotide of the invention, wherein the change in the expression level is indicative of a candidate therapeutic agent.