Prostate Cancer And Melanoma Antigens

Abstract

Methods for identifying a human subject as a candidate for further prostate cancer or melanoma examination are disclosed. Also disclosed are methods for determining whether an immune therapy has elicited a tumor-specific immune response in a prostate cancer or melanoma patient. Further disclosed are kits that can be used to practice the above methods. Methods for identifying candidate compounds for further testing as preventive or therapeutic agents for melanoma are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. Utility patent application Ser. No. 12/033,229, filed Feb. 19, 2008, and which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/890,590, filed Feb. 19, 2007. These documents are incorporated herein by reference as if set forth in its entirety.

BACKGROUND

[0003] Immune responses can be elicited to tumor-expressed antigens. For example, several groups have reported the detection of antibody responses to prostate tumor-associated antigens compared with control groups (Wang X, et al., N. Engl. J. Med. 353:1224-1235 (2005); McNeel D, et al., J. Urol. 164:1825-1829 (2000); Minelli A, et al., Anticancer Res. 25:4399-4402 (2005); Bradford T, et al., Urol Oncol. 24:237-242 (2006); and Shi F, et al., Prostate 63:252-258 (2005)). Cancer-testis antigens (CTA) are of particular interest as potential tumor antigens given that their expression is typically restricted to germ cells among normal tissues, but aberrantly expressed in tumor cells (Scanlan M, et al., Cancer Immun. 4:1 (2004)). The absence of MHC class I molecule expression on germ cells makes CTA essentially tumor-specific antigens in terms of potential CD8.sup.+ T-cell target antigens (Kowalik I, et al., Andrologia. 21:237-243 (1989)). Many of these CTA were discovered using antibody screening methods including the serological evaluation of recombinant cDNA expression libraries (SEREX) approach (Sahin U, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1005); Hoeppner L, et al., Cancer Immun. 6:1-7 (2006); and Tureci O, et al., Mol. Med. Today 3:342-349 (1997)).

[0004] Several CTA, including members of the MAGE and GAGE families, have been identified as antigens recognized by tumor-specific cytotoxic T-cells (CTL) (Van der Bruggen P, et al., Science 254:1643-1647 (1991); and Van den Eynde B, et al., J. Exp. Med. 182:689-698 (1995)). Detectable immune responses to these antigens are believed to be a result of their ectopic expression in MHC class I-expressing malignant cells. Several CTA have been shown to be recognized by both antibodies and CTL, thus providing validation for the original approach of using antibody screening to identify potential tumor-specific T cell antigens (Jager E, et al., J. Exp. Med. 187:265-270 (1998); and Monji M, et al., Clin. Cancer Res. 10:6047-6057 (2004)). Some CTA are expressed in several malignancies of different tissue origins (Scanlan et al., supra; and Mashino K, et al., Br. J. Cancer. 85:713-720 (2001)).

BRIEF SUMMARY

[0005] The present invention provides methods for identifying a human subject as a candidate for further prostate cancer or melanoma examination based on certain prostate cancer and melanoma antigens identified by the inventors. Methods of identifying immune responses elicited by an immune therapy directed at prostate cancer or melanoma are also provided. The present invention also provides kits that can be used to practice the above methods. Methods for identifying candidate compounds for further testing as preventive or therapeutic agents for melanoma are also disclosed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0006] FIG. 1 shows an immunoblot (probed with anti-SSX-2/4 specific antibody) that confirmed protein expression. Phage encoding SSX-2, SSX-1, SSX-4 and a negative control (empty phage construct) were spotted directly onto a bacterial lawn. Proteins were transferred to an isopropyl-beta-D-thiogalactopyranoside (IPTG)-infused nitrocellulose membrane, and mouse anti-SSX-2 mAb (clone E3AS, with known reactivity to SSX-2 and SSX-4, but not SSX-1) was used to probe the membrane.

[0007] FIG. 2 shows that high throughput immunoblot (HTI) detected CTA-specific IgG in patient sera. A) Purified glutathione-S-transferase (GST)-SSX-2 and GST were evaluated by Western blot with patient sera (patients 24, 19 or 35) or with monoclonal antibodies (positive) specific for SSX-2 or GST. B) HTI conducted with CTA phage array using sera from patients 24, 19 or 35. Positive refers to phage encoding IgG, and negative refers to empty phage control.

[0008] FIG. 3 shows that patients with melanoma have IgG specific for several CTA. Shown is the percentage of male patients with melanoma (n=44), or male control blood donors without cancer (n=50), with IgG specific for each of the 29 CTA tested.

[0009] FIG. 4 shows that MAD-CT-2, NY-ESO-1 and SSX-2 are expressed in melanoma cell lines. RT-PCR (reverse transcriptase-polymerase chain reaction) with primers specific for NY-ESO-1, SSX-2, MAD-CT-2 or .beta.-actin was conducted using mRNA from cell lines (e.g., MEL-5 and MEL-21), cDNA encoding each of the proteins (positive control), or no DNA template (negative control).

[0010] FIG. 5 shows that HTI identified IgG responses to 4 (i.e., SSX-2, NY-ESO-1, LAGE-1 and NFX2) of 29 known CTA using sera from prostate cancer patients.

[0011] FIG. 6 shows the expression of SSX-2 in 5 normal prostate epithelial cell lines (lanes 1-5: 1. PrEC1; 2. PrEC2; 3. PrEC3; 4. PrEC4; and 5. PZ-HPV7) and 6 prostate cancer cell lines (lanes 6-11: 6. SWPC1; 7. SWPC2; 8. SWNPC2; 9. LAPC4; 10. MDAPCa2b; and 11. MDAPCa2a) by RT-PCR. POS represents a cDNA positive control; whereas M represents molecular weight markers.

[0012] FIG. 7 shows the expression of SSX-2 in prostate cancer metastatic tissues. Prostate cancer metastatic tissue cDNA samples of 7 different prostate cancer patients (lanes 1-7) were analyzed for SSX-2 expression using PCR. Patients 1 and 2 showed relatively strong expression and patients 6 and 7 showed weak expression. POS and NEG are positive and negative controls, respectively. M represents molecular weight markers.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0013] The present invention is based on the inventors' identification of a number of antigens to which patients with prostate cancer or melanoma have developed antibody immune responses. The prostate cancer antigens identified include SSX-2, MAD-Pro-30, MAD-Pro-42, transgelin, ZCWCC3, ACAA1, actinin and NFX2; whereas the melanoma antigens identified include MAD-CT-2, MAD-CT-1 and PAGE-1. The inventors have also found that prostate cancer cells as well as metastatic tissues express several of the above newly identified prostate cancer antigens (e.g., SSX-2). In addition, melanoma cells express several of the newly identified melanoma antigens (e.g., MAD-CT-2). The identification provides new tools for assisting the diagnosis and the detection of recurrence of prostate cancer and melanoma, especially for men. The identification also provides new tools for determining whether an immune therapy elicited a tumor-specific immune response.

[0014] The cancer antigens disclosed herein are known proteins, and their DNA and amino acid sequences are available in the art (see, e.g., Table 1). While it is envisioned that a prostate cancer or melanoma antigen identified by the inventors can be used by itself for the screening of prostate cancer and melanoma, respectively, a panel of more than one antigen is preferred. Other prostate cancer and melanoma antigens known in the art can be included in the panel. For example, a prostate cancer antigen panel may include SSX-2, NY-ESO-1, NFX2, MAD-Pro-22, MAD-Pro-30, MAD-Pro-34, MAD-Pro-42, MAD-CaP-1, MAD-CaP-5, MAD-CaP-15, MAD-CaP-20, MAD-CT-1, MAD-CT-2, MAD-CT-3, MAD-CT-5, transgelin, ZCWCC3, ACAA1, androgen receptor (AR; or ligand-binding domain of AR) and actinin. A smaller prostate cancer antigen panel may include SSX-2, NY-ESO-1, NFX2, MAD-Pro-22, MAD-Pro-30, MAD-Pro-34, MAD-Pro-42, MAD-CaP-1, MAD-CaP-5, MAD-CaP-15, MAD-CaP-20, MAD-CT-1, MAD-CT-2, MAD-CT-3, MAD-CT-5, transgelin, ZCWCC3, ACAA1 and AR (or ligand-binding domain of AR). Another smaller prostate cancer antigen panel may include SSX-2, NY-ESO-1, NFX2, MAD-Pro-22, MAD-Pro-30, MAD-Pro-34, MAD-Pro-42, MAD-CaP-1, MAD-CaP-5, MAD-CaP-15, MAD-CaP-20, MAD-CT-1, MAD-CT-2, MAD-CT-3, MAD-CT-5, transgelin, ACAA1 and AR (or ligand-binding domain of AR). Another smaller prostate cancer antigen panel may include MAD-Pro-34, MAD-Pro-42 and MAD-CT-2. Another smaller prostate cancer antigen panel may include SSX-2, MAD-Pro-30, AR (or ligand-binding domain of AR) and MAD-Pro-22 (PSA). Another smaller prostate cancer antigen panel may include MAD-Pro-30, AR (or ligand-binding domain of AR) and PSA.

[0015] An example of a melanoma antigen panel includes MAD-CT-2, SSX-2, NY-ESO-1, MAD-CT-1 and PAGE-1. A smaller melanoma antigen panel may include MAD-CT-2, SSX-2, NY-ESO-1 and PAGE-1. Another smaller melanoma antigen panel may include MAD-CT-2, SSX-2 and NY-ESO-1.

TABLE-US-00001 TABLE 1 Prostate and Melanoma cancer antigens. Designation Herein Common Name GenBank ID MAD-Pro-22 Prostate specific antigen (PSA) NM_145864 MAD-Pro-30 Recombination signal binding protein NM_015874 (RBPJK) MAD-Pro-34 Nucleolar autoantigen; SC65; No55 NM 006455.1 MAD-Pro-42 NY-CO-7/STUB1 NM_005861 MAD-CaP-1 HMG17 NM_005517 MAD-CaP-5 KIAA1404 gene product/ZNFX1 NM_021035 MAD-CaP-15 CLL-associated antigen KW-12/ NM_000975 RPL11 MAD-CaP-20 Human bullous pemphigoid antigen NM_015548 MAD-CT-1 Protamine 2 NM_002762 MAD-CT-2 Hypothetical protein of unknown AK097414 function FLJ40095/FLJ36144 MAD-CT-3 Sorting nexin 13 (SNX13) NM_015132 MAD-CT-5 Sjogren's syndrome antigen B NM_003142 (autoantigen La) SSX-2 Synovial sarcoma, X breakpoint 2 Z49105 Transgelin Transgelin BC024296 ZCWCC3 Zinc-finger protein CW type with NM_015358 coiled domain 3 ACAA1 Acetyl-coenzyme A acyltransferase 1 NM_001607 AR or AR-LBD Androgen receptor (AR), or ligand- M20132 binding domain of AR Actinin Actinin alpha 1, 2, 4 D89980 NY-ESO-1 NY-ESO-1/CTAG1B NM_001327 PAGE-1 PAGE1 AF058989 NFX2 NFX2 AF332009 LAGE-1 LAGE-1 AJ223093

[0016] In one aspect, the present invention relates to a method for identifying a human subject as a candidate for further prostate cancer examination. The method includes the step of determining whether the human subject has developed an immune reaction to a prostate cancer antigen selected from SSX-2, MAD-Pro-30, MAD-Pro-42, transgelin, ZCWCC3, ACAA1, actinin and NFX2, wherein the presence of an immune reaction indicates that the human subject is a candidate for further prostate cancer examination. In one embodiment, a prostate cancer antigen panel as disclosed herein is used in the method and whether the human subject has developed an immune reaction to the antigens in the panel is determined. The presence of an immune reaction to any member of the panel indicates that the human subject is a candidate for further prostate cancer examination. For a subject who has already been indicated to have prostate cancer by other tests, the method here can be used to confirm the diagnosis or identify recurrence after treatment.

