KR20010101242A - Compositions and methods for therapy and diagnosis of ovarian cancer - Google Patents

Abstract

Compositions and methods are provided for the treatment and diagnosis of cancer, such as ovarian cancer. The composition may comprise one or more ovarian carcinoma proteins, immunogenic portions thereof, polynucleotides encoding such portions or antibodies or immune system cells specific for said proteins. Such compositions can be used for the prevention and treatment of diseases such as, for example, ovarian cancer. Also provided are methods for identifying tumor antigens secreted from ovarian carcinoma and / or other tumors. Polypeptides and polynucleotides as provided herein can also be used for the diagnosis and monitoring of ovarian cancer.

Description

Compositions and methods for the treatment and diagnosis of ovarian cancer {Compositions and methods for therapy and diagnosis of ovarian cancer}

Ovarian cancer is a major health problem for women worldwide, including in the United States. Although progress has been made in the detection and treatment of ovarian cancer, no vaccines or other universally successful methods for the prevention or treatment of ovarian cancer are currently used. Treatment of ovarian cancer currently relies on a combination of aggressive treatments that may include one or more of several therapies such as early diagnosis and surgery, radiotherapy, chemotherapy and hormonal therapy. Therapeutic procedures for specific cancers are often selected based on a variety of prognostic variables, including analysis of specific tumor markers. However, the use of established markers often leads to inexplicable consequences, and high mortality is consistently observed in many cancer patients.

Immunotherapy has the potential to substantially improve cancer treatment and survival. Such therapies may include the generation or augmentation of an immune response against ovarian carcinoma antigens. However, to date a relatively small number of ovarian carcinoma antigens are known and the generation of an immune response to such antigens has not been shown to be therapeutically beneficial.

Accordingly, there is a need in the art for improved methods for identifying ovarian cancer antigens and for using such antigens in the treatment of ovarian cancer. The present invention fulfills this need and further provides other related advantages.

Summary of the Invention

In brief, the present invention provides compositions and methods for the treatment of cancer, such as ovarian cancer. In one aspect, the present invention provides immunogenicity of ovarian carcinoma proteins or ovarian carcinoma protein variants that differ by one or more substitutions, deletions, additions and / or insertions but do not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum. Provided is a polypeptide comprising a moiety. In certain embodiments, the ovarian carcinoma protein comprises a sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-81, 313-331, 359, 366, 379, 385-387, 391 and the complement of these polynucleotides. .

The present invention also provides polynucleotides encoding the above-described polypeptides or portions thereof, expression vectors comprising such polynucleotides, and host cells transformed or transfected with such expression vectors.

In another aspect, the present invention provides pharmaceutical compositions and vaccines. The pharmaceutical composition may comprise a physiologically acceptable carrier or excipient encoded by a polynucleotide comprising (i) a sequence provided in any one of SEQ ID NOs: 1-81, 313-331, 359, 366, 379, 385-387, or 391. Ovarian carcinoma protein comprising an amino acid sequence or an immunogenic portion of an ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum A polypeptide that binds to the polypeptide, (ii) a polynucleotide encoding such a polypeptide, (iii) an antibody that specifically binds to the polypeptide, (iv) an antigen-presenting cell that expresses the polypeptide, and / or (v) such And in combination with one or more of the T cells that specifically react with the polypeptide. The vaccine comprises an ovarian carcinoma comprising a nonspecific immune response enhancer comprising (i) an amino acid sequence encoded by a polynucleotide comprising a sequence provided in any one of SEQ ID NOs: 1-81, 313-331, 359, 366, 379, 385-387, or 391. A polypeptide comprising an immunogenic portion of a protein or ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum, (ii) Polynucleotides encoding such polypeptides, (iii) antiidiotype antibodies that specifically bind to antibodies that specifically bind to these polypeptides, (iv) antigen-presenting cells expressing such polypeptides, and / or ( v) can be combined with one or more of the T cells that specifically react with such polypeptides. .

The invention also provides, in another aspect, fusion proteins comprising one or more of the polypeptides described above, as well as polynucleotides encoding such fusion proteins.

In another aspect, provided is a pharmaceutical composition comprising a fusion protein or polynucleotide encoding it in combination with a physiologically acceptable carrier.

In another aspect, there is provided a vaccine comprising a fusion protein or polynucleotide encoding it in combination with a nonspecific immune response enhancer.

In a further aspect, the present invention provides a method of inhibiting the progression of cancer from a patient, comprising administering to the patient the pharmaceutical composition or vaccine described above.

In another aspect, the present invention provides a T cell comprising (a) an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide comprising a sequence provided in any one of SEQ ID NOs: 1 to 387 or 391 or one or more substitutions, deletions, additions And / or a polypeptide comprising an immunogenic portion of an ovarian carcinoma protein variant which, by insertion, differs but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum, (b) a polynucleotide encoding such polypeptide, (c) providing a method of stimulating and / or proliferating T cells, including contacting antigen presenting cells expressing these polypeptides under conditions and for a time sufficient to permit stimulation and / or proliferation of the T cells. . Such polypeptides, polynucleotides and / or antigen presenting cells may be present in pharmaceutical compositions or vaccines for use in stimulating and / or proliferating T cells in a mammal.

In another aspect, the present invention provides a method of inhibiting the progression of ovarian cancer from a patient, comprising administering to the patient a T cell prepared as described above.

In a further aspect, the present invention provides an antibody comprising (a) a CD4 ⁺ and / or CD8 ⁺ T cell isolated from a patient, and (i) an amino acid sequence encoded by a polynucleotide comprising the sequence provided in any one of SEQ ID NOs: 1-387 or 391. Polypeptides comprising an ovarian carcinoma protein comprising or an immunogenic portion of an ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum , (ii) incubating with at least one of the polynucleotides encoding such polypeptides or (iii) the antigen-presenting cells expressing such polypeptides to proliferate T cells, and (b) administer to the patient an effective amount of proliferated cells. Inhibiting the progression of ovarian cancer from the patient, thereby inhibiting the progression of ovarian cancer from the patient Provide a method. Proliferated cells can be cloned prior to administration to the patient.

In another aspect, the present invention provides a method for identifying a secreted tumor antigen. This method comprises (a) transplanting tumor cells into an immunodeficient mammal, (b) obtaining serum from the immunodeficient mammal after sufficient time to allow tumor antigens to be secreted into the serum, and (c) immunocompetent Immunizing the mammal with serum, (d) obtaining antiserum from an immunocompetent mammal, and (e) selecting a tumor expression library with antiserum and identifying tumor antigens secreted therefrom. Preferred methods of identifying secreted ovarian carcinoma antigens include (a) transplanting ovarian carcinoma cells into SCID mice, (b) obtaining serum from SCID mice after sufficient time to allow the secretion of ovarian carcinoma antigens into the serum, and (c ) Immunizing the immunocompetent mouse with serum, (d) obtaining antiserum from the immunocompetent mammal, and (e) selecting an ovarian carcinoma expression library with antiserum and identifying the ovarian carcinoma antigen secreted therefrom.

These and other aspects of the invention will be apparent upon reference to the following detailed description and the accompanying drawings. All references described herein are incorporated by reference in their entirety.

The present invention generally relates to the treatment of ovarian cancer. More specifically, the present invention relates to polypeptides comprising at least a portion of ovarian carcinoma proteins and to polynucleotides encoding such polypeptides, as well as antibodies and immune system cells that specifically recognize said polypeptides. Such polypeptides, polynucleotides, antibodies and cells can be used in vaccines and pharmaceutical compositions for the treatment of ovarian cancer.

1A-1S (SEQ ID NOS: 1-71) show partial sequences of polynucleotides encoding exemplary secreted ovarian carcinoma antigens.

2A-2C show the total insert sequences for three of the clones of FIG. 1. 2A shows sequence 11731; SEQ ID NO: 72, designated O7E. FIG. 2B shows sequence (11785; SEQ ID NO: 73) named O9E and FIG. 2C shows sequence (13695; SEQ ID NO: 74) named O8E.

3 shows the results of microarray expression analysis of ovarian carcinoma sequences named O8E.

4 shows a partial sequence of a polynucleotide (named 3g; SEQ ID NO: 75) encoding the ovarian carcinoma sequence, a splice fusion between human T-cell leukemia virus type I carcinogenic protein TAX and osteonectin.

Figure 5 shows ovarian carcinoma polynucleotide (SEQ ID NO: 76) named 3f.

FIG. 6 shows an ovarian carcinoma polynucleotide (SEQ ID NO: 77) named 6b.

7A and 7B show ovarian carcinoma polynucleotides (SEQ ID NOS: 78 and 79, respectively) designated 8e and 8h.

FIG. 8 shows ovarian carcinoma polynucleotide (SEQ ID NO: 80) designated 12c.

9 shows ovarian carcinoma polynucleotide (SEQ ID NO: 81) named 12h.

10 shows the results of microarray expression analysis of ovarian carcinoma sequences named 3f.

FIG. 11 shows the results of microarray expression analysis of ovarian carcinoma sequence named 6b.

Figure 12 shows the results of microarray expression analysis of ovarian carcinoma sequence named 8e.

FIG. 13 shows the results of microarray expression analysis of the ovarian carcinoma sequence named 12c.

FIG. 14 shows the results of microarray expression analysis of the ovarian carcinoma sequence named 12h.

15A-15EEE show partial sequences of additional polynucleotides (SEQ ID NOs: 82-310) encoding exemplary secreted ovarian carcinoma antigens.

16 is a diagram illustrating the location of several partial O8E sequences within the full length sequence.

As noted above, the present invention generally relates to compositions and methods for the treatment of cancer, such as ovarian cancer. The compositions described herein may comprise immunogenic polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies that bind the polypeptides, antigen presenting cells (APCs) and / or immune system cells (eg T cells). Can be.

Polypeptides of the invention generally comprise at least an immunogenic portion of an ovarian carcinoma protein or variant thereof. Certain ovarian carcinoma proteins have been identified using immunoassay techniques and are referred to herein as ovarian carcinoma antigens. An "ovarian carcinoma antigen" is a protein expressed by ovarian cancer cells (preferably human cells) at levels two or more times higher than in normal ovarian cells. Certain ovarian carcinoma antigens react to detectable levels (in immunoassays such as ELISA or Western blot) with antisera generated against serum from immunodeficient animals transplanted with human ovarian cancer. These ovarian carcinoma antigens flow out or secrete from ovarian cancer into the serum of immunodeficient animals. Thus, certain ovarian carcinoma antigens provided herein are secreted antigens. Certain nucleic acid sequences of the invention generally include DNA or RNA sequences that encode all or a portion of such polypeptides, or sequences that are complementary to such sequences.

The invention also provides ovarian carcinoma sequences identified using techniques for assessing modified expression in ovarian cancer. The sequence can be a polynucleotide or a protein sequence. Ovarian carcinoma sequences are generally expressed at levels two or more, preferably five or more, higher than expression levels in normal ovarian tissue in ovarian cancer, as determined using the representative assays provided herein. Certain partial ovarian carcinoma polynucleotide sequences are provided herein. Proteins encoded by genes comprising such polynucleotide sequences (or complementary sequences thereof) are also contemplated as ovarian carcinoma proteins.

Antibodies are generally immune system proteins or antigen-binding fragments thereof capable of binding at least a portion of an ovarian carcinoma polypeptide as described herein. T cells that can be used in the compositions provided herein are T cells specific to such polypeptides (eg, CD4 ⁺ and / or CD8 ⁺ ). Certain methods described herein further utilize antigen-presenting cells (eg, dendritic cells or macrophages) expressing an ovarian carcinoma polypeptide as provided herein.

Ovarian Carcinoma Polynucleotide

All of the polynucleotides encoding an ovarian carcinoma protein or portion or other variant thereof as described herein are included in the present invention. Preferred polynucleotides comprise at least 15 contiguous nucleotides, preferably at least 30 contiguous nucleotides, and more preferably at least 45 contiguous nucleotides encoding a portion of an ovarian carcinoma protein. More preferably, the polynucleotides encode an immunogenic portion of an ovarian carcinoma protein, such as an ovarian carcinoma antigen. Any polynucleotide complementary to this sequence is also included in the present invention. The polynucleotides may be single stranded (coding or antisense) or double stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention, although not necessarily required, and the polynucleotides may be linked to other molecules and / or support materials, although not necessarily.

The polynucleotide may comprise a native sequence (ie, an endogenous sequence that encodes an ovarian carcinoma protein or portion thereof) or may comprise a variant of such sequence. The polynucleotide variant may comprise an immunogenicity of the encoded polypeptide in a native ovarian carcinoma protein. It may contain one or more substitutions, additions, deletions and / or insertions that do not decrease in comparison. The impact on the immunogenicity of the encoded polypeptide can generally be assessed as described herein. The variant preferably exhibits at least about 70% identity with a polynucleotide sequence encoding a native ovarian carcinoma protein or portion thereof, more preferably at least about 80%, and most preferably at least about 90%.

The percent identity for two polynucleotide or polypeptide sequences can be readily determined by comparing the sequences using computer algorithms well known to those skilled in the art, such as Megaalign, using default variables. Comparisons between two sequences are typically made by identifying and comparing local regions of sequence similarity by comparing sequences in a comparison window. As used herein, a “comparison window” is about 20 or more, usually 30 to about 75, or 40 to about at least two sequences that can be optimally aligned and then one sequence can be compared with the same number of consecutive reference sequences Points to fragments of 50 consecutive positions. Optimal alignment of sequences for comparison can be performed using the Megaline program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default variables. Can be. Preferably, the proportion of sequence identity is 20% or less, usually 5 to less than or equal to about 20 positions or more, wherein a portion of the polynucleotide or polypeptide sequence in the comparison window does not contain additions or deletions May comprise 15% or 10-12% additions or deletions (ie, gaps)]. The ratio of sequence identity determines the number of positions where identical nucleic acid bases or amino acid residues are present in two sequences to yield the number of positions matched, and the number of positions matched is the total number of positions in the reference sequence (ie, window size). It can be calculated by multiplying the result by 100 and calculating the proportion of sequence identity.

The variant may also, or alternatively, be substantially homologous to the native gene or portion or complement thereof. Such polynucleotide variants can hybridize under moderately stringent conditions with the native DNA sequence (or complementary sequence) encoding the native ovarian carcinoma protein. Suitable moderately stringent conditions were pre-washed in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0, hybridized overnight at 50 ° C.-65 ° C., 5 × SSC, then 20 at 65 ° C. each time. Washing twice with 2X, 0.5X and 0.2X SSC containing 0.1% SDS for minutes.

It will be understood by those skilled in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides exhibit minimal homology with the nucleotide sequences of all native genes. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are modified as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNAs and proteins are not necessary but may have a modified structure or function. Alleles can be identified using standard techniques (eg, hybridization, amplification, and / or database sequence comparison).

Polynucleotides can be prepared using any of a number of techniques. For example, ovarian carcinoma polynucleotides are generated for serum of immunocompetent mice following injection of a terminal ovarian cancer expression library into mice with serum from SCID mice transplanted with terminal ovarian cancer as described in more detail below. Can be identified by selection with antisera. Ovarian carcinoma polynucleotides can also be identified using any of several techniques designed to assess differential gene expression. Alternatively, polynucleotides can be amplified from cDNA prepared from ovarian cancer cells. Such polynucleotides can be amplified via polymerase chain reaction (PCR). For this method, sequence-specific primers can be designed, purchased or synthesized based on the sequences provided herein.

The amplified portion can be used to isolate full length genes from suitable libraries (eg, ovarian carcinoma cDNA libraries) using the techniques known. Within this technique, the library (cDNA or genomic) is selected using one or more polynucleotide probes or primers suitable for amplification. Preferably, libraries are screened by size to include larger molecules. Random primed libraries may also be desirable for identifying the 5 'and upstream regions of a gene. Genomic libraries are preferred for obtaining introns and extending 5 'sequences.

For hybridization techniques, a partial sequence, using the familiar techniques, for example, nick (nick) using the ³² P-end decryption or - it can be labeled by a cover. The bacterial or bacteriophage libraries are then screened by hybridization with a labeled probe containing a denatured bacterial colony (or a loaf containing phage plaques). Sambrook et al., Molecular Cloning: A Laboratory Manual. , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989]. Colonies or plaques that hybridize are selected and propagated, and then DNA is isolated for further analysis. The cDNA clone can be analyzed by determining the amount of additional sequence, for example, by PCR using primers from a vector and primers from partial sequences. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques, which can include generating a series of deletion clones. The resulting overlapping sequences are then aggregated into one continuous sequence. Full length cDNA molecules can be generated by linking suitable fragments using known techniques.

As an alternative, there are many amplification techniques for obtaining full length coding sequences from partial cDNA sequences. Within this technique, amplification is generally carried out via PCR. The amplification step can be performed using any of a variety of commercially available kits. Primers can be designed using, for example, software well known in the art. The primer is preferably 22 to 30 nucleotides in length, has a GC content of at least 50% and anneals to the target sequence at a temperature of about 68 ° C to 72 ° C. The amplified region can be sequenced as described above and the overlapping sequences can be aggregated into contiguous sequences.

One such amplification technique uses inverse PCR to generate fragments belonging to known regions of genes using restriction enzymes [Triglia et al., Nucl. Acids Res. 16: 8186, 1988. The fragment is then used as a template for ring closure by intramolecular ligation and for PCR with different primers derived from known regions. Alternatively, the sequence adjacent to the partial sequence can be repaired by amplification with a primer for the linker sequence and a primer specific for a known region. The amplified sequence is typically subjected to secondary amplification with the same linker primer and a second primer specific for a known region. Modifications to this method using two primers that initiate extension in opposite directions from known sequences are described in WO 96/38591. Further techniques are described in Capture PCR [Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991] and working PCR [Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991. Other methods using amplification can also be used to obtain full length cDNA sequences.

As a specific example, sequences provided in expressed sequence tag (EST) databases such as those available from GenBank can be analyzed to obtain full length cDNA sequences. Investigations of overlapping ESTs can generally be carried out using well known programs (eg, NCBI BLAST surveys), which can be used to generate continuous full length sequences.

Specific nucleic acid sequences of cDNA molecules encoding portions of ovarian carcinoma antigens are provided in FIGS. 1A-1S (SEQ ID NOS: 1-71) and FIGS. 15A-15EEE (SEQ ID NOs: 82-310). The sequences provided in FIGS. 1A-1S were identified as novel. For the sequences provided in FIGS. 15A-15EEE, database searches revealed a match with substantial identity. These polynucleotides were isolated by serological selection of ovarian cancer cDNA expression libraries using techniques designed to identify secreted tumor antigens. Briefly, terminal ovarian cancer expression libraries were prepared from SCID-induced human ovarian cancer (OV9334) on a vector λ-screen (Novagen). Serum used for selection was obtained by injecting sera from SCID mice implanted with terminal ovarian cancer into immunocompetent mice. It is possible to identify cDNA molecules encoding the immunogenic portion of the secreted tumor antigen by this technique.

Particularly included in the invention are polynucleotides provided herein, as well as full length polynucleotides comprising such sequences, other portions of such full length polynucleotides, and sequences complementary to all or part of such full length molecules. It will be apparent to those skilled in the art that the technique can also be applied to identify antigens secreted from other types of tumors.

Other nucleic acid sequences of cDNA molecules encoding portions of ovarian carcinoma proteins are provided in FIGS. 4-9 (SEQ ID NOS: 75-81) and SEQ ID NOs: 313-384. These sequences were identified by selecting microarrays of cDNA for tumor-associated expression (ie, expression at least 5 times higher in ovarian cancer compared to normal ovarian tissue as determined using the representative assays provided herein). Such selection is in accordance with the manufacturer's instructions and essentially as described in Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155, 1997, using a Synteni microarray (Palo Alto, Calif.). SEQ ID NOs: 311 and 391 are full length sequences incorporating some of these nucleic acid sequences.

Any of several well-known techniques can be used to assess tumor-associated expression of cDNA. For example, hybridization techniques using labeled polynucleotide probes can be used. As an alternative or in addition, amplification techniques such as real-time PCR can be used. See Gibson et al., Genome Research 6: 995-1001, 1996; Heid et al., Genome Research 6: 986-994, 1996. Real-time PCR is a technique for evaluating the accumulation level of PCR products during amplification. This technique allows for quantitative assessment of mRNA levels in multiple samples. Briefly, mRNA is extracted from tumors and normal tissues and cDNA is prepared using standard techniques. Real-time PCR can be performed using, for example, 7700 Prism equipment from the manufacturer (Perkin Elmer / Applied Biosystems, Foster City, CA). For example, matched primers and fluorescent probes can be designed for that gene using primer expression programs provided by the manufacturer (Perkin Elmer / Applied Biosystems, Foster City, CA). Optimal concentrations of primers and probes can be initially determined by one skilled in the art, and control (eg, β-actin) primers and probes can be purchased, for example, from the manufacturer (Perkin Elmer / Applied Biosystems, Foster City, CA). . To quantify the amount of specific RNA in the sample, a standard curve is generated side by side using the plasmid containing the gene. Standard curves can be generated using Ct values generated in real-time PCR that correlate with the initial cDNA concentration used in the assay. Standard dilution ranges from 10 to 10 ⁶ copies of the gene in question are generally sufficient. In addition, standard curves are generated for the control sequences. This allows for standardization of the initial RNA content of the tissue sample against the control amount for comparison purposes.

Polynucleotide variants can generally be prepared by any method known in the art, including chemical synthesis by solid phase phosphoramidite chemical synthesis. Modifications in the polynucleotide sequence can also be accomplished using standard mutagenesis techniques such as oligonucleotide-directed site-specific mutagenesis (Adelman et al., DNA 2: 183, 1983). Alternatively, the RNA molecule may be produced by in vitro or in vivo transcription of a DNA sequence encoding a ovarian carcinoma antigen or portion thereof, as long as the DNA is inserted into a vector with a suitable RNA polymerase promoter (T7 or SP6). have. As described herein, certain portions can be used to prepare encoded polypeptides. In addition, or alternatively, a portion may be administered to the patient to produce the encoded polypeptide in vivo.

Portions of sequences complementary to the coding sequence (ie, antisense polynucleotides) can also be used as probes or to control gene expression. CDNA constructs that can be transcribed into antisense RNA can also be introduced into cells or tissues to promote production of antisense RNA. Antisense polynucleotides can be used as described herein to inhibit the expression of ovarian carcinoma proteins. Antisense technology can be used to regulate gene expression through triple-helix formation, which inhibits the ability of the double helix to sufficiently cleave for binding of polymerases, transcription factors or regulatory molecules. Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY; 1994]. Alternatively, the antisense molecule can be designed to block translation by hybridizing with regulatory regions (eg, promoters, enhancers or transcription initiation sites) of the gene and blocking transcription of the gene or inhibiting binding of the transcript to ribosomes. have.

Any polynucleotide can be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' ends, the use of phosphorothioate or 2'O-methyl, but not phosphodiesterase bonds in the backbone, and / Or insertions of acetyl-, methyl-, thio- and other modified forms of adenine, cytidine, guanine and uridine as well as non-traditional bases such as inosine, quenocin and wibutosin.

The nucleotide sequences described herein can be linked to many other nucleotide sequences using established recombinant DNA techniques. For example, polynucleotides can be cloned into various cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, the vector contains a replication origin that functions in one or more organisms, a convenient restriction endonuclease site, and one or more selection markers. Other elements may be used depending on the intended use and will be apparent to those skilled in the art.

In a particular embodiment, the polynucleotides may be formulated to be introduced into the mammalian cells for expression. Such formulations are particularly useful for therapeutic purposes as described below. It will be understood by those skilled in the art that there are many ways to achieve expression of polynucleotides in a target cell, and any suitable method may be used. For example, polynucleotides can be inserted into viral vectors such as, but not limited to, adenoviruses, adeno-associated viruses, retroviruses or vaccinia or other varicella virus (eg, avian pox virus). Techniques for inserting DNA into such vectors are well known to those skilled in the art. Retroviral vectors may additionally be transferred to or inserted targeting moieties such as genes for selection markers (to aid in the identification or selection of transduced cells) and / or genes encoding ligands for receptors on specific target cells. Can be made specific to the target. Targeting can also be accomplished using antibodies by methods known to those of skill in the art.

Other formulations for therapeutic purposes include gelatinous dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. Preferred colloidal systems for use as delivery vehicles in vitro or in vivo are liposomes (ie artificial membrane vesicles). The preparation and use of such systems are well known in the art.