[0017] The method disclosed above may optionally include the step of subjecting a human subject to further prostate cancer examination if the human subject tests positive for at least one of the prostate cancer antigens. Any known test for assisting the diagnosis of prostate cancer can be used. Examples include serum prostate-specific antigen (PSA) blood test and standard pathological evaluation of prostate tissue specimen obtained from a biopsy. For subjects with a history of treated prostate cancer, radiographic scans can be conducted for detecting recurrent prostate cancer. In one embodiment, prostate or a prostate tissue specimen from the subject is examined for the presence of prostate cancer.

[0018] In a second aspect, the present invention relates to a method for determining whether an immune therapy elicited a tumor-specific immune response in a prostate cancer patient. In one embodiment, the immune therapy is an antigen-specific immune therapy. The new prostate cancer antigens identified herein can serve as targets for antigen-specific immune therapies and whether such an antigen-specific immune therapy has elicited a tumor-specific immune response can be determined by testing whether a patient has developed an immune reaction to the antigen. An antigen-specific immune therapy may ultimately elicit responses to other antigens (i.e., antigens other than the one the therapy is designed to specifically target). For example, a successful antigen-specific immune therapy causes immune-mediated tumor destruction, leading to recognition of other antigens. Therefore, response to another antigen (other than the one the therapy is designed to specifically target) in an antigen-specific immune therapy indicates indirectly that the therapy elicited an immune response to the targeted antigen and is therefore effective. To practice the method, the prostate cancer antigens disclosed herein can be used individually, in combination with each other, or in combination with other known prostate cancer antigens to determine whether an antigen-specific immune therapy has elicited a tumor-specific immune response. In this regard, whether the patient has developed an immune reaction to a prostate cancer antigen selected from SSX-2, MAD-Pro-30, MAD-Pro-42, transgelin, ZCWCC3, ACAA1, actinin and NFX2 can be analyzed, wherein the presence of an immune reaction indicates that the therapy elicited a tumor-specific immune response. Any panel of prostate cancer antigens disclosed herein can be used for this purpose, wherein the presence of an immune reaction to at least one member of the panel indicates that the therapy elicited an immune response. Preferably, the patient is also tested for immune responses to the antigen or antigens before therapy so that it can be confirmed that an immune response detected after the start of the therapy is elicited by the therapy. The method may optionally include the step of monitoring the status of the prostate cancer in the patient by, e.g., the prostate cancer examination techniques described above.

[0019] In another embodiment of the second aspect, the immune therapy is a non-antigen-specific immune therapy. As used herein, "non-antigen-specific immune therapy" means an immune therapy that does not specifically target a particular antigen or an immune therapy that does not identify the antigen targets. Examples of non-antigen-specific therapies include whole-cell-based therapies such as the GVAX vaccines (granulocyte macrophage colony-stimulating factor-secreting cancer cell immunotherapy) or whole-cell vaccines developed by Onyvax Ltd. (London, England) (Hege K, et al., Int. Rev. Immunol. 25:321-352 (2006); Nemunaitis J, et al., Cancer Gene Ther. 13:555-562 (2006); and Michael A, et al., Clin. Cancer Res. 11:4469-4478 (2005)), cytokine-based therapies such as interleukin-2 or interferon-gamma (King D, et al. J. Clin. Oncol. 22:4463-4473 (2004)) or other immunomodulatory therapies including anti-CTLA-4 therapies used either alone or in combination with another therapy (Thompson R, et al., Urol. Oncol. 24:442-427 (2006); Korman A, et al., Adv. Immunol. 90:297-339 (2006); and Maker A, et al., Ann. Surg. Oncol. 12:1005-1016 (2005)). Traditionally, it has been difficult to assess whether a non-antigen-specific immune therapy generated a tumor-specific immune response because the therapy does not specifically target a particular antigen or the identities of the antigen targets are unknown. The prostate cancer antigens disclosed herein can be used individually, in combination with each other, or in combination with other known prostate cancer antigens to determine whether a non-antigen-specific immune therapy has elicited a tumor-specific immune response. In this regard, whether the patient has developed an immune reaction to a prostate cancer antigen selected from SSX-2, MAD-Pro-30, MAD-Pro-42, transgelin, ZCWCC3, ACAA1, actinin and NFX2 can be analyzed, wherein the presence of an immune reaction indicates that the therapy elicited a tumor-specific immune response. Any panel of prostate cancer antigens disclosed here can be used for this purpose, wherein the presence of an immune reaction to at least one member of the panel indicates that the therapy elicited an immune response. Preferably, the patient is also tested for immune responses to the antigen or antigens before therapy to confirm that an immune response detected after the start of the therapy is elicited by the therapy. The method may optionally include the step of monitoring the status of the prostate cancer in the patient by, e.g., the prostate cancer examination techniques described above.

[0020] In one embodiment, the method is used in clinical trials of antigen-specific or non-antigen-specific immune therapies for determining whether the therapies elicited a tumor-specific immune response.

[0021] One of ordinary skill in the art is more than capable of determining whether a human subject developed an immune reaction to one of the prostate cancer antigens. One way is to determine whether the human subject has produced antibodies to the antigens. For example, one can take a blood sample or blood-derived sample (e.g., a serum sample, a plasma sample or any preparation thereof that preserves the activity of immunoglobulins) from the human subject and test whether it contains antibodies to the antigens. Examples of such tests include enzyme-linked immunosorbent assay (ELISA), Western blot, protein microarray or high-throughput immunoblot analysis (Sreekumar A, et al., J. Natl. Cancer Inst. 96:834-843 (2004)), phage array-type analysis (Wang et al., supra; and Dubovsky J, et al., J. Immunother. 30:675-683 (2007)), and other methods known in the art. Another way is to determine whether the human subject has developed antigen-specific T cells, which is also well-known to one of ordinary skill in the art. For example, antigen stimulation can be used to detect antigen-specific T cells for their ability to proliferate, secrete various cytokines or exhibit cytolytic function (see e.g., Olson B & McNeel D, Prostate 67:1729-1739 (2007)). Other examples include enzyme-linked immunsorbent spot (ELISPOT) assays, fluorescence cell sorting of cytokine-producing cells, and peptide MHC/HLA tetramer assays (see e.g., Hobeika A, et al., J. Immunother. 28:63-72 (2005)). Preferably, circulating antigen-specific T cells are detected as they are the most accessible ones. For example, peripheral blood mononuclear cells (PBMC) can be analyzed for this purpose.

[0022] In a third aspect, the present invention relates to a method for identifying a human subject as a candidate for further melanoma examination. The method includes the step of determining whether the human subject has developed an immune reaction to a melanoma antigen selected from MAD-CT-2, MAD-CT-1 and PAGE-1, wherein the presence of an immune reaction indicates that the human subject is a candidate for further melanoma examination. MAD-CT-2 is a preferred melanoma antigen. In one embodiment, a melanoma antigen panel disclosed herein is used in the method and whether the human subject has developed an immune reaction to the antigens in the panel is determined. The presence of an immune reaction to any member of the panel indicates that the human subject is a candidate for further melanoma examination. For a subject already known to have melanoma by other tests, the method can be used to confirm the diagnosis or suggest recurrent disease.

[0023] The method disclosed above may optionally include the step of subjecting a human subject to further melanoma examination if the human subject is tested positive for at least one of the melanoma antigens. Any known test for assisting the diagnosing of melanoma can be used. For example, a skin specimen from the subject can be examined for the presence of melanoma (e.g., by pathological analysis). Radiographic imaging studies may be employed to evaluate for the presence of metastatic lesions.

[0024] In a fourth aspect, the present invention relates to a method for determining whether an immune therapy elicited a tumor-specific immune response in a melanoma patient. The method is the same as that described above for prostate cancer except that it is practiced with melanoma patients and the melanoma antigens identified by the inventors. In one embodiment, the immune therapy is an antigen-specific immune therapy. In another embodiment, the immune therapy is a non-antigen-specific immune therapy. The melanoma antigens disclosed here can be used individually, in combination with each other, or in combination with other known melanoma antigens to determine whether an immune therapy has elicited a tumor-specific immune response. In this regard, whether the patient has developed an immune reaction to a melanoma antigen selected from MAD-CT-2, MAD-CT-1 and PAGE-1 can be analyzed, wherein the presence of an immune reaction indicates that the therapy has elicited a tumor-specific immune response. Any panel of melanoma antigens disclosed herein can be used for this purpose, wherein the presence of an immune reaction to at least one member of the panel indicates that the therapy has elicited an immune response. Preferably, the patient is also tested for immune responses to the antigen or antigens before therapy so that it can be confirmed that an immune response detected after the start of the therapy is elicited by the therapy. The method may optionally include the step of monitoring the status of melanoma in the patient by, e.g., the melanoma examination techniques described above. In one application, the method is used in clinical trials of antigen-specific or non-antigen-specific immune therapies for determining whether the therapies have elicited a tumor-specific immune response.

[0025] Similar to what has been discussed above with respect to the prostate cancer antigens, it is well within the capability of one of ordinary skill in the art to determine whether a human subject has developed an immune reaction to one of the melanoma antigens, and examples of applicable techniques have been described above in connection with the prostate cancer antigens.

[0026] In a fifth aspect, the present invention relates to yet another method for identifying a human subject as a candidate for further melanoma examination. The method includes the step of determining whether the cells in a region of the subject's skin suspected of being malignant express MAD-CT-2, wherein the expression of MAD-CT-2 indicates that the subject is a candidate for further melanoma examination. The expression of MAD-CT-2 can be determined at either the mRNA level or the protein level and one of ordinary skill in the art is familiar with the techniques for such determination. For example, antibodies directed to an epitope on an antigen can be used to detect the antigen at the protein level, and it is well within the capability of one of ordinary skill in the art to generate such antibodies if not already available. The presence of mRNA for an antigen can be measured using methods for hybridizing nucleic acids, including, without limitation, RNA, DNA and cDNA. Such methods are generally known to those skilled in the art (e.g., RT-PCR amplification, Northern blot and Southern blot).

[0027] Optionally, the method further includes the step of subjecting a human subject who is positive for the expression of MAD-CT-2 to further melanoma examination. For a subject already identified as having melanoma by other tests, the method provided herein can be used to confirm the diagnosis.

[0028] In sixth aspect, the present invention relates a method for identifying candidate compounds for further testing as preventive or therapeutic agents for melanoma. As MAD-CT-2 is expressed in melanoma cells, it can serve as a marker for melanoma drug screening because, presumably, an anti-melanoma agent can bring down the MAD-CT-2 mRNA and protein level in melanoma cells that express this marker. Accordingly, a compound that demonstrates such an activity may be a good candidate for further testing for anti-tumor efficacy. In this regard, animal or human cells that express MAD-CT-2 can be exposed to a test agent, and the effect of the test agent on MAD-CT-2 expression at the mRNA or protein level relative to that of corresponding untreated control cells can be measured, wherein a lower expression than that of the control cells indicates that the agent is a candidate for further testing as preventive or therapeutic agents for melanoma. The animal or human cells that express MAD-CT-2 can contain a DNA sequence encoding MAD-CT-2 under the control of an endogenous promoter such as a native promoter or another promoter following translocation to a new site. Preferably, human or animal melanoma cells that express MAD-CT-2 are used. In one embodiment, such human or animal melanoma cells are those of a cell line.

[0029] In seventh aspect, the invention relates to kits suitable for use in the methods disclosed herein. For the method of identifying a human subject as a candidate for further prostate cancer examination in connection with analyzing whether the subject has developed an immune response to prostate cancer antigens, the kit includes the proteins or suitable fragments thereof of a prostate cancer antigen panel disclosed herein or expression vectors/systems for expressing the proteins or suitable fragments (e.g., a phage system as described in Example 1 below, an expression vector containing a DNA sequence encoding an antigen protein operably linked to a promoter such as a non-native promoter, or a cell containing the expression vector). In this regard, a protein in the panel can be represented by the protein with additional amino acid sequences at one or both of the N- and C-terminal ends, as long as the additional sequences do not affect the function of the proteins in connection with the present invention (i.e., the ability to bind to the corresponding immunoglobulins). The additional amino acid sequences can, but do not have to, assist in the purification, detection, or stabilization of the proteins. Optionally, the kit also includes a positive control, a negative control or both. For example, a positive control may be a composition containing an antibody to one or more of the antigens in the panel (e.g., a blood/serum/plasma preparation from a patient or group of patients known to interact with one or more of the antigens). A negative control for the proteins in the panel and the expression vectors/systems for expressing the proteins may be a non-tumor antigen protein such as a housekeeping protein and an expression vector/system for expressing the non-tumor antigen. A negative control for the patient's serum or blood-derived sample may be a serum or blood-derived sample from a normal individual that does not interact with any of the proteins in the panel.

[0030] For the method of identifying a human subject as a candidate for further melanoma examination in connection with analyzing whether the subject developed an immune response to melanoma antigens, the kit includes the proteins or suitable fragments thereof of a melanoma antigen panel disclosed herein or expression vectors/systems for expressing the proteins or suitable fragments (e.g., a phage system as described in Example 1 below, an expression vector containing a DNA sequence encoding an antigen protein operably linked to a promoter such as a non-native promoter, or a cell containing the expression vector). In this regard, a protein in the panel can be represented by the protein with additional amino acid sequences at one or both of the N- and C-terminal ends, as long as the additional sequences do not affect the function of the proteins in connection with the present invention (i.e., the ability to bind to the corresponding immunoglobulins). The additional amino acid sequences can, but do not have to, assist in the purification, detection or stabilization of the proteins. Optionally, the kit also includes a positive control, a negative control or both. For example, a positive control may be a composition containing an antibody to one or more of the antigens in the panel (e.g., a blood/serum/plasma preparation from a patient known to interact with one or more of the antigens). A negative control for the proteins in the panel and the expression vectors/systems for expressing the proteins may be a non-tumor antigen protein, such as a housekeeping protein and an expression vector/system for expressing the non-tumor antigen. A negative control for the patient's serum or blood-derived sample may be a serum or blood-derived sample from a normal individual that does not interact with any of the proteins in the panel.

[0031] For the method of identifying a human subject as a candidate for further melanoma examination in connection with analyzing the expression of MAD-CT-2, the kit includes an antibody to MAD-CT-2 or an oligonucleotide set for amplifying and detecting MAD-CT-2 mRNA or cDNA. For example, the oligonucleotide set can contain a pair of PCR primers, preferably RT-PCR primers, which can be used for both amplifying and detecting the mRNA or cDNA. The set may also contain a separate oligonucleotide for detecting the amplified sequence. Optionally, the kit also includes a positive control, a negative control or both. For example, a positive control may be a composition that contains MAD-CT-2 or the corresponding mRNA or cDNA (e.g., melanoma cells or an extract thereof of either a cell line or a patient that are known to express MAD-CT-2). A negative control for the cells from a subject being tested may be cells that do not express MAD-CT-2 (e.g., skin cells or other types of cells from a normal individual). A negative control for the antibody or oligonucleotide set may be an antibody that does not interact with MAD-CT-2 or a set of oligonucleotides that does not amplify and detect the corresponding mRNA/cDNA (e.g., an antibody to a non-tumor antigen protein, such as a housekeeping protein or a set of oligonucleotides that amplifies and detects the mRNA/cDNA of the non-tumor antigen protein).

[0032] Any of the kits described above can optionally contain an instruction manual directing use of the kit according to the method of the present invention.

[0033] The invention will be more fully understood upon consideration of the following non-limiting examples.

Examples

Example 1

Antibody Responses to Cancer-Testis Antigens in Melanoma Patients

[0034] In this example, we show the construction of a panel of 29 CTA in lambda (.lamda.) phage, and implementation of a novel high throughput immunoscreening method using a panel of sera from patients with melanoma (n=44) and volunteer blood donors (n=50). We show that antibody responses occurred in 39% of patients with melanoma to at least one CTA antigen in a defined panel of 5 compared with 4% of controls (p<0.001). Moreover, antibody responses to one antigen, MAD-CT-2, occurred in 27% of patients compared with 0/50 controls (p<0.0001). We also show that MAD-CT-2 is expressed in melanoma cell lines.

[0035] Materials and Methods.

[0036] Subject Population: Sera were obtained from 44 male patients with metastatic melanoma, mean age 50 years (range 25-78 years). All patients had been treated with primary resection. 19/44 (43%) were treated with prior immunotherapy, and 9/44 (20%) were treated with prior chemotherapy. All subjects gave written institutional review board (IRB)-approved informed consent for their blood products to be used for immunological research. Blood was collected at the University of Wisconsin Hospital and Clinics (Madison, Wis.), and sera were stored in aliquots at -80.degree. C. until used. Control sera were obtained from volunteer male blood donors, mean age 34 years (range 18-57 years), who also gave IRB-approved written informed consent.

[0037] Phage Cloning: Plasmid DNA encoding full-length cDNAs for 29 CTA were either purchased as IMAGE clones (American Type Culture Collection (ATCC); Manassas, Va.), or were obtained from cDNA expression libraries from previous studies (Hoeppner et al., supra; and Stone B, et al., Int. J. Cancer 104:73-84 (2003)). Primers, specific for each CTA, were designed with the purpose of appending a single 5' EcoRI and a single 3' XhoI site for subcloning. In cases where the CTA had an internal EcoRI or XhoI site, an alternate 5' MfeI site or 3' Sail site was used. Table 2 shows the sequence of the primers and IMAGE clone identification for each CTA construct. Polymerase chain reaction (PCR) was performed using template cDNAs, gene specific primers, Taq polymerase (Promega; Madison, Wis.), and 30 amplification cycles optimized with respect to temperature for each primer pair. PCR products were gel purified (Qiaquick Gel Extraction Kit; Qiagen; Valencia, Calif.), digested with the appropriate restriction enzymes, and ligated into .lamda.-phage arms (Lambda ZAP express protocol; Stratagene; La Jolla, Calif.). Phage were amplified by standard methods and sequenced to confirm their identity and to detect any mutations introduced by PCR (Table 2).

TABLE-US-00002 TABLE 2 CTA phage construction. Shown are the names and GenBank identifiers for each CTA chosen for analysis. In addition, the 5' and 3' primers used for the gene-specific cDNA PCR amplifi- cation and subcloning are shown, and the IMAGE clone identi- fiers from which the genes were cloned. Variations from the published amino acid sequences identified after final clone sequencing are shown. Image Name GenBank ID Clone ID 5' Primer MAGE-A1.sup.1 NM_004988 GGAATTCATGTCTCTTGAGCAGAGGAGTC (SEQ ID NO: 1) SSX2.sup.2 BC007343 GGTGCTCAAATACCAGAGAAG * (SEQ ID NO: 3) NY-ESO-1.sup.3 AJ003149 GGAATTCCATGCAGGCCGAAGGCCGGGG (SEQ ID NO: 5) GAG-7.sup.4 NM_021123 GGAATTCATGAGTTGGCGAGGAAGATCGACC (SEQ ID NO: 7) SSX4.sup.5 U90841 GGAATTCATGAACGGAGACGACGCCTTG (SEQ ID NO: 9) NXF2.sup.6 BC015020 3921074 CCAATTGATGTGCTCTACTCTAAAGAAGTGTG (SEQ ID NO: 11) TPX1.sup.7 BC022011 4826427 GGAATTCAATGGCTTTACTACCGGTGTTGTTTC (SEQ ID NO: 13) XAGE-1.sup.8 BC009538 3893227 GGAATTCATGGAGAGCCCCAAAAAGAAGAACC (SEQ ID NO: 15) LAGE-1.sup.9 BC002833 3638129 GGAATTCATGCAGGCCGAAGGCCGGGGCAC (SEQ ID NO: 17) PAGE-1.sup.4 BC010897 4043535 GGAATTCGATGAGTGCACGAGTGAGATCAAG (SEQ ID NO: 19) MAGE-E1.sup.10 BC081566 6292139 CCAATTGCATGGCTGAGGGAAGCTTCAGCGTG (SEQ ID NO: 21) SPANXC.sup.11 BC054023 6648369 CCAATTGATGGACAAACAATCCAGTGCCGGCGG (SEQ ID NO23) ADAM2.sup.12 BC064547 5744846 GGAATTCATGTGGCGCGTCTTGTTTCTGCTC (SEQ ID NO: 25) TSP50.sup.13 BC037775 5272458 GGAATTCATGGGTCGCTGGTGCCAGACCGTC (SEQ ID NO: 27) NY-SAR-35.sup.14 BC034320 4836772 GGAATTCATGTCTTCACATAGGAGGAAAGCGAAG (SEQ ID NO: 29) FATE1.sup.15 BC022064 4826440 GGAATTCATGGCAGGAGGCCCTCCCAACACC (SEQ ID NO: 31) PAGE-5.sup.12 BC009230 3955765 GGATTCGTGATGCAGGCGCCATGGGCC (SEQ ID NO: 33) LIP1.sup.12 BC023635 4841470 GGAATTCATGTCTCTACGCTGCGGGGATGCAG (SEQ ID NO: 35) SPA17.sup.16 BC032457 5171849 CCAATTGATGTCGATTCCATTCTCCAACACC (SEQ ID NO: 37) MAGE-A8.sup.1 BE387798 GGAATTCATGCCTCTTGAGCAGAGGAGTCAG (SEQ ID NO: 39) MAGE-B1.sup.17 BE897525 GGAATTCATGCCTCGGGGTCAGAAGAG (SEQ ID NO: 41) MAGE-B2.sup.17 BC026071 GGAATTCATGCCTCGTGGTCAGAAGAGTAAG (SEQ ID NO: 43) MAGE-A4.sup.1 BC017723 GGAATTCATGTCTTCTGAGCAGAAGAGTC (SEQ ID NO: 45) SSX1.sup.2 BC001003 3445470 GGAATTCATGAACGGAGACAACACCTTTG (SEQ ID NO: 47) GAGE-2.sup.18 BC069397 7262151 GGAATTCATGAGTTGGCGAGGAAGATCGACC (SEQ ID NO: 49) GAGE-4.sup.18 BC069470 GGAATTCATGATTGGGCCTATGCGGCCCGAG (SEQ ID NO: 51) MAGE-A3.sup.1 BC016803 * MAD-CT-1.sup.19 NM_002762 GAGGAGCCTGAGCGAACG * (SEQ ID NO: 53) MAD-CT-2.sup.19 AK097414 GAGGATATGAGATCAGAAAGAGAAG * (SEQ ID NO: 55) Sequence Name 3' Primer variation: MAGE-A1.sup.1 CCGCTCGAGCTCAGACTCCCTCTTCCTCCTC (SEQ ID NO: 2) SSX2.sup.2 CTTTGGGTCCAGATCTCTCGTG * (SEQ ID NO: 4) NY-ESO-1.sup.3 CCGCTCGAGCTTAGCGCCTCTGCCCTGAGGG G to V mutation (SEQ ID NO: 6) at a.a.#41 GAG-7.sup.4 CCGCTCGAGTTACACTGTGAGCTTTTCACC (SEQ ID NO: 8) SSX4.sup.5 CCGCTCGAGCTACTCGTCATCTTCCTCAGGG (SEQ ID NO: 10) NXF2.sup.6 CCGCTCGAGTTAGGAGATTTGCTTGAAGGCCTCTG (SEQ ID NO: 12) TPX1.sup.7 CCGCTCGAGTCAGTAAATTTTGTTCTCACATAGG (SEQ ID NO: 14) XAGE-1.sup.8 CCGCTCGAGTTAAACTTGTTGCTCTTCACCTG (SEQ ID NO: 16) LAGE-1.sup.9 CCGCTCGAGCTAAATGAGAGGGGCAGAGAACATC (SEQ ID NO: 18) PAGE-1.sup.4 CCGCTCGAGTTATGGCTGCCCATCCCTGCTTC (SEQ ID NO: 20) MAGE-E1.sup.10 CCGCTCGAGTCAACGGTGCTGGATCCAGGAG (SEQ ID NO: 22) SPANXC.sup.11 CCGCTCGAGCTACTTTGCAGGTATTTCACATTATTTC Missing last 7 (SEQ ID NO: 24) a.a.'s ADAM2.sup.12 CGGCTCGAGACTACCCTTTAGGTTCACTCTCAC (SEQ ID NO: 26) TSP50.sup.13 CCGCTCGAGTCAGAGGGCAGCAAGGAGG (SEQ ID NO: 28) NY-SAR-35.sup.14 CCGCTCGAGCTACTCGTCACCATGTTCCTCAC (SEQ ID NO: 30) FATE1.sup.15 CCGCTCGAGATCACTGGTTCATCCACAGCCAC (SEQ ID NO: 32) PAGE-5.sup.12 CCGCTCGAGCTATAGTTGCCCTTCACCTGCTTGG (SEQ ID NO: 34) LIP1.sup.12 CCGCTCGAGTTTTAGAGGTCTTTTGTTTTTCTTTTAGCC (SEQ ID NO: 36) SPA17.sup.16 CCGCTCGAGTCACTTGTTTTCCTCTTTTTCCTC (SEQ ID NO: 38) MAGE-A8.sup.1 CCGCTCGAGACTCACTCTTCCCCCTCTCTCAA (SEQ ID NO: 40) MAGE-B1.sup.17 ACGCGTCGACTCACATGGGGTGGGAGGACCTG (SEQ ID NO: 42) MAGE-B2.sup.17 CCGCTCGAGCTCAGACTCCGGCTTTCTCTTC (SEQ ID NO: 44) MAGE-A4.sup.1 CCGCTCGAGCTCAGACTCCCTCTTCCTCCT (SEQ ID NO: 46) SSX1.sup.2 CCGCTCGAGTTACTCGTCATCTTCCTCAGGG (SEQ ID NO: 48) GAGE-2.sup.18 CCGCTCGAGTTAACACTGTGATTGCTTTTCACCTTC (SEQ ID NO: 50) GAGE-4.sup.18 CCGCTCGAGTTAACACTGTGATTGCCCTTCACCTTC (SEQ ID NO: 52) MAGE-A3.sup.1 * MAD-CT-1.sup.19 GGATTCTTAGTGCCTTCTGCATGTTCTCTT * (SEQ ID NO: 54) MAD-CT-2.sup.19 TCCACACTGCCAGTGTGGCTCAT * (SEQ ID NO: 56) * Phage encoding MAGE-A3 were obtained from previous unpublished studies. .sup.1De Plaen E et al. Immunogenetics 1994, 40: 360-369. .sup.2Crew AJ et al. Embo J. 1995, 14: 2333-2340. .sup.3Chen YT et al. Proc. Natl. Acad. Sci. U.S.A. 1997, 94: 1914-1918. .sup.4Chen ME et al. J Biol Chem. 1998, 273: 17618-17625. .sup.5Gure AO et al. Int J Cancer 1997, 72: 965-971. .sup.6Loriot A et al. Int J Cancer 2003, 105: 371-376. .sup.7Kasahara M et al. Immunogenetics 1989, 29: 61-63. .sup.8Brinkmann U et al. Cancer Res. 1999, 59: 1445-1448. .sup.9Chen YT et al. Proc Natl Acad Sci U S A. 1998, 95: 6919-6923. .sup.10Sasaki M et al. Cancer Res. 2001, 61: 4809-4814. .sup.11Zendman AJ et al. Cancer Res. 1999, 59: 6223-6229. .sup.12Scanlan MJ et al. Int J Cancer 2002, 98: 485-492. .sup.13Yuan L et al. Cancer Res. 1999, 59: 3215-3221. .sup.14Lee SY et al. Proc Natl Acad Sci U S A. 2003, 100: 2651-2656. .sup.15Dong XY et al. Br J Cancer 2003, 89: 291-297. .sup.16Lim SH et al. Blood 2001, 97: 1508-1510. .sup.17Lurquin C et al. Genomics 1997, 46: 397-408. .sup.18Van den Eynde B et al. J Exp Med. 1995, 182: 689-698. .sup.19Hoeppner LH et al. Cancer Immun. 2006, 6: 1-7.