Ovarian Carcinoma Polypeptide

Within the context of the present invention, a polypeptide may comprise at least an immunogenic portion of an ovarian carcinoma protein or variant thereof as described herein. As noted above, certain ovarian carcinoma proteins are ovarian carcinoma expressed by ovarian cancer cells and reacting detectably with antisera produced against serum from immunodeficient animals transplanted with ovarian carcinoma in an immunoassay (eg, ELISA). It is an antigen. Other ovarian carcinoma proteins are encoded by ovarian carcinoma polynucleotides provided herein. The length of the polypeptide as described herein can be any. There may be additional sequences and / or heterologous sequences derived from natural proteins, which sequences may retain additional immunogenic or antigenic properties, although not necessarily.

As used herein, an “immunogenic moiety” is a portion of an antigen that is recognized (ie, specifically bound) by a B-cell and / or T-cell surface antigen receptor. Such immunogenic moieties generally comprise at least 5 amino acid residues in an ovarian carcinoma protein or variant thereof, more preferably at least 10, and even more preferably at least 20 amino acid residues. Preferred immunogenic portions are encoded by isolated cDNA molecules as described herein. In addition, immunogenic moieties can generally be identified using known techniques as summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and the references cited herein. have. Such techniques include screening polypeptides for their ability to react with ovarian carcinoma protein-specific antibodies, antisera and / or T-cell lines or clones. As used herein, antisera and antibodies are those in which they specifically bind to ovarian carcinoma proteins (ie, they react with ovarian carcinoma proteins in ELISA or other immunoassays and do not react to detectable levels with irrelevant proteins). "Ovarian carcinoma protein-specific". Such antiserum, antibodies, and T cells can be prepared as described herein and using techniques known to the art. The immunogenic portion of the native ovarian carcinoma protein is the portion that reacts with such antiserum, antibodies and / or T-cells at a level substantially less than the reactivity of the full length polypeptide (eg, in ELISA and / or T-cell reactivity assays). to be. Such immunogenic moieties may respond at levels similar to or greater than the reactivity of full length proteins in such assays. Such screening can generally be carried out using methods known to those skilled in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the polypeptide may be immobilized on a solid support and contacted with the serum of the patient to allow antibodies in the serum to bind the immobilized polypeptide. The unbound serum can then be removed and the bound antibody can be detected using, for example, ¹²⁵ I-labeled Protein A.

As noted above, the composition may comprise variants of natural ovarian carcinoma proteins. As used herein, a polypeptide “variant” is a polypeptide that differs from a native ovarian carcinoma protein by one or more substitutions, deletions, additions and / or insertions, but without substantially reduced immunogenicity of the polypeptide. In other words, the ability of the variant to react with ovarian carcinoma protein-specific antiserum may be increased or invariant compared to native ovarian carcinoma protein, or may be reduced to less than 50%, preferably less than 20%, relative to native ovarian carcinoma protein. have. Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the reaction of the modified polypeptide with an ovarian carcinoma protein-specific antibody or antiserum as described herein. Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) is removed from the N- and / or C-terminus of the mature protein.

Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90%, and most preferably at least about 95% identity with the native polypeptide. Preferably, variants contain conservative substitutions. A “conservative substitution” is a substitution in which an amino acid is substituted with another amino acid having similar properties, and consequently one skilled in the art of peptide chemistry can expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions can generally be achieved based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphotericity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, Charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups exhibiting similar hydrophilicity values include leucine, isoleucine and valine; Glycine and alanine; Asparagine and glutamine; And serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may exhibit conservative modifications include (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; And (5) phe, tyr, trp, his. Variants may also or alternatively contain non-conservative modifications. Variants can also be modified, or alternatively, for example, by deletion or addition of amino acids with minimal impact on the immunogenicity, secondary structure and degree of hydration of the polypeptide.

As noted above, the polypeptide may include a signal (or leader) sequence at the N-terminus of the protein that directs the transfer of the protein concurrently with or after translation. The polypeptide may also be linked to a linker or other sequence for its convenient synthesis, purification or identification (eg poly-His), or to increase binding of the polypeptide to a solid support. For example, the polypeptide can be bound to an immunoglobulin Fc region.

Polypeptides can be prepared using any of several well known techniques. Recombinant polypeptides encoded by DNA sequences as described above can be readily prepared from DNA sequences using any of a variety of expression vectors known to those skilled in the art. Expression can be achieved in any suitable host cell transformed or transfected with an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast and higher eukaryotic cells. Preferably, the host cell used is E. coli. E. coli, yeast or mammalian cell lines (e.g. COS or CHO). Supernatants from suitable host / vector systems that secrete recombinant proteins or polypeptides into the culture medium may first be concentrated using commercially available filters. After concentration, the concentrate can be applied to a suitable purification matrix, such as an affinity matrix or an ion exchange resin. Finally, one or more reversed phase HPLC steps can be used to further purify the recombinant polypeptide.

Portions and other variants having up to about 100 amino acids, typically up to about 50 amino acids, can also be produced by synthetic means using techniques known to those skilled in the art. For example, such polypeptides can be synthesized using any of the commercially available solid phase techniques, such as the Merrifield solid phase synthesis method in which amino acids are added to sequentially growing amino acid chains. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Automated synthesis of polypeptides is available from suppliers such as the manufacturer (Applied Biosystems, Inc., Foster City, CA) and can be operated according to the manufacturer's instructions.

In certain embodiments, the polypeptide may be a fusion protein comprising a plurality of polypeptides as described herein or a known tumor antigen, such as one polypeptide and an ovarian carcinoma protein or a variant of such a protein as described herein. Can be. The fusion partner may, for example, assist in providing a T helper epitope, preferably a T helper epitope recognized by humans (immunological fusion partner), or assist in expressing the protein at a higher level than the native recombinant protein. (Expression enhancer). Preferred specific fusion partners are fusion partners that are immunological and enhance expression. Other fusion partners can choose to increase the solubility of the protein or to allow the protein to target the desired intracellular compartments. Still further fusion partners include affinity tags that facilitate purification of the protein.

Fusion proteins can generally be prepared using standard techniques, including chemical bonding. Preferably, the fusion protein is expressed as a recombinant protein, thereby allowing it to be produced at increased levels compared to unfused proteins in the expression system. In brief, DNA sequences encoding polypeptide components can be collected separately and linked into appropriate expression vectors. The 3 'end of the DNA sequence encoding one polypeptide component is linked with or without a peptide linker to the read frame of the sequence at the 5' end of the DNA sequence encoding the second polypeptide component. This allows translation into one fusion protein that maintains the biological activity of both component polypeptides.

Peptide linker sequences can be used to separate the first and second polypeptide components at sufficient intervals to ensure that each polypeptide folds securely into its secondary and tertiary structures. Such peptide linker sequences are inserted into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) the ability to accept flexible extended structures; (2) the inability to accept secondary structures capable of interacting with functional epitopes on the first and second polypeptides; And (3) lack of hydrophobic or charged residues that can react with the polypeptide functional epitope. Preferred peptide linker sequences contain Gly, Asn and Ser residues. In addition, other neutral amino acids, such as Thr and Ala, may be used in the linker sequence. Amino acid sequences that may be usefully used as linkers include those described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8562, 1986; And US Pat. No. 4,935,233 and US Pat. No. 4,751,180. The linker sequence may be 1 to about 50 amino acids in length. The linker sequence is not necessary if the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric hindrance.

The linked DNA sequence is operably linked to a suitable transcriptional or translational regulatory element. The regulatory element responsible for the expression of the DNA is located only 5 'of the DNA sequence encoding the first polypeptide. Similarly, the stop codons required to terminate the translational and transcription termination signals are present only at 3 'of the DNA sequence encoding the second polypeptide.

Also provided is a fusion protein comprising a polypeptide of the invention with an unrelated immunogenic protein. Preferably, the immunogenic protein may exhibit an immune memory response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins. See, eg, Stoute et al., New Engl. J. Med., 336: 86-91, 1997].

In a preferred embodiment, the immunological fusion partner is derived from protein D, which is the surface protein of Gram-negative bacterial Haemophilus influenza B (WO 91/18926). Preferably, the protein D derivative comprises the first about one third of the protein (eg, the first N-terminal 100-110 amino acids), and the protein D derivative may be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included at the N-terminus to provide a polypeptide having additional exogenous T-cell epitopes. Increase the expression level in E. coli, thus acting as an expression enhancer. Lipid tails ensure optimal presentation of antigen to antigen presenting cells. Other fusion partners include NS1 (hemagglutinin), a nonstructural protein from influenza viruses. Although different fragments containing T-helper epitopes can be used, typically, N-terminal 81 amino acids are used.

In another embodiment, the immunological fusion partner is a protein known as LYTA or a portion thereof (preferably C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alanine amidase, known as amidase LYTA, encoded by the LytA gene [Gene 43: 265-292, 1986]. LYTA is an automelting agent that specifically cleaves specific bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is involved in affinity for choline or for some choline analogs such as DEAE. This property allows E. coli to express plasmids useful for expression of fusion proteins. It has been studied for the development of E. coli C-LYTA. Purification of hybrid proteins containing C-LYTA fragments at the amino terminus has been described (Biotechnology 10: 795-798, 1992). In a preferred embodiment, repeating portions of LYTA can be inserted into the fusion protein. The repeat portion is found in the C-terminal region starting at residue 178. Particularly preferred repeat moieties contain residues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. A "isolated" polypeptide or polynucleotide has been removed from its natural environment. For example, natural proteins are isolated if they are separated from some or all of the substances present in nature together. Such polypeptides preferably have a purity of at least about 90%, more preferably at least about 95%, and most preferably at least about 99%. Polynucleotides are considered isolated, for example, when they are cloned into a vector that is not part of the natural environment.

Binder

The invention also provides substances such as antibodies and antigen-binding fragments thereof that specifically bind to ovarian carcinoma proteins. As used herein, an antibody or antigen-binding fragment thereof may cause an ovarian carcinoma if it reacts with detectable levels (eg, in an ELISA) with an ovarian carcinoma protein and does not react to detectable levels with an unrelated protein under similar conditions. It is said to "specifically bind" to a protein. As used herein, "bond" refers to a non-covalent bond between two separate molecules that cause a "complex" to form. Binding capacity can be assessed, for example, by determining the binding constant for the formation of the complex. The binding constant is the concentration of the complex divided by the value of the component concentration. In general, the two components are referred to herein as "binding" when the binding constant of the complex formation exceeds about 10 ³ L / mol. Binding constants can be determined using methods known in the art.

Binding agents can also be used to distinguish patients suffering from or without cancer, such as ovarian cancer. In other words, an antibody or other binding agent that binds to an ovarian carcinoma antigen generates a signal indicative of the presence of cancer in about 20% or more of patients with the disease, and in about 90% or more of individuals who do not have cancer Generates a negative signal indicating absence. Biological samples (eg, blood, serum, leukocytes, urine and / or tumor biopsies) from patients without cancer and individuals with cancer determined using standard clinical trials to determine whether the binder meets these requirements Assays can be assayed as described herein for the presence of a polypeptide that binds to the binder. It is clear that patients suffering from disease and individuals not suffering from disease should be tested in a statistically significant number. Each binder must meet the above criteria. However, those skilled in the art will understand that a combination of binders may be used to enhance the sensitivity.

Any material that meets the above requirements can be a binder. For example, the binding agent may be ribosomes with or without peptide components, RNA molecules or polypeptides. In a preferred embodiment, the binding agent is an antibody or antigen-binding fragment thereof. Antibodies can be prepared by any of a variety of techniques known to those skilled in the art (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). . In general, antibodies can be prepared via cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfection of the antibody gene into a bacterial or mammalian cell host suitable for the production of recombinant antibodies. have. As one technique, immunogens, including polypeptides, are initially injected into a variety of mammals (eg, mice, rats, rabbits, sheep or goats). In this step, the polypeptide of the present invention can act as an immunogen without modification. Alternatively, especially for relatively short polypeptides, the polypeptides may show good immune responses when linked to carrier proteins such as bovine serum albumin or scavenger hemocyanin. The immunogen is preferably injected into the animal host and periodically drawn from the animal according to a predetermined schedule including one or more boosters. Polyclonal antibodies specific for the polypeptide can then be purified from the serum, for example, by affinity chromatography using the polypeptide linked to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976, and improved techniques thereof. In summary, these methods include the preparation of immortal cell lines capable of producing antibodies with the desired specificity (ie, reactivity with the polypeptide in question). Such cell lines can be generated, for example, from spleen cells obtained from an immunized animal as described above. The spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably one that is genetically associated with the immunized animal. Various fusion techniques can be used. For example, splenocytes and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density in a selection medium that supports the growth of hybrid cells but not myeloma cells. Preferred selection techniques use HAT (hypoxanthine, aminopterin, thymidine) selection. After sufficient time, colonies of hybrids are usually observed after about 1 to 2 weeks. Single colonies are selected and their culture supernatants tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, several techniques can be used to increase yield, such as injecting hybridoma cell lines into the abdominal cavity of a suitable vertebrate host such as a mouse. Monoclonal antibodies can then be harvested from ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction. Polypeptides of the invention can be used, for example, in the purification of affinity chromatography steps.