[0038] Reverse transcriptase-PCR (RT-PCR): E. coli cultures (XL-1 blue MRF strain; Stratagene) were transduced with 10.sup.6 pfu of individual phage and grown overnight in LB media+20 mM MgSO.sub.4+0.2% maltose+2.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37.degree. C. Total RNA was prepared from centrifugally pelleted cultures (RNeasy Mini Columns; Qiagen). RT-PCR reactions were conducted using the Qiagen One-Step RT-PCR Kit (Qiagen) and transcript-specific primers (T7 Promoter and T3 Promoter; Invitrogen; Carlsbad, Calif.). To control for the possible amplification of contaminating genomic or phage DNA in the total RNA preparations, duplicate reactions were first heated to 100.degree. C. for 10 minutes immediately prior to the normal RT-PCR reaction to attenuate reverse transcriptase activity. PCR amplification reactions were resolved on agarose gels and the size of the amplified transcript confirmed by comparison with DNA size markers (1 Kb ladder; Promega).

[0039] High Throughput Immunoblot: XL-1 blue MRF E. coli were grown overnight at 31.degree. C. in LB medium supplemented with 20 mM MgSO.sub.4 and 0.2% maltose. Cultured cells were then collected by centrifugation, resuspended in 10 mM MgSO.sub.4, and poured in top agarose (LB broth/10 mM MgSO.sub.4/0.2% maltose/0.7% agarose) over LB agar in Omniwell plates (Nunc). 9,000 pfu of individual phage were then spotted in a 0.9 .mu.l volume in replicates onto multiple bacterial agar lawns using a liquid handling robot (Beckman; Biomek FX; Fullerton, Calif.). Spotted plates were allowed to sit undisturbed for 15 minutes at room temperature, and then overlaid with nitrocellulose membranes impregnated with 10 mM IPTG. Plates were incubated overnight at 37.degree. C. The next day, filters were gently peeled from bacterial lawns and washed twice in TBST (50 mM Tris pH 7.2, 100 mM NaCl, 0.5% Tween-20) for 5 minutes and once in TBS (50 mM Tris pH 7.2, 100 mM NaCl) for an additional 5 minutes. The filters were then blocked in TBS+1% BSA for 1 hour shaking at room temperature.

[0040] Membranes were then probed with human serum preadsorbed to E. coli proteins and the empty phage construct diluted 1:100 in blocking solution at 4.degree. C. overnight. Following this, the membranes were again washed twice in TBST and once in TBS, and human IgG was detected with anti-human IgG-AP (Sigma; St. Louis, Mo.) diluted 1:25,000 in blocking solution for 1 hour shaking at room temperature. The filters were again washed and then developed with 0.3 mg/ml nitro-blue tetrazolium chloride (NBT)+0.15 mg/ml 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP). After development, filters were washed with deionized water, and then dried prior to evaluation. Immunoreactive plaques were recorded for each filter by visual comparison with an internal positive (phage encoding human IgG) and negative (empty phage encoding beta-galactosidase) control plaques. A plaque was defined as positive if replicate plaques were read as positive and there was accordance among at least six of nine independent observers, to reduce the possibility of subjective interpretation (Dunphy E, et al., J. Clin. Immunol. 24:492-501 (2004)). Comparison of immunoreactive spots among subject groups was made by chi-square analysis.

[0041] Western blot: Amino-terminal glutathione-S-transferase (GST) linked SSX-2 and GST (control) were purified from IPTG-induced overnight E. coli (Rosetta Gami expression strain) cultures transformed with pET41b plasmid (EMD Biosciences; San Diego, Calif.) containing full length SSX-2 cloned in frame with the ATG start codon. Purified proteins were stored at -80.degree. C. and were thawed immediately before mixing 1:1 with 2.times.SDS Laemmli's loading buffer (0.04 M Tris pH 6.8, 12% glycerol, 1.25% sodium docecyl sulfate (SDS), 3% .beta.-mercaptoethanol, 0.06% bromophenol blue) and boiling for 10 minutes at 100.degree. C. The proteins were then resolved on 15% SDS-polyacrylamide gels and were electrophoretically transferred to nitrocellulose membranes. Membranes were then probed, using standard immunoblot techniques, with preadsorbed patient sera diluted 1:200 in blocking solution or protein-specific antibodies. Detection antibodies included goat anti-SSX-2 polyclonal antibody (N-16; Santa Cruz Biotechnology; Santa Cruz, Calif.), and anti-GST monoclonal antibody (rabbit A5800; Invitrogen; Carlsbad, Calif.).

[0042] Results.

[0043] Cloning and validation of phage encoding CTA: We wished to develop a tool permitting simultaneous analysis of antibody responses to multiple antigens within a single serum sample. Given the ease of .lamda. phage cloning and expression compared with standard methods of protein purification, we chose to use a phage expression array methodology, similar to what we have previously described (Dunphy E J et al. J. Clin. Immunol. 2004, 24:492-501). Consequently, cDNA encoding 25 unique CTA were amplified by PCR, ligated into .lamda. phage arms, packaged into phage particles, and sequenced to confirm their identity. In addition, phage encoding SSX-2, MAGE-A3, MAD-CT-1 and MAD-CT-2 were available from prior studies (Hoeppner et al., supra; Crew A, et al., Embo. J. 14:2333-2340 (1995); and De Plaen E, et al., Immunogenetics 40:360-369 (1994), each of which is incorporated herein by reference as if set forth in its entirety). Gene transcription from each CTA phage construct was evaluated using RT-PCR reactions on total RNA purified from phage-transduced E. coli. In all cases, CTA phage produced mRNA transcripts of the correct predicted size. Similarly, immunoblot confirmed protein expression of CTA for which antibody reagents were available. For example, FIG. 1 demonstrates that a monoclonal antibody recognizing SSX2 and SSX4 could identify protein expression from phage-transduced E. coli.

[0044] Antigen-specific IgG to a panel of CTA were detected by high throughput immunoblot (HTI): Phage encoding individual CTA were directly spotted onto bacterial lawns grown in top agarose using a Biomek FX liquid handling robot. Optimization studies showed that a volume of 0.9 .mu.l of 10.sup.4 pfu/.mu.l phage produced reproducibly dense plaques. After plating and transfer to nitrocellulose membranes, the filters were probed with human sera that had been preadsorbed for antibodies to E. coli and diluted 1:100. Specifically, sera were obtained from 44 male patients with metastatic melanoma, and 50 male controls without cancer, and used to probe 94 individual membranes. Human IgG was then detected and visualized as described. Within each spotted array, phage encoding human IgG were included as a positive control, and empty phage were included as a negative control. In our hands, by using the liquid handling robot to uniformly array phage, we could easily screen at least 100 sera at the same time and accomplish the entire screening in three days.

[0045] Given the presence of internal positive and negative control phage plaques on each membrane, several methods were initially evaluated to objectively evaluate the final immunoblot membranes in an automated fashion. These methods included using fluorescent analysis of membranes following the use of fluorescent-tagged secondary antibodies, and automated densitometric analysis following colorimetric staining (not shown). Unfortunately, given variable backgrounds due to the E. coli bacterial lawns, these methods were unreliable and frequently "missed" plaques that were clearly immunoreactive, and false positive plaques were often observed. Consequently, we found that visual inspection was more accurate. To eliminate subjectivity from this evaluation, and reduce the possibility of false positive interpretation, each filter was reviewed and scored by a panel of independent readers. Plaques were scored as immunoreactive if duplicate plaques were each scored positive by the same observer, and if there was concordance among at least 6 of 9 independent observers, similar to what we have previously reported (Dunphy E, et al., J. Clin. Immunol. 24:492-501 (2004)). A subset of sera was re-evaluated in similar fashion to confirm the reproducibility of the findings, and immunoreactive plaques were similar to those observed in the initial screening (data not shown).

[0046] The presence of IgG specific for SSX-2 was confirmed by Western blot analysis: Western blot analysis was used when possible to confirm responses to CTA identified by HTI. As shown in FIG. 2A, IgG specific for SSX-2 were detectable by Western blot using sera from patient 24, a patient found by HTI to have IgG specific for SSX-2 (FIG. 2B). Conversely, no response was detectable using sera from patients 19 or 35 from whom no detectable HTI response to SSX-2 was detected, indicating that HTI screening provided data consistent with that by Western blot analysis.

[0047] Patients with melanoma have frequent antibody responses to SSX-2, NY-ESO-1, and MAD-CT-2: As shown in FIG. 3, IgG responses to SSX-2 and NY-ESO-1 were detected in 2/44 (5%, p=0.13) and 3/44 (7%, p=0.061) patients, respectively, compared with 0/50 male controls. In addition, IgG responses to MAD-CT-2 were identified in 12/44 (27%) of patients compared with 0/50 controls (p<0.001). IgG responses were also detected at lower frequencies to PAGE-1 and MAD-CT-1. Overall, IgG responses to at least one of the CTA were detected in 17/44 (39%) of male patients with melanoma, compared with 2/50 (4%) of controls (p<0.001) (FIG. 3 and Table 3). There was no apparent association with whether patients had been previously treated with immunotherapy, as 7/17 patients with antibody responses had been previously treated with immunotherapy (41%) compared with 12/27 patients who had no CTA-specific antibody responses (44%, p=0.83). IgG responses to MAD-CT-2 were detected in patients who did not demonstrate reactivity to SSX-2 or NY-ESO-1 (Table 3). This did not appear to be due to differences in patterns of gene expression, as mRNA encoding all three gene products were detectable in two different melanoma cell lines tested (FIG. 4). The identification of MAD-CT-2 transcripts in melanoma cell lines, however, demonstrates that it is a melanoma CTA.

TABLE-US-00003 TABLE 3 IgG responses to at least one CTA were detectable in multiple patients with melanoma. Shown are the patients with serum IgG specific for each CTA (shaded box) arrayed against the CTA tested. ##STR00001##

Example 2

Antibody Responses to Cancer-Testis Antigens in Prostate Cancer Patients

[0048] Using techniques similar to those described in Example 1 above or an ELISA assay, the inventors have found that antibody responses occurred at a higher rate in prostate cancer patients than in control individuals for the following antigens: SSX-2, NY-ESO-1, LAGE-1, NFX2, MAD-Pro-22, MAD-Pro-30, MAD-Pro-34, MAD-Pro-42, MAD-CaP-1, MAD-CaP-5, MAD-CaP-15, MAD-CaP-20, MAD-CT-1, MAD-CT-2, MAD-CT-3, MAD-CT-5, transgelin, ZCWCC3, ACAA1, AR (or ligand-binding domain of AR) and actinin. The results are summarized in Table 4 with the results obtained by either ELISA assays (labeled) or using the techniques described in Example 1. The ELISA assays were conducted using purified antigen proteins which were probed with sera from prostate cancer patients or normal individuals followed by detection of antigen-antibody interaction using enzyme-conjugated anti-human Ig antibody and the corresponding enzyme substrate.

[0049] FIG. 5 shows a specific example of the studies conducted with the techniques described in Example 1 in which SSX-2, NY-ESO-1, LAGE-1, and NFX2 were found to be expressed at a higher rate in prostate cancer patients than in control individuals.

[0050] Using RT-PCR and SSX-2 specific antibody, the inventors found that SSX-2 was expressed in at least three prostate cancer cell lines: LAPC4 (FIG. 6), MDAPCa2b (FIG. 6), and LNCap (data not shown). The inventors also found that SSX-2 was expressed in prostate cancer metastatic tissue samples (FIG. 7).

TABLE-US-00004 TABLE 4 Antigen-specific IgG Responses Patients with Control Male Antigen Prostate Cancer Blood Donors SSX-2 1/100 0/50 NY-ESO-1 3/100 0/50 MAD-Pro-22 16/100 9/64 (PSA) 22/200 by ELISA 3/100 by ELISA MAD-Pro-30 18/100 3/64 MAD-Pro-34 5/100 0/64 MAD-Pro-42 5/100 0/64 MAD-CaP-1 2/100 0/50 MAD-CaP-5 5/100 1/50 MAD-CaP-15 3/100 0/50 MAD-CaP-20 5/100 1/50 MAD-CT-1 5/109 1/52 MAD-CT-2 3/109 0/52 MAD-CT-3 2/109 0/52 MAD-CT-5 5/109 1/52 Transgelin 2/27 0/25 ZCWCC3 5/26 4/25 ACAA1 13/26 9/25 AR LBD 18/105 by ELISA 0/41 by ELISA Actinin 1/1

[0051] Although the invention has been described in connection with specific embodiments, it is understood that the invention is not limited to such specific embodiments but encompasses all such modifications and variations apparent to a skilled artisan that fall within the scope of the appended claims.