In certain embodiments, it may be desirable to use antigen-binding fragments of antibodies. Such fragments include Fab fragments that can be prepared using standard techniques. In summary, immunoglobulins can be purified from rabbit serum by affinity chromatography on a Protein A bead column [Harlow and Lane, Antiodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988] to yield Fab and Fc fragments. have. Fab and Fc fragments can be separated by affinity chromatography on a Protein A bead column.

Monoclonal antibodies of the invention may bind to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducing agents, drugs, toxins and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At and ²¹² Bi. Preferred drugs include methotrexate and pyrimidine and purine analogs. Preferred differentiation inducing agents include phorbol esters and butyric acid. Preferred toxins include lysine, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, shigella toxin, and the US pore antiviral protein.

The therapeutic agent may be linked (eg, covalently) directly or indirectly (eg via a linker group) to a suitable monoclonal antibody. Direct reaction between the agent and the antibody is possible when each possesses a substituent that can react with the other. For example, nucleophilic groups such as amino or sulfhydryl groups on one side may react with alkyl groups containing good leaving groups (eg halides) on the other side, or carbonyl-containing groups such as anhydrides or acid halides. Can be.

Alternatively, it may be desirable to bind the therapeutic agent and antibody through a linker group. The linker group can act as a spacer to isolate the antibody from the agent to avoid conflict with binding forces. In addition, the linker group may act to increase the chemical reactivity of the substituent on the agent or antibody, thereby increasing the binding capacity. Increasing chemical reactivity can also promote the use of agents or functional groups on agents that are otherwise impossible.

It will be apparent to those skilled in the art that a variety of di- or multi-functional reagents, such as those described in the catalog of Pierce Chemical Company, Rockford, Ill., May be used as linker groups, regardless of homo and hetero functionality. The linkage may be made, for example, via an amino group, carboxyl group, sulfhydryl group or oxidized carbohydrate moiety. There are many references to this method, for example, US Pat. No. 4,671,958 to Rodwell et al.

If the therapeutic agent is more effective than when liberated from the antibody portion of the immunobinding agent of the invention, it may be desirable to use a cleavable linker group during internalization or when internalizing. Many different cleavable linker groups are disclosed. Mechanisms for intracellular release of agents from these linker groups include reduction of disulfide bonds (e.g., US Pat. No. 4,489,710 to Spitler), radiation of photodegradable bonds (e.g., US Pat. No. 4,625,014 to Center et al.), Derivatization Hydrolysis of purified amino acid side chains (eg US Pat. No. 4,638,045 to Kohmon et al.), Serum complement-mediated hydrolysis [eg US Pat. No. 4,671,958 to Rodwell et al.] And acid-catalyzed hydrolysis [eg Bla US Pat. No. 4,569,789 to Tler et al.

It may be desirable to bind one or more agents to the antibody. In one embodiment, multiple molecules of the agent are bound to one antibody molecule. In another embodiment, more than one type of agent may be bound to one antibody. Regardless of certain embodiments, immunocombinants with one or more agents can be prepared by a variety of methods. For example, one or more agents may be directly bound to the antibody molecule, or linkers may be used that provide multiple binding sites. Alternatively, a carrier can be used.

The carrier may contain the agent in several ways, including covalent bonds, either directly or through a linker group. Suitable carriers include proteins, peptides such as albumin (eg, US Pat. No. 4,507,234 to Kato et al.) And polysaccharides such as aminodextran (eg, US Pat. No. 4,699,784 to Sich et al.). The carrier may also contain the agent by encapsulation or non-covalent linkage, such as in liposome vesicles (eg, US Pat. Nos. 4,429,008 and 4,873,088). Carriers specific to radionuclide preparations include radiohalogenated small molecules and chelated compounds. For example, US Pat. No. 4,735,792 describes representative radiohalogenated small molecules and their synthesis. Radionuclide chelating agents can be formed from chelating compounds, including those containing nitrogen and sulfur atoms as donor atoms for binding metals or metal oxides, radionuclides. For example, US Pat. No. 4,673,562 to Davidson et al. Describes exemplary chelated compounds and their synthesis.

Several routes for administering antibodies and immunoconjugates can be used. Typically, the administration can be intravenous, intramuscular, subcutaneous or incised tumors. It is evident that the exact dose of antibody / immunoconjugate may vary depending on the antibody used, antigen density on the tumor and the removal rate of the antibody.

Also provided herein are anti-idiotype antibodies that mimic the immunogenic portion of ovarian carcinoma protein. Such antibodies can be derived for antibodies or antigen-binding fragments thereof that specifically bind to the antigenic portion of the ovarian carcinoma protein using techniques known to the art. An anti-idiotype antibody that mimics an immunogenic portion of an ovarian carcinoma protein is an antibody that specifically binds to an immunogenic portion of an ovarian carcinoma protein or an antigen-binding fragment thereof.

T cell

The immunotherapeutic composition also includes, or alternatively, T cells specific for ovarian carcinoma protein. Such cells can generally be prepared in vitro or in vivo using standard procedures. For example, T cells may be obtained from bone marrow, peripheral blood, or mammalian animals such as patients using cell isolation system commercially available products such as the CEPRATE ^™ system, available from the manufacturer (Selfuro Inc., Botel, Washington). May be present in or separated from the fractions (see US Pat. No. 5,240,856, US Pat. No. 5,215,926, WO 89/06280, WO 91/16116 and WO 92/07243). Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines or cultures.

T cells can be stimulated with ovarian carcinoma polypeptides, polynucleotides encoding them and / or antigen presenting cells (APCs) expressing such polypeptides. This stimulation is carried out under conditions and for a time sufficient to produce T cells specific for the polypeptide. Preferably, the ovarian carcinoma polypeptide or polynucleotide is present in a delivery vehicle such as microspheres to promote the production of specific T cells.

T cells are considered to be specific for ovarian carcinoma polypeptides if they kill target cells coated with ovarian carcinoma polypeptides or target cells that express genes encoding such polypeptides. T cell specificity can be assessed using any of a variety of standard techniques. For example, in chromium release assays or proliferation assays, the stimulation index of at least two-fold increase relative to negative control in lysis and / or proliferation indicates T cell specificity. Such assays are described, for example, in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of proliferation of T cells can be accomplished by various known techniques. For example, T cell proliferation can be detected by measuring the rate of increase in DNA synthesis (eg, by pulse-labeling a culture of T cells with tritium thymidine and measuring the amount of tritium thymidine inserted into the DNA). Can be. Contact with an ovarian carcinoma polypeptide (200 ng / ml to 100 μg / ml, preferably 100 ng / ml to 25 μg / ml) for 3 to 7 days should increase the proliferation of T cells by more than two times As noted, contacting for 2-3 days results in activation of T cells, as measured using standard cytokine assays, with a two-fold increase in the level of cytokine (eg, TNF or IFN-γ) release. Indicates T cell activation. Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). T cells activated in response to ovarian carcinoma polypeptide, polynucleotide or ovarian carcinoma polypeptide-expressing APC may be CD4 ⁺ and / or CD8 ⁺ . Ovarian carcinoma polypeptide-specific T cells can be propagated using standard techniques. In a preferred embodiment, T cells are derived from the patient or related or irrelevant donor and administered to the patient after stimulation and proliferation.

For therapeutic purposes, CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to ovarian carcinoma polypeptide, polynucleotide or APC can be proliferated numerically in vitro or in vivo. In vitro proliferation of such T cells can be achieved in several ways. For example, T cells can be reexposed to ovarian carcinoma polypeptides with or without the addition of stimulatory cells that synthesize ovarian carcinoma polypeptides and / or T cell growth factors such as interleukin-2. Alternatively, one or more T cells proliferating in the presence of an ovarian carcinoma polypeptide can be proliferated numerically by cloning. Methods for cloning cells are known in the art and include limiting dilution. After proliferation, cells are described, for example, in Chang et al., Crit Rev. Oncol. Hemato. 22: 213, 1996, may be re-administered to the patient.

Pharmaceutical Compositions and Vaccines

Within certain embodiments, polypeptides, polynucleotides, binders and / or immune system cells as described herein can be incorporated into pharmaceutical compositions or vaccines. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier. The vaccine may comprise one or more such compounds or cells and nonspecific immune response enhancers. Nonspecific immune response enhancers can be any substance that enhances an immune response against foreign antigens. Examples of nonspecific immune response enhancers include adjuvants, biodegradable microspheres (eg polylactic galactide), and liposomes, and compounds are incorporated therein (US Pat. No. 4,235,877 to Fullerton). Vaccine formulations are generally described, for example, in M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)", Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds that may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be incorporated into the fusion polypeptide in the composition or vaccine or exist as separate compounds.

The pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is produced in situ. As noted above, DNA may be present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the DNA sequences necessary for expression in a patient (eg, a suitable promoter and termination signal). Bacterial delivery systems include the administration of bacteria (eg, Bacillus-Calmette-Guerrin) that express an immunogenic portion of the polypeptide on the cell surface. In a preferred embodiment, the DNA can be introduced using a viral expression system (eg vaccinia or other varicella virus, retrovirus or adenovirus) that can include the use of nonpathogenic (defective) replication competent viruses. Suitable systems are described, for example, in Fisher-Hoch et al., PNAS 86: 317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patents 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; US Patent No. 4,777,127; British Patent 2,200,651; European Patent No. 0,345,242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., PNAS 91: 215-219, 1994; Kass-Eisler et al., PNAS 90: 11498-11502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993. Techniques for incorporating DNA into such expression systems are known to those skilled in the art. In addition, DNA is "naked" as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and as reviewed in Cohen, Science 259: 1691-1692, 1993. Can be. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads that are efficiently transported into the cell.

Any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the present invention, while the type of carrier can vary depending on the mode of administration. The compositions of the present invention may be formulated for a suitable mode of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, the above carriers or solid carriers (eg mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate) can be used. In addition, biodegradable microspheres (eg, polylactate polyglycolate) can be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268 and 5,075,109.

Such compositions may also contain buffers (e.g. neutral buffered saline or phosphate buffered saline), carbohydrates (e.g. glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids (e.g. glycine), antioxidants, Chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide) and / or preservatives. Alternatively, the composition of the present invention may be formulated as a lyophilisate. The compounds may also be encapsulated into liposomes using techniques known to the art.

Various nonspecific immune response enhancers can be used in the vaccines of the present invention. For example, an adjuvant may be included. Most adjuvants are substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and stimulants of immune responses (eg, lipid A, Bortadella pertussis or Mycobacterium tubercules). Rossis (Mycobacterium tuberculosis induced protein). Suitable adjuvants include, for example, Freund's incomplete and complete adjuvants (Diffco Laboratories, Detroit, Mich.), Merck Aid 65 (Merck & Company, Raway, New Jersey, Inc.), Commercially available as alum, biodegradable microspheres, monophosphoryl lipid A and quill A. GM-CSF or cytokines such as interleukin-2, -7 or -12 can also be used as adjuvants.

In the vaccines provided herein, adjuvant compositions can be designed to predominantly induce an Th1 type immune response. High levels of Th1-type cytokines (eg, IFN-γ, IL-2 and IL-12) tend to assist in the induction of cell mediated immune responses to antigens administered. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to assist in the induction of humoral immune responses. After application of the vaccine as provided herein, the patient will maintain an immune response including Th1- and Th2-type responses. In a preferred embodiment where the response is predominantly Th1-type, the level of Th1-type cytokines will increase to a level greater than the level of Th2-type cytokines. The levels of these cytokines can be easily assessed using standard assays. The class of cytokines is described in Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Preferred adjuvants for use to induce a predominant Th1-type reaction are, for example, monophosphonyl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with aluminum salts. Formulation of MPL adjuvants are commercially available from Liby Immunome Research Inc., Hamilton, Montana. US Pat. No. 4,436,727; 4,877,611; 4,877,611; 4,866,034 and 4,912,094]. Also preferred is AS-2 (manufactured by Smithklein). CpG-containing oligonucleotides in which CpG dinucleotides are unmethylated also induce a predominant Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555. Another preferred adjuvant is saponin, preferably QS21, which can be used alone or in combination with other adjuvant. For example, the enhanced system may be a combination of monophosphoryl lipid A with a saponin derivative, for example, a combination of QS21 with 3D-MPL as described in WO 94/00153 or WO 96 / 33739] include QS21 lower reactogenic compositions inhibited by cholesterol. Other preferred formulations include oil-in-water emulsions and tocophenols. Particularly potent adjuvant formulations including QS21, 3D-MPL and tocopherol in oil-in-water emulsions are described in WO 95/17210. Any of the vaccines provided herein can be prepared using known methods to provide a mixture of antigens, immune response enhancers and suitable carriers or adjuvants.

The compositions described herein may be administered as part of a sustained release formulation (such as a capsule or sponge that slowly releases the compound after administration). Such formulations may generally be prepared using known techniques and may be administered, for example, by oral, rectal or subcutaneous transplantation, or by implantation into a target site of interest. Sustained release formulations may contain polypeptides, polynucleotides or antibodies contained in a reservoir dispersed in a carrier matrix and / or surrounded by a rate controlling membrane. Carriers for use in such formulations may be biocompatible and biodegradable. Preferably, the formulation provides a relatively constant level of active ingredient release. The amount of active compound contained in a sustained release preparation depends on the site of implantation, the rate of release and the expected sustained release and the nature of the condition to be treated or prevented.

Several delivery vehicles can be used in pharmaceutical compositions and vaccines to facilitate the generation of antigen-specific immune responses that target tumor cells. Delivery vehicles include antigen presenting cells (APCs) such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to be efficient APCs. Such cells are not necessary but may increase the ability to present antigens, enhance the activation and / or maintenance of T cell responses, have anti-tumor effects and / or be immunologically suitable for inoculators (ie , Matched HLA haplotype). APCs can generally be isolated from various biological fluids and organs, and can be autologous, allogeneic, genotype, or heterologous cells.

Certain preferred embodiments of the invention utilize dendritic cells or progenitor cells thereof as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as physiological aids to demonstrate prophylactic or therapeutic anti-tumor immunity (Timmerman and Levy, Ann. Rev. Med. 50: 507-529, 1999]. In general, dendritic cells are B cells (CD19 and CD20), T cells (CD3) as a result of determination using their typical shape (radial in situ, distinct cytoplasmic processes (dendritic processes) in vitro) and standard assays. ), Lack of differentiation markers of monocytes (CD14) and natural killer cells (CD56). Dendritic cells can of course be engineered to express specific cell-surface receptors or ligands not commonly found in dendritic cells in vivo or ex vivo, and such modified dendritic cells are included in the present invention. As an alternative to dendritic cells, secreted vesicle antigen-loaded dendritic cells (so-called exosomes) can be used in the vaccine. See Zitvogel et al., Nature Med. 4: 594-600, 1998].

Dendritic cells and progenitor cells may be harvested from peripheral blood, bone marrow, tumor-infiltrating cells, peri-tumoral tissue-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or other suitable tissues or body fluids. For example, dendritic cells can be differentiated in vitro by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to a culture of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow may be synthesized with GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compounds that induce maturation and proliferation of dendritic cells. The combination can be differentiated into dendritic cells by adding to the culture medium.

Dendritic cells are typically classified into "mature" and "mature" cells, which provide a simple way to distinguish two well-characterized phenotypes. However, this classification should not be construed as excluding all possible intermediate steps of differentiation. Immature dendritic cells are characterized as APCs with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptors, mannose receptors and DEC-205 markers. Mature phenotypes are typically cells responsible for the lower expression of these markers and T cell activation, such as type I and II MHCs, adsorption molecules (eg CD54 and CD11) and costimulatory molecules (eg CD40, CD80 and CD86). Characterized by high expression of surface molecules.

APCs can generally be transfected with polynucleotides encoding an ovarian carcinoma antigen (or a portion or other variant thereof), such that the antigen or immunogenic portion thereof is expressed on the cell surface. Such transfection can occur ex vivo, and compositions or vaccines containing such transfected cells can be used for treatment as described herein. Alternatively, gene delivery vehicles targeting dendritic or other antigen presenting cells can be administered to the patient, resulting in transfection in vivo. In vivo or ex vivo transfection of dendritic cells is generally described, for example, by methods or methods described in WO 97/24447, Mahvi et al., Immunology and cell Biology 75: 456-460, 1997. It can be carried out using a method known in the art, such as the gene gun method described in. Antigen loading of dendritic cells can be used to incubate dendritic cells or progenitor cells with polypeptides, DNA (extracted or in plasmid vectors) or RNA, or antigen-expressing recombinant bacteria or viruses (eg vaccinia, chicken pox, adenovirus or lentiviral vectors). Can be achieved by culturing. Prior to loading, the polypeptide may be covalently linked to an immunological partner (eg, a carrier molecule) that provides T cell help. Alternatively, dendritic cells can be pulsed separately or in the presence of a polypeptide as a non-bound immunological partner.

Cancer therapy

As a further aspect of the invention, the compositions described herein can be used for immunotherapy of cancer such as ovarian cancer. Within this method, pharmaceutical compositions and vaccines are typically administered to a patient. As used herein, "patient" refers to all warm-blooded animals, preferably humans. The patient may or may not have cancer. Thus, the pharmaceutical compositions and vaccines can be used to prevent the progression of cancer or to treat cancer patients. In certain preferred embodiments, the patient is an ovarian cancer patient. Such cancers can be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors. The pharmaceutical compositions and vaccines may be administered before or after surgery to remove the initial tumor and / or before or after treatment, such as the administration of radiotherapy or conventional chemotherapeutic drugs.

In certain embodiments, immunotherapy is an active immunotherapy in which the treatment relies on in vivo stimulation of the endogenous host immune system in response to tumors by administration of immune response-modifying agents (eg, tumor vaccines, bacterial adjuvants and / or cytokines). Can be.

In another embodiment, the immunotherapy may be passive immunotherapy, wherein the treatment comprises the delivery of a reagent (eg, agonist cell or antibody) with an established tumor immune reactivity that can directly or indirectly mediate antitumor effects. However, it is not necessarily governed by a complete host immune system. Examples of main effect cells include T lymphocytes (eg, CD8 + cytotoxic T-lymphocytes, CD4 + T-helper tumor-infiltrating lymphocytes) expressing a polypeptide provided herein, killer cells (eg, natural killer cells, lymphokine-activated). Killer cells), B cells or antigen presenting cells (eg dendritic cells and macrophages). T cell receptors and antibody receptors specific for the polypeptides provided herein can be cloned, expressed and transferred into other vectors or effector cells for quantum immunotherapy. In addition, the polypeptides provided herein for passive immunotherapy can be used to generate antibodies or antiidiotype antibodies (as described above and in US Pat. No. 4,918,164).

Root cells can generally be obtained in sufficient amounts for quantum immunotherapy by in vitro growth as described herein. Culture conditions for proliferating billions of single antigen-specific effector cells while maintaining antigen recognition in vivo are well known in the art. These in vitro culture conditions typically utilize intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides provided herein can be used to rapidly propagate antigen-specific T cell cultures to produce a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells such as dendritic cells, macrophages, or B-cells can be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotide sequences using standard techniques known in the art. For example, antigen presenting cells can be transfected with polynucleotides having promoters suitable for increasing expression in recombinant viruses or other expression systems. Cultured effector cells for use in therapy should be able to grow and distribute widely and be able to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and survive long term while maintaining a substantial number upon repeated stimulation with an antigen supplemented with IL-2. See, eg, Cheever, et al., Immunological Reviews, 157: 177, 1997].

Alternatively, a vector expressing a polypeptide provided herein can be introduced into stem cells taken from a patient and cloned in vitro for autologous transplantation into the same patient.

The dosage as well as the route and frequency of administration vary from person to person and can be easily established using standard techniques. In general, pharmaceutical compositions and vaccines may be administered by injection (eg intradermal, intramuscular, intravenous or subcutaneous), intranasal (eg inhalation) or orally or incised tumors. One to ten doses may be administered for a 52 week period. Preferably six doses are administered at intervals of one month, followed by periodic boosting. Another protocol may be appropriate for individual patients. Suitable doses are those amounts that can promote an anti-tumor immune response when administered as described above and are at least 10-50% above the base (ie untreated) level. This response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent production of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of eliciting an immune response that results in improved clinical outcomes (eg, more frequent alleviation, complete, partial or longer term disease-free survival) in vaccinated patients compared to non-vaccinated patients. Generally, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in the dose is from about 100 μg to about 5 mg per kg of host. Suitable dosage sizes vary depending on the size of the patient, but typical ranges are from about 0.1 ml to about 5 ml.

In general, appropriate dosages and treatment regimens provide the active compounds in an amount sufficient to provide therapeutic and / or prophylactic benefits. Such responses can be monitored by establishing improved clinical outcomes (eg, more frequent alleviation, complete, partial or longer term disease-free survival) in treated patients compared to untreated patients. Increases in the already present immune response to ovarian carcinoma antigens are generally correlated with improved clinical outcomes. Such immune responses can generally be assessed using standard proliferation, cytotoxicity, or cytokine assays, which can be performed using samples taken from patients before and after treatment.

Screening to Identify Secreted Ovarian Carcinoma Antigens

The present invention provides a method for identifying secreted tumor antigens. Within this method, tumors are transplanted into immunodeficient animals, such as SCID mice, and maintained for a time sufficient for tumor antigens to be secreted into serum. In general, tumors can be maintained for 1 to 9 months, preferably 1 to 4 months, implanted subcutaneously or into germline pads in immunodeficient animals. Transplantation can generally be performed as described in WO 97/18300. Antisera are then prepared in immunocompetent mice using standard techniques and using serum containing secreted antigens as described herein. Briefly, 50-100 μl of serum (each set was collected from three sets of immunodeficiency mice containing different SCID-induced human ovarian cancer) was either RIBI-MPL or MPL + TDM (St., Missouri) One-to-one (vol: vol) can be mixed with an appropriate adjuvant such as Sigma Chemical Company of Lewis) and injected intraperitoneally into a winter-based immunocompetent animal for 5 months. Antisera from animals immunized in this manner can be obtained by collecting blood after tertiary, quaternary and error immunization. The antisera produced is usually not. Safety is validated for E. coli and phage antigens and used in serological expression screens (typically after dilution such as 1: 200).

The library is typically an expression library containing cDNA from one or more tumors of the type implanted in SCID mice. Such expression libraries can be prepared with any suitable vector, such as λ-screen (Novagen). CDNAs encoding polypeptides that react with antiserum can be identified and sequenced using standard techniques. Such cDNA molecules can be further characterized to assess expression in tumors and normal tissues and to assess antigen secretion in patients.

The methods provided herein have advantages over other methods for tumor antigen development. In particular, all antigens identified by this method must be secreted or released through necrosis of tumor cells. Such antigens may be present on the surface of tumor cells for a time sufficient to be targeted and killed by the immune system after vaccination.

Detection method of cancer

In general, cancer is detected from a patient based on the presence of one or more ovarian carcinoma proteins and / or polynucleotides encoding such proteins in biological samples taken from the patient (eg, blood, serum, urine and / or tumor biopsies). can do. In other words, such proteins can be used as markers indicating the presence or absence of cancers such as ovarian cancer. Such proteins may also be useful for the detection of other cancers. The binding agents provided herein generally allow for the detection of the level of protein binding to the agent in a biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding a tumor protein, which also indicates the presence or absence of cancer. In general, ovarian carcinoma-associated sequences should be present at levels three or more times higher in tumor tissue than in normal tissue.