Sequence CWU 1

1

70129DNAARTIFICIAL SEQUENCEPCR PRIMER 1ggaattcatg tctcttgagc agaggagtc 29231DNAARTIFICIAL SEQUENCEPCR PRIMER 2ccgctcgagc tcagactccc tcttcctcct c 31321DNAARTIFICIAL SEQUENCEPCR PRIMER 3ggtgctcaaa taccagagaa g 21422DNAARTIFICIAL SEQUENCEPCR PRIMER 4ctttgggtcc agatctctcg tg 22528DNAARTIFICIAL SEQUENCEPCR PRIMER 5ggaattccat gcaggccgaa ggccgggg 28631DNAARTIFICIAL SEQUENCEPCR PRIMER 6ccgctcgagc ttagcgcctc tgccctgagg g 31731DNAARTIFICIAL SEQUENCEPCR PRIMER 7ggaattcatg agttggcgag gaagatcgac c 31831DNAARTIFICIAL SEQUENCEPCR PRIMER 8ccgctcgagt taacactgtg agcttttcac c 31928DNAARTIFICIAL SEQUENCEPCR PRIMER 9ggaattcatg aacggagacg acgccttg 281031DNAARTIFICIAL SEQUENCEPCR PRIMER 10ccgctcgagt tactcgtcat cttcctcagg g 311132DNAARTIFICIAL SEQUENCEPCR PRIMER 11ccaattgatg tgctctactc taaagaagtg tg 321235DNAARTIFICIAL SEQUENCEPCR PRIMER 12ccgctcgagt taggagattt gcttgaaggc ctctg 351333DNAARTIFICIAL SEQUENCEPCR PRIMER 13ggaattcaat ggctttacta ccggtgttgt ttc 331434DNAARTIFICIAL SEQUENCEPCR PRIMER 14ccgctcgagt cagtaaattt tgttctcaca tagg 341532DNAARTIFICIAL SEQUENCEPCR PRIMER 15ggaattcatg gagagcccca aaaagaagaa cc 321632DNAARTIFICIAL SEQUENCEPCR PRIMER 16ccgctcgagt taaacttgtt gctcttcacc tg 321730DNAARTIFICIAL SEQUENCEPCR PRIMER 17ggaattcatg caggccgaag gccggggcac 301834DNAARTIFICIAL SEQUENCEPCR PRIMER 18ccgctcgagc taaatgagag gggcagagaa catc 341931DNAARTIFICIAL SEQUENCEPCR PRIMER 19ggaattcgat gagtgcacga gtgagatcaa g 312032DNAARTIFICIAL SEQUENCEPCR PRIMER 20ccgctcgagt tatggctgcc catccctgct tc 322132DNAARTIFICIAL SEQUENCEPCR PRIMER 21ccaattgcat ggctgaggga agcttcagcg tg 322231DNAARTIFICIAL SEQUENCEPCR PRIMER 22ccgctcgagt caacggtgct ggatccagga g 312333DNAARTIFICIAL SEQUENCEPCR PRIMER 23ccaattgatg gacaaacaat ccagtgccgg cgg 332437DNAARTIFICIAL SEQUENCEPCR PRIMER 24ccgctcgagc tactttgcag gtatttcaca ttatttc 372531DNAARTIFICIAL SEQUENCEPCR PRIMER 25ggaattcatg tggcgcgtct tgtttctgct c 312633DNAARTIFICIAL SEQUENCEPCR PRIMER 26cggctcgaga ctacccttta ggttcactct cac 332731DNAARTIFICIAL SEQUENCEPCR PRIMER 27ggaattcatg ggtcgctggt gccagaccgt c 312828DNAARTIFICIAL SEQUENCEPCR PRIMER 28ccgctcgagt cagagggcag caaggagg 282934DNAARTIFICIAL SEQUENCEPCR PRIMER 29ggaattcatg tcttcacata ggaggaaagc gaag 343032DNAARTIFICIAL SEQUENCEPCR PRIMER 30ccgctcgagc tactcgtcac catgttcctc ac 323131DNAARTIFICIAL SEQUENCEPCR PRIMER 31ggaattcatg gcaggaggcc ctcccaacac c 313232DNAARTIFICIAL SEQUENCEPCR PRIMER 32ccgctcgaga tcactggttc atccacagcc ac 323328DNAARTIFICIAL SEQUENCEPCR PRIMER 33ggaattcgtg atgcaggcgc catgggcc 283434DNAARTIFICIAL SEQUENCEPCR PRIMER 34ccgctcgagc tatagttgcc cttcacctgc ttgg 343532DNAARTIFICIAL SEQUENCEPCR PRIMER 35ggaattcatg tctctacgct gcggggatgc ag 323639DNAARTIFICIAL SEQUENCEPCR PRIMER 36ccgctcgagt tttagaggtc ttttgttttt cttttagcc 393731DNAARTIFICIAL SEQUENCEPCR PRIMER 37ccaattgatg tcgattccat tctccaacac c 313833DNAARTIFICIAL SEQUENCEPCR PRIMER 38ccgctcgagt cacttgtttt cctctttttc ctc 333931DNAARTIFICIAL SEQUENCEPCR PRIMER 39ggaattcatg cctcttgagc agaggagtca g 314032DNAARTIFICIAL SEQUENCEPCR PRIMER 40ccgctcgaga ctcactcttc cccctctctc aa 324127DNAARTIFICIAL SEQUENCEPCR PRIMER 41ggaattcatg cctcggggtc agaagag 274232DNAARTIFICIAL SEQUENCEPCR PRIMER 42acgcgtcgac tcacatgggg tgggaggacc tg 324331DNAARTIFICIAL SEQUENCEPCR PRIMER 43ggaattcatg cctcgtggtc agaagagtaa g 314431DNAARTIFICIAL SEQUENCEPCR PRIMER 44ccgctcgagc tcagactccg gctttctctt c 314529DNAARTIFICIAL SEQUENCEPCR PRIMER 45ggaattcatg tcttctgagc agaagagtc 294630DNAARTIFICIAL SEQUENCEPCR PRIMER 46ccgctcgagc tcagactccc tcttcctcct 304729DNAARTIFICIAL SEQUENCEPCR PRIMER 47ggaattcatg aacggagaca acacctttg 294831DNAARTIFICIAL SEQUENCEPCR PRIMER 48ccgctcgagt tactcgtcat cttcctcagg g 314931DNAARTIFICIAL SEQUENCEPCR PRIMER 49ggaattcatg agttggcgag gaagatcgac c 315036DNAARTIFICIAL SEQUENCEPCR PRIMER 50ccgctcgagt taacactgtg attgcttttc accttc 365131DNAARTIFICIAL SEQUENCEPCR PRIMER 51ggaattcatg attgggccta tgcggcccga g 315236DNAARTIFICIAL SEQUENCEPCR PRIMER 52ccgctcgagt taacactgtg attgcccttc accttc 365318DNAARTIFICIAL SEQUENCEPCR PRIMER 53gaggagcctg agcgaacg 185431DNAARTIFICIAL SEQUENCEPCR PRIMER 54ggaattctta gtgccttctg catgttctct t 315525DNAARTIFICIAL SEQUENCEPCR PRIMER 55gaggatatga gatcagaaag agaag 255623DNAARTIFICIAL SEQUENCEPCR PRIMER 56tccacactgc cagtgtggct cat 2357104PRTHomo sapiensMAD-Pro-22 57Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly1 5 10 15Glu Arg Gly His Gly Trp Gly Asp Ala Gly Glu Gly Ala Ser Pro Asp20 25 30Cys Gln Ala Glu Ala Leu Ser Pro Pro Thr Gln His Pro Ser Pro Asp35 40 45Arg Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro Leu Gln Ala His50 55 60Thr Pro Ser Pro Ser Ile Leu Gln Gln Ser Ser Leu Pro His Gln Val65 70 75 80Pro Ala Pro Ser His Leu Pro Gln Asn Phe Leu Pro Ile Ala Gln Pro85 90 95Ala Pro Cys Ser Gln Leu Leu Tyr10058487PRTHomo sapiensMAD-Pro-30 58Met Ala Pro Val Val Thr Gly Lys Phe Gly Glu Arg Pro Pro Pro Lys1 5 10 15Arg Leu Thr Arg Glu Ala Met Arg Asn Tyr Leu Lys Glu Arg Gly Asp20 25 30Gln Thr Val Leu Ile Leu His Ala Lys Val Ala Gln Lys Ser Tyr Gly35 40 45Asn Glu Lys Arg Phe Phe Cys Pro Pro Pro Cys Val Tyr Leu Met Gly50 55 60Ser Gly Trp Lys Lys Lys Lys Glu Gln Met Glu Arg Asp Gly Cys Ser65 70 75 80Glu Gln Glu Ser Gln Pro Cys Ala Phe Ile Gly Ile Gly Asn Ser Asp85 90 95Gln Glu Met Gln Gln Leu Asn Leu Glu Gly Lys Asn Tyr Cys Thr Ala100 105 110Lys Thr Leu Tyr Ile Ser Asp Ser Asp Lys Arg Lys His Phe Met Leu115 120 125Ser Val Lys Met Phe Tyr Gly Asn Ser Asp Asp Ile Gly Val Phe Leu130 135 140Ser Lys Arg Ile Lys Val Ile Ser Lys Pro Ser Lys Lys Lys Gln Ser145 150 155 160Leu Lys Asn Ala Asp Leu Cys Ile Ala Ser Gly Thr Lys Val Ala Leu165 170 175Phe Asn Arg Leu Arg Ser Gln Thr Val Ser Thr Arg Tyr Leu His Val180 185 190Glu Gly Gly Asn Phe His Ala Ser Ser Gln Gln Trp Gly Ala Phe Phe195 200 205Ile His Leu Leu Asp Asp Asp Glu Ser Glu Gly Glu Glu Phe Thr Val210 215 220Arg Asp Gly Tyr Ile His Tyr Gly Gln Thr Val Lys Leu Val Cys Ser225 230 235 240Val Thr Gly Met Ala Leu Pro Arg Leu Ile Ile Arg Lys Val Asp Lys245 250 255Gln Thr Ala Leu Leu Asp Ala Asp Asp Pro Val Ser Gln Leu His Lys260 265 270Cys Ala Phe Tyr Leu Lys Asp Thr Glu Arg Met Tyr Leu Cys Leu Ser275 280 285Gln Glu Arg Ile Ile Gln Phe Gln Ala Thr Pro Cys Pro Lys Glu Pro290 295 300Asn Lys Glu Met Ile Asn Asp Gly Ala Ser Trp Thr Ile Ile Ser Thr305 310 315 320Asp Lys Ala Glu Tyr Thr Phe Tyr Glu Gly Met Gly Pro Val Leu Ala325 330 335Pro Val Thr Pro Val Pro Val Val Glu Ser Leu Gln Leu Asn Gly Gly340 345 350Gly Asp Val Ala Met Leu Glu Leu Thr Gly Gln Asn Phe Thr Pro Asn355 360 365Leu Arg Val Trp Phe Gly Asp Val Glu Ala Glu Thr Met Tyr Arg Cys370 375 380Gly Glu Ser Met Leu Cys Val Val Pro Asp Ile Ser Ala Phe Arg Glu385 390 395 400Gly Trp Arg Trp Val Arg Gln Pro Val Gln Val Pro Val Thr Leu Val405 410 415Arg Asn Asp Gly Ile Ile Tyr Ser Thr Ser Leu Thr Phe Thr Tyr Thr420 425 430Pro Glu Pro Gly Pro Arg Pro His Cys Ser Ala Ala Gly Ala Ile Leu435 440 445Arg Ala Asn Ser Ser Gln Val Pro Pro Asn Glu Ser Asn Thr Asn Ser450 455 460Glu Gly Ser Tyr Thr Asn Ala Ser Thr Asn Ser Thr Ser Val Thr Ser465 470 475 480Ser Thr Ala Thr Val Val Ser48559919PRTHomo sapiensAR or AR-LBD 59Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser1 5 10 15Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu20 25 30Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala35 40 45Pro Pro Gly Ala Ser Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln50 55 60Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr65 70 75 80Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro Gln85 90 95Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln100 105 110Gln Pro Ser Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu Arg Gly115 120 125Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu Pro130 135 140Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro Ser145 150 155 160Thr Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser165 170 175Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu Leu180 185 190Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly Arg195 200 205Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr Leu210 215 220Gly Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys Ala225 230 235 240Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser245 250 255Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu Gly260 265 270Val Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu Cys275 280 285Lys Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp Thr290 295 300Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu Gly305 310 315 320Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly Thr325 330 335Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp340 345 350Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala355 360 365Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala Arg370 375 380Ile Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala385 390 395 400Ala Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Ala Gly405 410 415Ala Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser Ser420 425 430Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Cys435 440 445Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly450 455 460Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala Pro Tyr465 470 475 480Gly Tyr Thr Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Ser Asp Phe485 490 495Thr Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg Val Pro500 505 510Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp Met Asp515 520 525Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg Asp530 535 540His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu545 550 555 560Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys565 570 575Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys580 585 590Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg595 600 605Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met610 615 620Thr Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln625 630 635 640Glu Glu Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr Thr645 650 655Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile660 665 670Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly675 680 685His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu690 695 700Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys705 710 715 720Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val725 730 735Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg740 745 750Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu755 760 765Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys770 775 780Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile Thr785 790 795 800Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile805 810 815Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg Met820 825 830Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn835 840 845Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp850 855 860Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu865 870 875 880Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala885 890 895Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys900 905 910Pro Ile Tyr Phe His Thr Gln915601309DNAHomo sapiensSSX-2 60actttctctc tctttcgatt cttccatact cagagtacgc acggtctgat tttctctttg 60gattcttcca aaatcagagt cagactgctc ccggtgccat gaacggagac gacgcctttg 120caaggagacc cacggttggt gctcaaatac cagagaagat ccaaaaggcc ttcgatgata 180ttgccaaata cttctctaag gaagagtggg aaaagatgaa agcctcggag aaaatcttct 240atgtgtatat gaagagaaag tatgaggcta tgactaaact aggtttcaag gccaccctcc 300cacctttcat gtgtaataaa cgggccgaag acttccaggg gaatgatttg gataatgacc 360ctaaccgtgg gaatcaggtt gaacgtcctc agatgacttt cggcaggctc cagggaatct 420ccccgaagat catgcccaag aagccagcag aggaaggaaa tgattcggag gaagtgccag 480aagcatctgg cccacaaaat gatgggaaag agctgtgccc cccgggaaaa ccaactacct 540ctgagaagat tcacgagaga tctggaccca aaagggggga acatgcctgg acccacagac 600tgcgtgagag aaaacagctg gtgatttatg aagagatcag cgaccctgag gaagatgacg 660agtaactccc ctcagggata cgacacatgc ccatgatgag aagcagaacg tggtgacctt 720tcacgaacat gggcatggct gcggacccct cgtcatcagg tgcatagcaa gtgaaagcaa 780gtgttcacaa cagtgaaaag ttgagcgtca tttttcttag tgtgccaaga gttcgatgtt 840agcgtttacg ttgtattttc ttacactgtg tcattctgtt agatactaac attttcattg 900atgacgcaag ccatacttaa tgcatatttt ggtttgggta tccatgaacc taccnnnnga 960aaccaagnat tgccggttac ctctgcatgg accagcatta ccctcctctc tccccagatg 1020tgactactga ggcagttctg agtgtttaat ttcagatttt ttcctctgca tttacacaca 1080cacgacacaa accacaccac acacacacac acacacacac acacacacac acacacacca 1140agtaccagta