Various assay methods for detecting polypeptide markers in a sample using a binder are known to those skilled in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of cancer in a patient includes (a) contacting a biological sample taken from the patient with a binder, (b) detecting the level of polypeptide binding to the binder in the sample, and (c) adjusting the level of the polypeptide to a predetermined level. It can be determined by comparing with the cutoff value.

In a preferred embodiment, the assay comprises using a binder immobilized on a solid support to bind and remove the polypeptide from the residue of the sample. The bound polypeptide can then be detected using a detector containing a reporter group and specifically binding to the binder / polypeptide complex. Such detection reagents may include, for example, a binding agent that specifically binds to a polypeptide or an antibody or other agent that specifically binds to a binding agent such as anti-immunoglobulin, protein G, protein A or lectin. Alternatively, competition assays can be used, wherein the polypeptide is labeled with a reporter group and binding to the immobilized binder after incubation of the sample and the binder. The degree to which the components of the sample inhibit the binding of the labeled polypeptide to the binder indicates the reactivity of the sample with the fixed binder. Suitable polypeptides for use in this assay include full length ovarian carcinoma proteins to which the binder binds as described above, and portions thereof.

Solid supports are known to those skilled in the art and can be any material as long as the tumor protein can be attached thereto. For example, the solid support may be a test well in a microtiter plate or nitrocellulose or other suitable membrane. Alternatively, the support may be beads or discs such as glass, fiber glass, latex or plastic materials (eg polystyrene or polyvinylchloride). The support may also be a magnetic particle or fiber optical sensor, for example as described in US Pat. No. 5,359,681. The binder can be bound to a solid support using a variety of techniques known to those skilled in the art, which are extensively described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to non-covalent bonds and covalent attachments, such as adsorption, which may be a direct link between the agent and a functional group on the support or a crosslinking agent. Immobilization by adsorption to wells or membranes in microtiter plates is preferred. In this case, adsorption can be achieved by contacting the binder in a suitable buffer with a solid support for a suitable time. Contact time varies with temperature, but is typically about 1 hour to 1 day. In general, contacting the amount of binder from about 10 ng to about 10 μg, preferably from about 100 ng to about 1 μg, with the wells of the plastic microtiter plate (e.g., polystyrene or polyvinylchloride) is sufficient to immobilize the appropriate amount of binder. Suffice.

Covalent bonding of the binder to the solid support can generally be accomplished by first reacting the support with a bifunctional reagent that reacts with both the support and a functional group such as a hydroxyl or amino group on the binder. For example, the binder may be covalently bound to the support with the appropriate polymer coating by using benzoquinone or by condensing aldehyde groups on the support with active hydrogens and amines on the binding partner. See, for example, Pierce Immunotechnology Catalog and Handbook, 1991, at A 12-A 13].

In certain embodiments, the assay is a two-antibody sandwich assay. Such assays can be carried out by first contacting an antibody immobilized on a well of a solid support, often a microtiter plate, with the polypeptide to bind the immobilized antibody. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a detection reagent containing a reporter group (preferably a second antibody capable of binding other sites on the polypeptide) is added. The amount of detection reagent that remains bound on the solid support is then determined using a method suitable for the particular reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding site on the support is typically blocked. Any suitable blocking agent known to those skilled in the art may be used, such as bovine serum albumin (BSA) or Tween 20 ^™ (Sigma Chemical Company, St. Louis, MO). The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample taken from an ovarian cancer patient. Preferably, the contact time is sufficient to achieve a binding level that is at least 95% of the binding level reached in equilibrium between the bound polypeptide and the unbound polypeptide. Those skilled in the art will appreciate that the time required to achieve equilibrium can be readily determined by testing the level of binding that occurs at any given time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound samples can then be removed by washing the solid support with a suitable buffer such as PBS containing 0.1% Tween 20 ^™ . Subsequently, a second antibody containing a reporter group can be added to the solid support. Preferred reporter groups include the groups described above.

The detection reagent is then incubated for a time sufficient to detect the polypeptide bound to the immobilized antibody-polypeptide complex. The appropriate amount of time can generally be determined by assaying the level of binding that occurs over time. The unbound detection reagent is then removed and the bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation coefficients or autoradiography are generally appropriate. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin linked to different reporter groups (often radioactive or fluorescent groups or enzymes). Enzyme reporter groups generally can add a substrate, usually for a certain period of time, and then detect the reaction product by spectroscopy or other analysis.

To determine the presence or absence of a cancer, such as an ovarian cancer, a signal detected from a group of reporters bound to a solid support is generally compared with a signal corresponding to a predetermined cutoff value. In one preferred embodiment, the cutoff value for detection of cancer is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without cancer. In general, a sample that generates a signal that exhibits three standard deviations above a predetermined cutoff value is considered to be positive for cancer. In another preferred embodiment, the cutoff value is described in Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107] to determine using the receiver operator curve. In summary, in this aspect, the cutoff value can be determined from the plot of true positive rate (ie sensitivity) and gastric positive rate (100% -specificity) corresponding to each possible cutoff for diagnostic test results. A cutoff value close to the top left corner (ie, the value that accounts for the highest immunity) is the most precise cutoff value and a signal that generates a signal larger than the cutoff value determined by this method can be considered positive. Alternatively, the cutoff value can be moved to the left along the plot when minimizing the above positive ratio, or to the right when minimizing the negative ratio. In general, samples that generate signals greater than the cutoff value determined by this method are judged positive for cancer.

In a related aspect, the assay is carried out by a flow-through or strip test method in which the binder is anchored to a membrane such as nitrocellulose. In the flow-through test, the polypeptide in the sample binds to the fixed binder as the sample passes through the membrane. The second labeled binder then binds to the binder-polypeptide complex while the solution containing the second labeled binder passes through the membrane. The detection of the bound second binder can then be carried out as described above. In the strip test method, one end of the membrane to which the binder is bound is immersed in the solution containing the sample. The sample travels along the membrane through the region containing the second binder and into the region of the fixed binder. The concentration of the second binder in the region of the immobilized antibody indicates the presence of cancer. Typically, the concentration of the second binder at this site forms a line-like pattern that is visually readable. The absence of such a pattern indicates a negative result. In general, the amount of binder immobilized on the membrane is such that the biological sample forms a visually identifiable pattern when the biological sample contains a level of polypeptide sufficient to generate a positive signal in a two-antibody sandwich assay, i. Is selected. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane is from about 25 ng to about 1 μg and more preferably from about 50 ng to about 500 ng. Such testing can typically be done with very small amounts of biological samples.

Of course, there are many other assay protocols suitable for use with the tumor proteins or binders of the invention. The above description is merely illustrative. For example, it will be apparent to one skilled in the art that the protocol can be readily modified to detect antibodies that bind the polypeptide in biological samples using ovarian carcinoma polypeptides. Detection of such ovarian carcinoma protein specific antibodies may be correlated with the presence of cancer.

Cancer can also be detected, or alternatively, based on the presence of T cells that specifically react with ovarian carcinoma proteins in biological samples. As a specific method, a biological sample comprising CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient is subjected to an ovarian carcinoma protein, a polynucleotide encoding such a polypeptide, and / or an APC expressing at least an immunogenic portion of such polypeptide. Incubate with and detect the presence of specific activation of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by conventional techniques (eg, picol / highpark density gradient centrifugation of peripheral blood lymphocytes). T cells can be cultured in vitro with ovarian carcinoma protein (eg, 5-25 μg / ml) at 37 ° C. for 2-9 days (typically 4 days). As a control, it may be necessary to culture other aliquots of T cell samples in the absence of ovarian carcinoma proteins. In the case of CD4 ⁺ T cells, activation is preferably detected by assessing the proliferation of T cells. In the case of CD8 ⁺ T cells, activation is preferably detected by assessing cytolytic activity. At least two-fold higher proliferation levels and / or at least 20% greater cytolytic activity than in disease-free patients indicate the presence of cancer in the patient.

As noted above, cancer may also be detected, or alternatively, based on the level of mRNA encoding ovarian carcinoma protein in a biological sample. For example, two or more oligonucleotide primers, wherein one or more of the oligonucleotide primers are specific for a polynucleotide encoding an ovarian carcinoma protein (ie, hybridize) are based on the polymerase chain reaction (PCR) basis. The assay can be used to amplify a portion of ovarian carcinoma protein cDNA derived from a biological sample. The amplified cDNA is then isolated and detected using techniques known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to polynucleotides encoding ovarian carcinoma proteins can be used in hybridization assays to detect the presence of polynucleotides encoding tumor proteins in biological samples.

In order for hybridization to occur under assay conditions, the oligonucleotide primers and probes are at least about 60%, preferably at least about 75%, with a portion of the ovarian carcinoma protein encoding polynucleotide that is at least 10 nucleotides in length, preferably at least 20 nucleotides in length. Above, and more preferably at least about 90% identity, should comprise an oligonucleotide sequence. Preferably, the oligonucleotide primers and / or probes hybridize to polynucleotides encoding polypeptides described herein under moderately stringent conditions as defined herein. Oligonucleotide primers and / or probes that can be usefully used in the diagnostic methods described herein are preferably at least 10 to 40 nucleotides in length. In a preferred embodiment, the oligonucleotide primer comprises at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides of a DNA molecule having a sequence provided herein. Techniques for PCR basic assays and hybridization assays are well known in the art. See, eg, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989].

One preferred assay uses RT-PCR where PCR is applied in combination with reverse transcription. Typically, RNA is extracted from a biological sample such as biopsy tissue and reverse transcribed to produce cDNA molecules. PCR amplification with one or more specific primers results in cDNA molecules, which can be isolated and visualized using, for example, gel electrophoresis. Amplification can be performed on biological samples taken from the subject and the subject not suffering from cancer. Amplification reactions can be performed on several cDNA dilutions within a 2-fold range. It is typically considered positive if expression increases more than twofold in several dilutions of the test patient sample relative to the same dilution of the non-cancer sample.

In another embodiment, ovarian carcinoma proteins and polynucleotides encoding such proteins can be used as markers to monitor cancer progression. In such embodiments, the assays described above for diagnosing cancer can be performed over time and can assess changes in the level of reactive polypeptide. For example, the assay may be performed every 24 to 72 hours for a period of six months to one year, and thereafter, as needed. In general, cancer is progressing in patients in which the level of polypeptide detected by the binder increases over time. In contrast, cancer does not progress when the level of reactive polypeptide is constant or decreases over time.

Certain in vivo diagnostic assays can be performed directly on tumors. One such assay involves contacting tumor cells with a binder. The bound binder can then be detected either directly or indirectly through the reporter group. Such binders may also be applied to histology. Alternatively, polynucleotide probes can be used for this application.

As noted above, to improve sensitivity, multiple ovarian carcinoma protein markers can be assayed in a given sample. It is apparent that binders specific for the different proteins provided herein can be combined in a single assay. In addition, multiple primers or probes may be used simultaneously. The selection of tumor protein markers can be made based on routine experimentation to determine the combination that leads to optimal sensitivity. Additionally or alternatively, assays for tumor proteins provided herein can be combined with assays for other known tumor antigens.

Diagnostic kit

The present invention also provides a kit for use in the above diagnostic method. Such kits typically contain two or more components necessary to conduct a diagnostic assay. The component may be a compound, a reagent, a container and / or equipment. For example, one container in a kit may contain a monoclonal antibody or fragment thereof that specifically binds to an ovarian carcinoma protein. Such antibodies or fragments may be provided attached to the support material described above. One or more additional containers may contain elements such as reagents or buffers to be used in the assay. Such kits may also, or alternatively, contain the detection reagents described above containing a reporter group suitable for direct or indirect detection of antibody binding.

Alternatively, the kit can be designed to detect the level of mRNA encoding an ovarian carcinoma protein in a biological sample. Such kits generally comprise one or more oligonucleotide probes or primers as described above that hybridize to a polynucleotide encoding an ovarian carcinoma protein. Such oligonucleotides can be used, for example, in PCR or hybridization assays. Additional components that may be present in such kits include a second oligonucleotide and / or diagnostic reagent or container for facilitating a polynucleotide encoding an ovarian carcinoma protein.

The invention is illustrated by the following examples. However, these examples do not limit the present invention.

Example 1

Identification of Representative Ovarian Carcinoma Protein cDNA

This example illustrates the identification of cDNA molecules encoding ovarian carcinoma proteins.

Anti-SCID mouse serum (generated for serum from SCID mice with terminal ovarian carcinoma); Safety was previously validated for E. coli and phage antigens and used at a 1: 200 dilution in serological expression screens. A library selected from SCID-induced human ovarian cancer (OV9334) was prepared using the directional RH oligo (dT) priming cDNA library construction kit and λScreen vector from Novagen. Bacteriophage lambda screens were used. Approximately 400,000 pfu of amplified OV9334 library was selected.

196 positive clones were isolated. Particular sequences that appear novel are provided in FIGS. 1A-1S and SEQ ID NOs: 1-71. Three complete insert sequences are shown in FIGS. 2A-2C (SEQ ID NOS: 72-74). Other clones with known sequences are shown in Figures 15A-15EEE (SEQ ID NOs: 82-310). Database investigations identified the following sequences that are substantially identical to the sequences provided in FIGS. 15A-15EEE.

These clones were further characterized by microarray technology to determine mRNA expression levels in various tumors and normal tissues. This analysis was carried out using a Sinteni (Palo Alto, CA) microarray according to the manufacturer's instructions. PCR amplification products were aligned on slides and each product occupied a specific position in the alignment. MRNA was extracted from the tissue sample and reverse transcribed to produce a fluorescently-labeled cDNA probe. Microarrays were detected with labeled cDNA probes and the slides were scanned to measure fluorescence intensity. The data was analyzed using GEMtools software provided by Synteny. Results for one clone (named 13695 or O8E) are shown in FIG. 3.

Example 2

Identification of Ovarian Carcinoma cDNA Using Microarray Technology

This example illustrates the identification of ovarian carcinoma polynucleotides by PCR subtraction and microarray analysis. Microarrays of cDNA were synthesized for ovarian cancer-specific expression using a Syntheni (Palo Alto, Calif.) microarray according to the manufacturer's instructions and essentially as described in Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155, 1997].

PCR subtraction was performed using a tester containing cDNA of four ovarian cancers (three of which are metastatic tumors) and a driver of cDNA from five normal tissues (adrenal, lung, pancreatic, spleen and brain). The cDNA fragments recovered from this subtraction were subjected to DNA microarray analysis, where the fragments were PCR amplified and adsorbed onto the chip and hybridized with fluorescently labeled probes derived from mRNA of human ovarian cancer and several normal human tissues. In this analysis, slides were scanned, fluorescence intensity was measured, and then analyzed using Synthene GEMtools software. In general, sequences that exhibit a five-fold or more increase in expression (relative to normal cells) in tumor cells are considered ovarian cancer antigens. Fluorescence results were analyzed and clones showing increased expression in ovarian cancer were further characterized by DNA sequencing and database searches to determine the novelty of the sequences.

Using this assay, the ovary is a splice fusion between human T cell leukemia virus type I carcinogenic protein TAX (Jin et al., Cell 93: 81-91, 1998) and an extracellular matrix protein called osteonectin. Cancer antigens were identified. The splice conjugation sequence is at the fusion point. The sequence of this clone is shown in Figure 4 and SEQ ID NO: 75. Osteonectin that was not spliced independently and unmodified from this assay was also identified.

Additional clones identified by this method are named 3f, 6b, 8e, 8h, 12c and 12h herein. The sequences of these clones are shown in FIGS. 5-9 and SEQ ID NOs: 76-81. Microarray analysis was performed as described above and the results are shown in FIGS. 10-14. Ovarian cancer (SCID-derived) cDNA libraries were selected to obtain full length sequences including clones 3f, 6b, 8e and 12h. This 2996 base pair sequence (named O772P) is provided in SEQ ID NO: 311 and the encoded 914 amino acid protein sequence is provided in SEQ ID NO: 312. PSORT assay is a type 1a transmembrane protein located on the plasma membrane.

In addition to the specific sequences described above, the following sequences were identified by this selection.

order Explanation OV4vG11 (SEQ ID NO: 313) Human clone 1119D9 on chromosome 20p12 OV4vB11 (SEQ ID NO: 314) Human UWGC from Chromosome 6p21: y14c094 OV4vD9 (SEQ ID NO: 315) Human clone 1049G16 on chromosomes 20q12 to 13.2 OV4vD5 (SEQ ID NO: 316) Human KIAA0014 Gene OV4vC2 (SEQ ID NO: 317) Human KIAA0084 Gene OV4vF3 (SEQ ID NO: 318) Human Chromosome 19 Cosmid R31167 OV4VC1 (SEQ ID NO: 319) new OV4vH3 (SEQ ID NO: 320) new OV4vD2 (SEQ ID NO: 321) new O815P (SEQ ID NO: 322) new OV4vC12 (SEQ ID NO: 323) new OV4vA4 (SEQ ID NO: 324) new OV4vA3 (SEQ ID NO: 325) new OV4v2A5 (SEQ ID NO: 326) new O819P (SEQ ID NO: 327) new O818P (SEQ ID NO: 328) new O817P (SEQ ID NO: 329) new O816P (SEQ ID NO: 330) new OV4vC5 (SEQ ID NO: 331) new 21721 (SEQ ID NO: 332) Human lumican 21719 (SEQ ID NO: 333) Human Retinoic Acid-binding Protein II 21717 (SEQ ID NO: 334) Human 26S Proteasome ATPase Subunit 21654 (SEQ ID NO: 335) Person Copine I 21627 (SEQ ID NO: 336) Human Neuron Specific Gamma-2 Enolase 21623 (SEQ ID NO: 337) Human Geranylgeranyl Transferase II 21621 (SEQ ID NO: 338) Human cyclin-dependent protein kinases 21616 (SEQ ID NO: 339) Human prepro-megakaryotonic promoter 21612 (SEQ ID NO: 340) Person UPH1 21558 (SEQ ID NO: 341) Human RalGDS-Type 2 (RGL2) 21555 (SEQ ID NO: 342) Human autoantigen P542

order Explanation 21548 (SEQ ID NO: 343) Human Actin-Related Protein (ARP2) 21462 (SEQ ID NO: 344) Human Huntingtin Interacting Proteins 21441 (SEQ ID NO: 345) Human 90K product (tumor related antigen) 21439 (SEQ ID NO: 346) Human guanine nucleotide regulatory protein (tim1) 21438 (SEQ ID NO: 347) Human Ku autoimmune (p70 / p80) antigens 21237 (SEQ ID NO: 348) Person S-Laminin 21436 (SEQ ID NO: 349) Person Ribophorin I 21435 (SEQ ID NO: 350) Human cytoplasmic chaperonin hTRiC5 21425 (SEQ ID NO: 351) Person EMX2 21423 (SEQ ID NO: 352) Human p87 / p89 gene 21419 (SEQ ID NO: 353) Person HPBRII-7 21252 (SEQ ID NO: 354) Person T1-227H 21251 (SEQ ID NO: 355) Person Coolin I 21247 (SEQ ID NO: 356) Kunitz type protease inhibitory factor (KOP) 21244-1 (SEQ ID NO: 357) Human protein tyrosine phosphatase receptor F (PTPRE) 21718 (SEQ ID NO: 358) Human LTR repeats OV2-90 (SEQ ID NO: 359) new SEQ ID NO: 360 Man zinc fingered SEQ ID NO: 361 Human polyA binding protein SEQ ID NO: 362 Man plate tropine SEQ ID NO: 363 Human PAC Clone 278C19 SEQ ID NO: 364 Person LLRep3 SEQ ID NO: 365 Human Kunitz Type Protease Inhibitor SEQ ID NO: 366 Human KIAA0106 gene SEQ ID NO: 367 Human keratin SEQ ID NO: 368 Human HIV-1TAR SEQ ID NO: 369 Human glial induced nexin SEQ ID NO: 370 Human Probronectin SEQ ID NO: 371 Human ECM Pro BM40 SEQ ID NO: 372 Human collagen SEQ ID NO: 373 Human alpha enolase SEQ ID NO: 374 Human aldolase SEQ ID NO: 375 Human Transition Growth Factor BIG H3 SEQ ID NO: 376 Human SPARC Osteonectin SEQ ID NO: 377 Human SLP1 Leukocyte Protease SEQ ID NO: 378 Human Mitochondrial ATP Synthetase SEQ ID NO: 379 Human DNA Sequence Clone 461P17 SEQ ID NO: 380 Man dpbB pro y box SEQ ID NO: 381 Person 40kDa keratin SEQ ID NO: 382 Human Arginosuccinate Synthetase SEQ ID NO: 383 Human acid ribosomal phosphoprotein SEQ ID NO: 384 Human colon carcinoma laminin binding protein

The screening further identified multiple forms of clone O772P (named 21013, 21003 and 21008 herein). PSORT analysis indicates that 21003 (SEQ ID NO: 386; translated into SEQ ID NO: 389) and 21008 (SEQ ID NO: 387; translated into SEQ ID NO: 390) represent the type 1a transmembrane protein form of O772P. 21013 (SEQ ID NO: 385; translated into SEQ ID NO: 388) appears to be a truncated form of protein and is predicted to be a secreted protein by PSORT analysis.

Further sequencing gave a full length clone for O8E (2627 bp consistent with the message size observed by Northern analysis; SEQ ID NO: 391). This nucleotide sequence was obtained as follows: The original O8E sequence (OrigO8Econs) was found to overlap by 33 nucleotides with the sequence from the EST clone (IMAGE # 1987589). This clone provides 1042 additional nucleotides upstream of the original O8E sequence. The link between EST and O8E was verified by sequencing multiple PCR fragments generated from ovarian early tumor libraries using primers for specific EST and O8E sequences (ESTxO8EPCR). When anchored PCR from an ovarian cancer library provided several clones terminated upstream of putative starting methionine (AnchoredPCR cons), a full length state was additionally shown but no further sequence information was obtained. . 16 shows a diagram illustrating the location of each subsequence within the full length O8E sequence.

Both protein sequences can be translated from full length O8E. “a” (SEQ ID NO: 393) starts with putative starting methionine. The second form “b” (SEQ ID NO: 392) comprises 27 additional upstream residues at the 5 ′ end of the nucleotide sequence.

Although certain aspects of the invention have been described herein for purposes of illustration, it is apparent that various modifications may be made from the above without departing from the spirit and scope of the invention. Accordingly, the invention is defined within the scope of the appended claims.

Summary of Sequence Listing

SEQ ID NOs: 1-71 are ovarian carcinoma antigen polynucleotides shown in FIGS. 1A-1S.

SEQ ID NOs: 72-74 are ovarian carcinoma antigen polynucleotides shown in Figures 2A-2C.

SEQ ID NO: 75 is 3 g of ovarian carcinoma polynucleotide (FIG. 4).

SEQ ID NO: 76 is Ovarian carcinoma polynucleotide 3f (FIG. 5).

SEQ ID NO: 77 is Ovarian carcinoma polynucleotide 6b (Figure 6).

SEQ ID NO: 78 is Ovarian carcinoma polynucleotide 8e (FIG. 7A).

SEQ ID NO: 79 is Ovarian carcinoma polynucleotide 8h (FIG. 7B).

SEQ ID NO: 80 is Ovarian carcinoma polynucleotide 12e (FIG. 8).

SEQ ID NO: 81 is Ovarian carcinoma polynucleotide 12h (FIG. 9).

SEQ ID NOs: 82-310 are ovarian carcinoma antigen polynucleotides set forth in FIGS. 15A-15EEE.

SEQ ID NO: 311 is Ovarian carcinoma polynucleotide O772P.

SEQ ID NO: 312 is an O772P amino acid sequence.

SEQ ID NOs: 313 to 384 are ovarian carcinoma antigen polynucleotides.

SEQ ID NOs: 385-390 provide sequences in the form of O772P.

SEQ ID NO: 391 is the full length sequence of ovarian carcinoma polynucleotide O8E.

SEQ ID NOs: 392-393 are protein sequences encoded by O8E.