taagcatctg ccatctgctt ttcccattgc catgcgtcct ggtcaagctc 1200ccctcactct gtttcctggt cagcatgtac tcccctcatc cgattcccct gtagcagtca 1260ctgcacagtt aataaacctt tgcaaacgtt aaaaaaaaaa aaaaaaaaa 130961303PRTHomo sapiensMAD-Pro-42 61Met Lys Gly Lys Glu Glu Lys Glu Gly Gly Ala Arg Leu Gly Ala Gly1 5 10 15Gly Gly Ser Pro Glu Lys Ser Pro Ser Ala Gln Glu Leu Lys Glu Gln20 25 30Gly Asn Arg Leu Phe Val Gly Arg Lys Tyr Pro Glu Ala Ala Ala Cys35 40 45Tyr Gly Arg Ala Ile Thr Arg Asn Pro Leu Val Ala Val Tyr Tyr Thr50 55 60Asn Arg Ala Leu Cys Tyr Leu Lys Met Gln Gln His Glu Gln Ala Leu65 70 75 80Ala Asp Cys Arg Arg Ala Leu Glu Leu Asp Gly Gln Ser Val Lys Ala85 90 95His Phe Phe Leu Gly Gln Cys Gln Leu Glu Met Glu Ser Tyr Asp Glu100 105 110Ala Ile Ala Asn Leu Gln Arg Ala Tyr Ser Leu Ala Lys Glu Gln Arg115 120 125Leu Asn Phe Gly Asp Asp Ile Pro Ser Ala Leu Arg Ile Ala Lys Lys130 135 140Lys Arg Trp Asn Ser Ile Glu Glu Arg Arg Ile His Gln Glu Ser Glu145 150 155 160Leu His Ser Tyr Leu Ser Arg Leu Ile Ala Ala Glu Arg Glu Arg Glu165 170 175Leu Glu Glu Cys Gln Arg Asn His Glu Gly Asp Glu Asp Asp Ser His180 185 190Val Arg Ala Gln Gln Ala Cys Ile Glu Ala Lys His Asp Lys Tyr Met195 200 205Ala Asp Met Asp Glu Leu Phe Ser Gln Val Asp Glu Lys Arg Lys Lys210 215 220Arg Asp Ile Pro Asp Tyr Leu Cys Gly Lys Ile Ser Phe Glu Leu Met225 230 235 240Arg Glu Pro Cys Ile Thr Pro Ser Gly Ile Thr Tyr Asp Arg Lys Asp245 250 255Ile Glu Glu His Leu Gln Arg Val Gly His Phe Asp Pro Val Thr Arg260 265 270Ser Pro Leu Thr Gln Glu Gln Leu Ile Pro Asn Leu Ala Met Lys Glu275 280 285Val Ile Asp Ala Phe Ile Ser Glu Asn Gly Trp Val Glu Asp Tyr290 295 300622166DNAHomo sapiensTransgelin 62cttttggaag tctcttcaga caaacactgg agagaaggca cagcacccta gggtgacatc 60agtggacagg tcagtttcct gttcctgttc ccagccaccc ctgtctgtct gcccagggac 120ctacctgcct ggcccactcc tgctgccacc cctctccatg agtgggactc ctgagcagtg 180ccaggcccag gccctcaggg tggcagttgc tggatggggc caggcttccc attccctggc 240aggcagactc ttggctctgg aagatccctg ggggactctg acttcttggt tactcaggct 300actccgtaag ggtagcctta cgagagcgta agggcatgag gggaagtcga tgggtagacc 360aggtgtctct gatttctctg aggaaatgca gcttctttgc caagaactct tgggctttgg 420tggcttcgtc gcccttgcct ggggtgggcg agttttagtc tcttcagctg cagaccagaa 480gctgagctga cctgaaaaga gctggagttc tggagttcgg gcttccaggc cagggcctgc 540ttctgggtga gagtgggagg taatggttgc ttgaggcaga ggcagtaaac cctcacaccc 600gcagctggac aaaggcacca gctggctgta ttctgtgggg gcaatgtggc ttctggaagg 660tctctccagt ggctctgtag ctggtcttgg caaagaatgt tctagaccaa gggttgtcaa 720actacagctc atgggccaaa tccagcctgc tgtctgcttt tgtaagtaaa gttttattgg 780aacaaagcca tgttcatttg ttacatattg tctatggctg ctttcacact gcaacagcag 840agaccatgtg tcctgcaaag cttaaaatat gttctgtctt atcctttaca aaaacagttt 900actgacccct gctctagacc tccaaagacc tggtatcctc tttcctggtg ttcaagttca 960gagaggtgtc tggagtaggg gctgagcctg gcctggatgg gcagtgctgt gacaagtgtc 1020taggatggcc gggtatcctg cacagagcta gaaggctgcc tggcacgggt gaaagcagag 1080ctgctccctg accctctgcc cctccctcct ccaccctggc ctgctttagc tttccccaga 1140catggccaac aagggtcctt cctatggcat gagccgcgaa gtgcagtcca aaatcgagaa 1200gaagtatgac gaggagctgg aggagcggct ggtggagtgg atcatagtgc agtgtggccc 1260tgatgtgggc cgcccagacc gtgggcgctt gggcttccag gtctggctga agaatggcgt 1320gattctgagc aagctggtga acagcctgta ccctgatggc tccaagccgg tgaaggtgcc 1380cgagaaccca ccctccatgg tcttcaagca gatggagcag gtggctcagt tcctgaaggc 1440ggctgaggac tatggggtca tcaagactga catgttccag actgttgacc tctttgaagg 1500caaagacatg gcagcagtgc agaggaccct gatggctttg ggcagcttgg cagtgaccaa 1560gaatgatggg cactaccgtg gagatcccaa ctggtttatg aagaaagcgc aggagcataa 1620gagggaattc acagagagcc agctgcagga gggaaagcat gtcattggcc ttcagatggg 1680cagcaacaga ggggcctccc aggccggcat gacaggctac ggacgacctc ggcagatcat 1740cagttagagc ggagagggct agccctgagc ccggccctcc cccagctcct tggctgcagc 1800catcccgctt agcctgcctc acccacaccc gtgtggtacc ttcagccctg gccaagcttt 1860gaggctctgt cactgagcaa tggtaactgc acctgggcag ctcctccctg tgcccccagc 1920ctcagcccaa cttcttaccc gaaagcatca ctgccttggc ccctccctcc cggctgcccc 1980catcacctct actgtctcct ccctgggcta agcaggggag aagcgggctg ggggtagcct 2040ggatgtgggc caagtccact gtcctccttg gcggcaaaag cccattgaag aagaaccagc 2100ccagcctgcc ccctatcttg tcctggaata tttttggggt tggaactcaa aaaaaaaaaa 2160aaaaaa 216663939PRTHomo sapiensZCWCC3 63Met Ala Ala Gln Pro Pro Arg Gly Ile Arg Leu Ser Ala Leu Cys Pro1 5 10 15Lys Phe Leu His Thr Asn Ser Thr Ser His Thr Trp Pro Phe Ser Ala20 25 30Val Ala Glu Leu Ile Asp Asn Ala Tyr Asp Pro Asp Val Asn Ala Lys35 40 45Gln Ile Trp Ile Asp Lys Thr Val Ile Asn Asp His Ile Cys Leu Thr50 55 60Phe Thr Asp Asn Gly Asn Gly Met Thr Ser Asp Lys Leu His Lys Met65 70 75 80Leu Ser Phe Gly Phe Ser Asp Lys Val Thr Met Asn Gly His Val Pro85 90 95Val Gly Leu Tyr Gly Asn Gly Phe Lys Ser Gly Ser Met Arg Leu Gly100 105 110Lys Asp Ala Ile Val Phe Thr Lys Asn Gly Glu Ser Met Ser Val Gly115 120 125Leu Leu Ser Gln Thr Tyr Leu Glu Val Ile Lys Ala Glu His Val Val130 135 140Val Pro Ile Val Ala Phe Asn Lys His Arg Gln Met Ile Asn Leu Ala145 150 155 160Glu Ser Lys Ala Ser Leu Ala Ala Ile Leu Glu His Ser Leu Phe Ser165 170 175Thr Glu Gln Lys Leu Leu Ala Glu Leu Asp Ala Ile Ile Gly Lys Lys180 185 190Gly Thr Arg Ile Ile Ile Trp Asn Leu Arg Ser Tyr Lys Asn Ala Thr195 200 205Glu Phe Asp Phe Glu Lys Asp Lys Tyr Asp Ile Arg Ile Pro Glu Asp210 215 220Leu Asp Glu Ile Thr Gly Lys Lys Gly Tyr Lys Lys Gln Glu Arg Met225 230 235 240Asp Gln Ile Ala Pro Glu Ser Asp Tyr Ser Leu Arg Ala Tyr Cys Ser245 250 255Ile Leu Tyr Leu Lys Pro Arg Met Gln Ile Ile Leu Arg Gly Gln Lys260 265 270Val Lys Thr Gln Leu Val Ser Lys Ser Leu Ala Tyr Ile Glu Arg Asp275 280 285Val Tyr Arg Pro Lys Phe Leu Ser Lys Thr Val Arg Ile Thr Phe Gly290 295 300Phe Asn Cys Arg Asn Lys Asp His Tyr Gly Ile Met Met Tyr His Arg305 310 315 320Asn Arg Leu Ile Lys Ala Tyr Glu Lys Val Gly Cys Gln Leu Arg Ala325 330 335Asn Asn Met Gly Val Gly Val Val Gly Ile Ile Glu Cys Asn Phe Leu340 345 350Lys Pro Thr His Asn Lys Gln Asp Phe Asp Tyr Thr Asn Glu Tyr Arg355 360 365Leu Thr Ile Thr Ala Leu Gly Glu Lys Leu Asn Asp Tyr Trp Asn Glu370 375 380Met Lys Val Lys Lys Asn Thr Glu Tyr Pro Leu Asn Leu Pro Val Glu385 390 395 400Asp Ile Gln Lys Arg Pro Asp Gln Thr Trp Val Gln Cys Asp Ala Cys405 410 415Leu Lys Trp Arg Lys Leu Pro Asp Gly Met Asp Gln Leu Pro Glu Lys420 425 430Trp Tyr Cys Ser Asn Asn Pro Asp Pro Gln Phe Arg Asn Cys Glu Val435 440 445Pro Glu Glu Pro Glu Asp Glu Asp Leu Val His Pro Thr Tyr Glu Lys450 455 460Thr Tyr Lys Lys Thr Asn Lys Glu Lys Phe Arg Ile Arg Gln Pro Glu465 470 475 480Met Ile Pro Arg Ile Asn Ala Glu Leu Leu Phe Arg Pro Thr Ala Leu485 490 495Ser Thr Pro Ser Phe Ser Ser Pro Lys Glu Ser Val Pro Arg Arg His500 505 510Leu Ser Glu Gly Thr Asn Ser Tyr Ala Thr Arg Leu Leu Asn Asn His515 520 525Gln Val Pro Pro Gln Ser Glu Pro Glu Ser Asn Ser Leu Lys Arg Arg530 535 540Leu Ser Thr Arg Ser Ser Ile Leu Asn Ala Lys Asn Arg Arg Leu Ser545 550 555 560Ser Gln Phe Glu Asn Ser Val Tyr Lys Gly Asp Asp Asp Asp Glu Asp565 570 575Val Ile Ile Leu Glu Glu Asn Ser Thr Pro Lys Pro Ala Val Asp His580 585 590Asp Ile Asp Met Lys Ser Glu Gln Ser His Val Glu Gln Gly Gly Val595 600 605Gln Val Glu Phe Val Gly Asp Ser Glu Pro Cys Gly Gln Thr Gly Ser610 615 620Thr Ser Thr Ser Ser Ser Arg Cys Asp Gln Gly Asn Thr Ala Ala Thr625 630 635 640Gln Thr Glu Val Pro Ser Leu Val Val Lys Lys Glu Glu Thr Val Glu645 650 655Asp Glu Ile Asp Val Arg Asn Asp Ala Val Ile Leu Pro Ser Cys Val660 665 670Glu Ala Glu Ala Lys Ile His Glu Thr Gln Glu Thr Thr Asp Lys Ser675 680 685Ala Asp Asp Ala Gly Cys Gln Leu Gln Glu Leu Arg Asn Gln Leu Leu690 695 700Leu Val Thr Glu Glu Lys Glu Asn Tyr Lys Arg Gln Cys His Met Phe705 710 715 720Thr Asp Gln Ile Lys Val Leu Gln Gln Arg Ile Leu Glu Met Asn Asp725 730 735Lys Tyr Val Lys Lys Glu Thr Cys His Gln Ser Thr Glu Thr Asp Ala740 745 750Val Phe Leu Leu Glu Ser Ile Asn Gly Lys Ser Glu Ser Pro Asp His755 760 765Met Val Ser Gln Tyr Gln Gln Ala Leu Glu Glu Ile Glu Arg Leu Lys770 775 780Lys Gln Cys Ser Ala Leu Gln His Val Lys Ala Glu Cys Ser Gln Cys785 790 795 800Ser Asn Asn Glu Ser Lys Ser Glu Met Asp Glu Met Ala Val Gln Leu805 810 815Asp Asp Val Phe Arg Gln Leu Asp Lys Cys Ser Ile Glu Arg Asp Gln820 825 830Tyr Lys Ser Glu Val Glu Leu Leu Glu Met Glu Lys Ser Gln Ile Arg835 840 845Ser Gln Cys Glu Glu Leu Lys Thr Glu Val Glu Gln Leu Lys Ser Thr850 855 860Asn Gln Gln Thr Ala Thr Asp Val Ser Thr Ser Ser Asn Ile Glu Glu865 870 875 880Ser Val Asn His Met Asp Gly Glu Ser Leu Lys Leu Arg Ser Leu Arg885 890 895Val Asn Val Gly Gln Leu Leu Ala Met Ile Val Pro Asp Leu Asp Leu900 905 910Gln Gln Val Asn Tyr Asp Val Asp Val Val Asp Glu Ile Leu Gly Gln915 920 925Val Val Glu Gln Met Ser Glu Ile Ser Ser Thr930 93564424PRTHomo sapiensACAA1 64Met Gln Arg Leu Gln Val Val Leu Gly His Leu Arg Gly Pro Ala Asp1 5 10 15Ser Gly Trp Met Pro Gln Ala Ala Pro Cys Leu Ser Gly Ala Pro Gln20 25 30Ala Ser Ala Ala Asp Val Val Val Val His Gly Arg Arg Thr Ala Ile35 40 45Cys Arg Ala Gly Arg Gly Gly Phe Lys Asp Thr Thr Pro Asp Glu Leu50 55 60Leu Ser Ala Val Met Thr Ala Val Leu Lys Asp Val Asn Leu Arg Pro65 70 75 80Glu Gln Leu Gly Asp Ile Cys Val Gly Asn Val Leu Gln Pro Gly Ala85 90 95Gly Ala Ile Met Ala Arg Ile Ala Gln Phe Leu Ser Asp Ile Pro Glu100 105 110Thr Val Pro Leu Ser Thr Val Asn Arg Gln Cys Ser Ser Gly Leu Gln115 120 125Ala Val Ala Ser Ile Ala Gly Gly Ile Arg Asn Gly Ser Tyr Asp Ile130 135 140Gly Met Ala Cys Gly Val Glu Ser Met Ser Leu Ala Asp Arg Gly Asn145 150 155 160Pro Gly Asn Ile Thr Ser Arg Leu Met Glu Lys Glu Lys Ala Arg Asp165 170 175Cys Leu Ile Pro Met Gly Ile Thr Ser Glu Asn Val Ala Glu Arg Phe180 185 190Gly Ile Ser Arg Glu Lys Gln Asp Thr Phe Ala Leu Ala Ser Gln Gln195 200 205Lys Ala Ala Arg Ala Gln Ser Lys Gly Cys Phe Gln Ala Glu Ile Val210 215 220Pro Val Thr Thr Thr Val His Asp Asp Lys Gly Thr Lys Arg Ser Ile225 230 235 240Thr Val Thr Gln Asp Glu Gly Ile Arg Pro Ser Thr Thr Met Glu Gly245 250 255Leu Ala Lys Leu Lys Pro Ala Phe Lys Lys Asp Gly Ser Thr Thr Ala260 265 270Gly Asn Ser Ser Gln Val Ser Asp Gly Ala Ala Ala Ile Leu Leu Ala275 280 285Arg Arg Ser Lys Ala Glu Glu Leu Gly Leu Pro Ile Leu Gly Val Leu290 295 300Arg Ser Tyr Ala Val Val Gly Val Pro Pro Asp Ile Met Gly Ile Gly305 310 315 320Pro Ala Tyr Ala Ile Pro Val Ala Leu Gln Lys Ala Gly Leu Thr Val325 330 335Ser Asp Val Asp Ile Phe Glu Ile Asn Glu Ala Phe Ala Ser Gln Ala340 345 350Ala Tyr Cys Val Glu Lys Leu Arg Leu Pro Pro Glu Lys Val Asn Pro355 360 365Leu Gly Gly Ala Val Ala Leu Gly His Pro Leu Gly Cys Thr Gly Ala370 375 380Arg Gln Val Ile Thr Leu Leu Asn Glu Leu Lys Arg Arg Gly Lys Arg385 390 395 400Ala Tyr Gly Val Val Ser Met Cys Ile Gly Thr Gly Met Gly Ala Ala405 410 415Ala Val Phe Glu Tyr Pro Gly Asn42065884PRTHomo sapiensActinin 65Met Gly Asp Tyr Met Ala Gln Glu Asp Asp Trp Asp Arg Asp Leu Leu1 5 10 15Leu Asp Pro Ala Trp Glu Lys Gln Gln Arg Lys Thr Phe Thr Ala Trp20 25 30Cys Asn Ser His Leu Arg Lys Ala Gly Thr Gln Ile Glu Asn Ile Asp35 40 45Glu Asp Phe Arg Asp Gly Leu Lys Leu Met Leu Leu Leu Glu Val Ile50 55 60Ser Gly Glu Arg Leu Pro Lys Pro Glu Arg Gly Lys Met Arg Val His65 70 75 80Lys Ile Asn Asn Val Asn Lys Ala Leu Asp Phe Ile Ala Ser Lys Gly85 90 95Val Lys Leu Val Ser Ile Gly Ala Glu Glu Ile Val Asp Gly Asn Ala100 105 110Lys Met Thr Leu Gly Met Ile Trp Thr Ile Ile Leu Arg Phe Ala Ile115 120 125Gln Asp Ile Ser Val Glu Glu Thr Ser Ala Lys Glu Gly Leu Leu Leu130 135 140Trp Cys Gln Arg Lys Thr Ala Pro Tyr Lys Asn Val Asn Val Gln Asn145 150 155 160Phe His Ile Ser Trp Lys Asp Gly Leu Ala Phe Asn Ala Leu Ile His165 170 175Arg His Arg Pro Glu Leu Ile Glu Tyr Asp Lys Leu Arg Lys Asp Asp180 185 190Pro Val Thr Asn Leu Asn Asn Ala Phe Glu Val Ala Glu Lys Tyr Leu195 200 205Asp Ile Pro Lys Met Leu Asp Ala Glu Asp Ile Val Asn Thr Ala Arg210 215 220Pro Asp Glu Lys Ala Ile Met Thr Tyr Val Ser Ser Phe Tyr His Ala225 230 235 240Phe Ser Gly Ala Gln Lys Ala Glu Thr Ala Ala Asn Arg Ile Cys Lys245 250 255Val Leu Ala Val Asn Gln Glu Asn Glu His Leu Met Glu Asp Tyr Glu260 265 270Lys Leu Ala Ser Asp Leu Leu Glu Trp Ile Arg Arg Thr Ile Pro Trp275 280 285Leu Glu Asp Arg Val Pro Gln Lys Thr Ile Gln Glu Met Gln Gln Lys290 295 300Leu Glu Asp Phe Arg Asp Tyr Arg Arg Val His Lys Pro Pro Lys Val305 310 315 320Gln Glu Lys Cys Gln Leu Glu Ile Asn Phe Asn Thr Leu Gln Thr Lys325 330 335Leu Arg Leu Ser Asn Arg Pro Ala Phe Met Pro Ser Glu Gly Lys Met340 345 350Val Ser Asp Ile Asn Asn Gly Trp Gln His Leu Glu Gln Ala Glu Lys355 360 365Gly Tyr Glu Glu Trp Leu Leu Asn Glu Ile Arg Arg Leu Glu Arg Leu370 375 380Asp His Leu Ala Glu Lys Phe Arg Gln Lys Ala Ser Ile His Glu Ala385 390 395 400Trp Thr Asp Gly Lys Glu Ala Met Leu Lys His Arg Asp Tyr Glu Thr405 410 415Ala Thr Leu Ser Asp Ile Lys Ala Leu Ile Arg Lys His Glu Ala Phe420 425 430Glu Ser Asp Leu Ala Ala His Gln Asp Arg Val Glu Gln Ile Ala Ala435 440 445Ile Ala Gln Glu Leu Asn Glu Leu Asp Tyr Tyr Asp Ser His Asn Val450 455 460Asn Thr Arg Cys Gln Lys Ile Cys Asp Gln Trp Asp Ala Leu Gly Ser465 470 475 480Leu Thr His Ser Arg Arg Glu Ala Leu Glu Lys Thr Glu Lys Gln Leu485 490 495Glu Ala Ile Asp Gln Leu His Leu Glu Tyr Ala Lys Arg Ala Ala Pro500 505 510Phe Asn Asn Trp Met Glu Ser Ala Met Glu Asp Leu Gln Asp Met Phe515 520 525Ile Val His Thr Ile Glu Glu Ile Glu Gly Leu Ile Ser Ala His Asp530 535 540Gln Phe Lys Ser Thr Leu Pro Asp Ala Asp Arg Glu Arg Glu Ala Ile545