Claims (68)

an amino acid encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 81, 313 to 331, 359, 366, 379, 385 to 387, or 391, and (b) the complement of the polynucleotide. Isolation comprising at least an immunogenic portion of an ovarian carcinoma protein comprising a sequence or an ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce its ability to react with antigen-specific antiserum Polypeptides. The polynucleotide of claim 1 wherein the polynucleotide is selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1-81, 313-331, 359, 366, 379, 385-387, or 391, and (b) the complement of said polynucleotide. A polypeptide comprising an amino acid sequence encoded by the sequence. a amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 81, 319 to 331, 359, 385 to 387, or 391, and (b) the complement of the polynucleotide. Five of the polypeptides comprising at least an immunogenic portion of an ovarian carcinoma protein or ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with antigen-specific antiserum An isolated polynucleotide encoding at least amino acid residues. The polynucleotide of claim 3, wherein the polynucleotide encodes an immunogenic portion of the polypeptide. The polynucleotide of claim 3 comprising a sequence provided as one of SEQ ID NOs: 1-81, 319-331, 359, 385-387, or 391 or the complement of the sequence. An isolated polynucleotide complementary to the polynucleotide according to claim 3. An expression vector comprising the polynucleotide according to claim 3 or 6. A host cell transformed or transfected with the expression vector according to claim 7. A pharmaceutical composition comprising the polypeptide of claim 1 in combination with a physiologically acceptable carrier. The pharmaceutical composition of claim 9, wherein the polypeptide comprises an amino acid sequence encoded by a polynucleotide comprising a sequence provided as one of SEQ ID NOs: 1-81, 313-331, 359, 366,379, 385-387, or 391. A vaccine comprising the polypeptide of claim 1 in combination with a nonspecific immune response enhancer. The vaccine of claim 11, wherein the polypeptide comprises an amino acid sequence encoded by a polynucleotide comprising a sequence provided as one of SEQ ID NOs: 1-81, 313-331, 359, 366, 379, 385-387, or 391. an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 81, 319 to 331, 359, 385 to 387 or 391 and (ii) the complement of the polynucleotide. Ovarian carcinoma comprising at least an immunogenic portion of an ovarian carcinoma protein comprising or an ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with antigen-specific antiserum. A pharmaceutical composition comprising a polynucleotide encoding a polypeptide and (b) a physiologically acceptable carrier. The pharmaceutical composition of claim 13, wherein the polynucleotide comprises a sequence provided as one of SEQ ID NOs: 1-81, 319-331,359, 385-387, or 391 or the complement of the sequence. by a polynucleotide sequence selected from the group consisting of: (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 81, 313 to 331, 359, 366, 379, 385 to 387 or 391 and (ii) the complement of said polynucleotides. At least an immunogenic portion of an ovarian carcinoma protein comprising an amino acid sequence encoded or at least one ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce its ability to react with antigen-specific antiserum A polynucleotide encoding a ovarian carcinoma polypeptide comprising and a vaccine comprising. The vaccine of claim 15, wherein the polynucleotide comprises a sequence provided as one of SEQ ID NOs: 1-81, 319-331, 359, 385-387, or 391. by a polynucleotide sequence selected from the group consisting of: (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 81, 313 to 331, 359, 366, 379, 385 to 387 or 391 and (ii) the complement of said polynucleotides. A pharmaceutical composition comprising an antibody that specifically binds an ovarian carcinoma protein comprising an amino acid sequence to be encoded and (b) a physiologically acceptable carrier. Ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide , An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs in deletion, addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (b) in (a) A polynucleotide encoding a provided polypeptide and (c) an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide. Ovarian Carcinoma Stages Containing How to from a patient by administering an effective amount of an agent selected from the group consisting of antibody specifically binding to be a patient including inhibited the progression of ovarian cancer, inhibiting the progression of ovarian cancer in a patient. 19. The method of claim 18, wherein the agent is present in the pharmaceutical composition according to any one of claims 9, 13 and 17. The method of claim 18, wherein the agent is present in the vaccine according to any one of claims 11, 15 and 18. A fusion protein comprising at least one polypeptide according to claim 1. A polynucleotide encoding the fusion protein according to claim 21. A pharmaceutical composition comprising the fusion protein according to claim 21 in combination with a physiologically acceptable carrier. A vaccine comprising the fusion protein according to claim 21 in combination with a nonspecific immune response enhancer. A pharmaceutical composition comprising the polynucleotide according to claim 22 in combination with a physiologically acceptable carrier. A vaccine comprising the polynucleotide according to claim 22 in combination with a nonspecific immune response enhancer. A method of inhibiting the progression of ovarian cancer in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition according to claim 23. A method of inhibiting the progression of ovarian cancer in a patient, comprising administering to the patient an effective amount of a vaccine according to claim 23. an ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391, and (ii) a complement of said polynucleotide, Antigen presenting cells expressing an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant which differs by deletion, addition and / or insertion but has not substantially reduced its ability to react with antigen-specific antiserum and (b ) A pharmaceutical composition comprising a physiologically acceptable carrier or excipient. Ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide Antigen presenting cells expressing an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by deletion, addition and / or insertion but has not substantially reduced the ability to react with antigen-specific antiserum and ( b) vaccines comprising nonspecific immune response enhancers. specifically for an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide. A vaccine comprising an anti-idiotype antibody or antigen-binding fragment thereof specifically bound by a binding antibody and (b) a nonspecific immune response enhancer. 32. The vaccine of claim 30 or 31 wherein the immune response enhancer is an adjuvant. Ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide , T cells that specifically react with an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by deletion, addition, and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum And (b) a physiologically acceptable carrier. Ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide , T cells that specifically react with an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by deletion, addition, and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum And (b) a nonspecific immune response enhancer. 34. A method of inhibiting the progression of ovarian cancer in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition according to claim 29 or 33. 35. A method of inhibiting the progression of ovarian cancer in a patient, comprising administering to the patient an effective amount of a vaccine according to any one of claims 30, 31 and 34. Ovarian carcinoma protein or one or more substitutions comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) (i) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotide , An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by deletion, addition and / or insertion but has not substantially reduced the ability to react with antigen-specific antiserum, (b) the polypeptide Polynucleotides encoding and / or (c) contacting the T cells with antigen presenting cells expressing the polypeptide under conditions and for a time sufficient to permit stimulation and / or proliferation of the T cells. Methods of stimulation and / or propagation. The method of claim 37, wherein the T cells are cloned prior to proliferation. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the polynucleotides provided as one of SEQ ID NOs: 1 to 387 or 391 and the complement of said polynucleotides; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide Stimulating T cells in a mammal by administering to the mammal a pharmaceutical composition comprising a polynucleotide or (iii) at least one antigen-presenting cell expressing an ovarian carcinoma polypeptide and (b) a physiologically acceptable carrier or excipient Including and / or multiplying , Stimulating and / or proliferating T cells in a mammal. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and a complement of the polynucleotide; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide Stimulating and / or propagating T cells in the mammal by administering to the mammal a vaccine comprising at least one antigen-presenting cell expressing a polynucleotide or (iii) an ovarian carcinoma polypeptide and (b) a nonspecific immune response enhancer. To grow T cells in mammals And / or how to multiply. 41. A method of inhibiting the progression of ovarian cancer in a patient, comprising administering to the patient a T cell prepared according to the method according to claim 39 or 40. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and a complement of the polynucleotide; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide Proliferating T cells by culturing at least one antigen-presenting cell expressing a polynucleotide or (iii) an ovarian carcinoma polypeptide and CD4 ⁺ T cells isolated from the patient, and (b) administering an effective amount of the proliferated T cells to the patient Thereby inhibiting the progression of ovarian cancer in the patient. A method of inhibiting the progression of ovarian cancer in a patient, including the system. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and a complement of the polynucleotide; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide Proliferating T cells by culturing at least one antigen-presenting cell expressing a polynucleotide or (iii) an ovarian carcinoma polypeptide and CD4 ⁺ T cells isolated from the patient, (b) cloning one or more proliferated cells, ( c) administering to the patient an effective amount of cloned T cells. Methods of inhibiting the progression of ovarian cancer in a patient. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and a complement of the polynucleotide; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide T cells are grown by culturing at least one antigen-presenting cell expressing a polynucleotide or (iii) an ovarian carcinoma polypeptide and CD8 ⁺ T cells isolated from the patient, and (b) administering an effective amount of the proliferated T cells to the patient, Thereby inhibiting the progression of ovarian cancer in the patient Including the method of inhibiting the progression of ovarian cancer patients. (a) (i) an ovarian carcinoma protein or one or more substitutions, deletions, comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and a complement of the polynucleotide; An ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein variant that differs by addition and / or insertion but does not substantially reduce its ability to react with antigen-specific antiserum, (ii) encodes an ovarian carcinoma polypeptide Proliferating T cells by culturing at least one antigen-presenting cell expressing a polynucleotide or (iii) an ovarian carcinoma polypeptide and CD8 ⁺ T cells isolated from the patient, (b) cloning one or more proliferated cells, ( c) administering an effective amount of the cloned T cells to the patient. In addition, the method of inhibiting the progression of ovarian cancer in a patient. (a) transplanting tumor cells into immunodeficient mammals, (b) serum is obtained from the immunodeficient mammals after a time sufficient to allow tumor antigens to be secreted into the serum, and (c) immunizing the immunocompetent mammal with serum. And (d) obtaining antiserum from an immunocompetent mammal, and (e) selecting a tumor expression library with antiserum to identify tumor antigen secreted therefrom. The method of claim 46, wherein the immunodeficient mammal is a SCID mouse and the immunocompetent mammal is an immunocompetent mouse. (a) ovarian carcinoma cells are transplanted into SCID mice, (b) serum is obtained from SCID mice after sufficient time to allow ovarian carcinoma antigens to be secreted into the serum, (c) immunocompetent mice are immunized with serum, ( d) obtaining antisera from immunocompetent mice, and (e) selecting an ovarian carcinoma expression library with antisera to identify ovarian carcinoma antigens secreted therefrom. a binding agent that binds to an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides Contacting a biological sample taken from the patient with (b) detecting the amount of polypeptide binding to the binder in the sample, and (c) comparing the amount of polypeptide to a predetermined cutoff value from there Determining the presence or absence of cancer in the patient. The method of claim 49, wherein the binder is an antibody. The method of claim 50, wherein the antibody is a monoclonal antibody. The method of claim 49, wherein the cancer is ovarian cancer. a binding agent that binds to an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides Contacting the biological sample taken from the patient at the first time point, (b) detecting the amount of polypeptide binding to the binder in the sample, and (c) using the biological sample taken from the patient at a later time point (a) And (b) repeating, (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b), thereby monitoring the progression of cancer in the patient. How to monitor cancer progression. The method of claim 53, wherein the binder is an antibody. The method of claim 54, wherein the antibody is a monoclonal antibody. The method of claim 53, wherein the cancer is ovarian cancer. a poly encoding an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides Contacting the biological sample taken from the patient with an oligonucleotide that hybridizes to the nucleotide, (b) detects the amount of polynucleotide hybridizing with the oligonucleotide in the sample, and (c) the amount of polynucleotide that hybridizes with the oligonucleotide Comparing the pre-determined cutoff value therefrom to determine the presence or absence of cancer in the patient. The method of claim 57, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined by polymerase chain reaction. The method of claim 57, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined by a hybridization assay. a poly encoding an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides Contacting the biological sample taken from the patient with an oligonucleotide that hybridizes to the nucleotide, (b) detecting the amount of polynucleotide hybridizing with the oligonucleotide in the sample, and (c) the biological sample taken from the patient at a later time point. Repeating steps (a) and (b), and (d) comparing the amount of polynucleotides detected in step (c) with the amount detected in step (b) to monitor the progression of cancer in the patient therefrom. Room to monitor cancer progression in patients, including . 61. The method of claim 60, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined by polymerase chain reaction. The method of claim 60, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined by a hybridization assay. specifically an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides A diagnostic kit comprising a detection reagent comprising at least one binding antibody or antigen-binding fragment thereof and (b) a reporter group. The kit of claim 63, wherein the antibody is immobilized on a solid support. 64. The kit of claim 63, wherein the solid support comprises nitrocellulose, latex or plastic material. 64. The kit of claim 63, wherein the detection reagent comprises anti-immunoglobulin, protein G, protein A or lectin. 64. The kit of claim 63, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles. a poly encoding an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (a) a polynucleotide provided as one of SEQ ID NOs: 1 to 387 or 391 and (ii) a complement of said polynucleotides A diagnostic kit comprising an oligonucleotide comprising 10 to 40 nucleotides that hybridize with nucleotides under moderately stringent conditions, and (b) a diagnostic reagent for use in a polymerase chain reaction or hybridization assay.