550 555 560Leu Ala Ile His Lys Glu Ala Gln Arg Ile Ala Glu Ser Asn His Ile565 570 575Lys Leu Ser Gly Ser Asn Pro Tyr Thr Thr Val Thr Pro Gln Ile Ile580 585 590Asn Ser Lys Trp Glu Lys Val Gln Gln Leu Val Pro Lys Arg Asp His595 600 605Ala Leu Leu Glu Glu Gln Ser Lys Gln Gln Ser Asn Glu His Leu Arg610 615 620Arg Gln Phe Ala Ser Gln Ala Asn Val Val Gly Pro Trp Ile Gln Thr625 630 635 640Lys Met Glu Glu Ile Gly Arg Ile Ser Ile Glu Met Asn Gly Thr Leu645 650 655Glu Asp Gln Leu Ser His Leu Lys Gln Tyr Glu Arg Ser Ile Val Asp660 665 670Tyr Lys Pro Asn Leu Asp Leu Leu Glu Gln Gln His Gln Leu Ile Gln675 680 685Glu Ala Leu Ile Phe Asp Asn Lys His Thr Asn Tyr Thr Met Glu His690 695 700Ile Arg Val Gly Trp Glu Gln Leu Leu Thr Thr Ile Ala Arg Thr Ile705 710 715 720Asn Glu Val Glu Asn Gln Ile Leu Thr Arg Asp Ala Lys Gly Ile Ser725 730 735Gln Glu Gln Met Gln Glu Phe Arg Ala Ser Phe Asn His Phe Asp Lys740 745 750Asp His Gly Gly Ala Leu Gly Pro Glu Glu Phe Lys Ala Cys Leu Ile755 760 765Ser Leu Gly Tyr Asp Val Glu Asn Asp Arg Gln Gly Glu Ala Glu Phe770 775 780Asn Arg Ile Met Ser Leu Val Asp Pro Asn His Ser Gly Leu Val Thr785 790 795 800Phe Gln Ala Phe Ile Asp Phe Met Ser Arg Glu Thr Thr Asp Thr Asp805 810 815Thr Ala Asp Gln Val Ile Ala Ser Phe Lys Val Leu Ala Gly Asp Lys820 825 830Asn Phe Ile Thr Ala Glu Glu Leu Arg Arg Glu Leu Pro Pro Asp Gln835 840 845Ala Glu Tyr Cys Ile Ala Arg Met Ala Pro Tyr Gln Gly Pro Asp Ala850 855 860Val Pro Gly Ala Leu Asp Tyr Lys Ser Phe Ser Thr Ala Leu Tyr Gly865 870 875 880Glu Ser Asp Leu661024PRTHomo sapiensNFX2 66Met Ala Glu Ala Pro Pro Val Ser Gly Thr Phe Lys Phe Asn Thr Asp1 5 10 15Ala Ala Glu Phe Ile Pro Gln Glu Lys Lys Asn Ser Gly Leu Asn Cys20 25 30Gly Thr Gln Arg Arg Leu Asp Ser Asn Arg Ile Gly Arg Arg Asn Tyr35 40 45Ser Ser Pro Pro Pro Cys His Leu Ser Arg Gln Val Pro Tyr Asp Glu50 55 60Ile Ser Ala Val His Gln His Ser Tyr His Pro Ser Gly Ser Lys Pro65 70 75 80Lys Ser Gln Gln Thr Ser Phe Gln Ser Ser Pro Cys Asn Lys Ser Pro85 90 95Lys Ser His Gly Leu Gln Asn Gln Pro Trp Gln Lys Leu Arg Asn Glu100 105 110Lys His His Ile Arg Val Lys Lys Ala Gln Ser Leu Ala Glu Gln Thr115 120 125Ser Asp Thr Ala Gly Leu Glu Ser Ser Thr Arg Ser Glu Ser Gly Thr130 135 140Asp Leu Arg Glu His Ser Pro Ser Glu Ser Glu Lys Glu Val Val Gly145 150 155 160Ala Asp Pro Arg Gly Ala Lys Pro Lys Lys Ala Thr Gln Phe Val Tyr165 170 175Ser Tyr Gly Arg Gly Pro Lys Val Lys Gly Lys Leu Lys Cys Glu Trp180 185 190Ser Asn Arg Thr Thr Pro Lys Pro Glu Asp Ala Gly Pro Glu Ser Thr195 200 205Lys Pro Val Gly Val Phe His Pro Asp Ser Ser Glu Ala Ser Ser Arg210 215 220Lys Gly Val Leu Asp Gly Tyr Gly Ala Arg Arg Asn Glu Gln Arg Arg225 230 235 240Tyr Pro Gln Lys Arg Pro Pro Trp Glu Val Glu Gly Ala Arg Pro Arg245 250 255Pro Gly Arg Asn Pro Pro Lys Gln Glu Gly His Arg His Thr Asn Ala260 265 270Gly His Arg Asn Asn Met Gly Pro Ile Pro Lys Asp Asp Leu Asn Glu275 280 285Arg Pro Ala Lys Ser Thr Cys Asp Ser Glu Asn Leu Ala Val Ile Asn290 295 300Lys Ser Ser Arg Arg Val Asp Gln Glu Lys Cys Thr Val Arg Arg Gln305 310 315 320Asp Pro Gln Val Val Ser Pro Phe Ser Arg Gly Lys Gln Asn His Val325 330 335Leu Lys Asn Val Glu Thr His Thr Gly Ser Leu Ile Glu Gln Leu Thr340 345 350Thr Glu Lys Tyr Glu Cys Met Val Cys Cys Glu Leu Val Arg Val Thr355 360 365Ala Pro Val Trp Ser Cys Gln Ser Cys Tyr His Val Phe His Leu Asn370 375 380Cys Ile Lys Lys Trp Ala Arg Ser Pro Ala Ser Gln Ala Asp Gly Gln385 390 395 400Ser Gly Trp Arg Cys Pro Ala Cys Gln Asn Val Ser Ala His Val Pro405 410 415Asn Thr Tyr Thr Cys Phe Cys Gly Lys Val Lys Asn Pro Glu Trp Ser420 425 430Arg Asn Glu Ile Pro His Ser Cys Gly Glu Val Cys Arg Lys Lys Gln435 440 445Pro Gly Gln Asp Cys Pro His Ser Cys Asn Leu Leu Cys His Pro Gly450 455 460Pro Cys Pro Pro Cys Pro Ala Phe Met Thr Lys Thr Cys Glu Cys Gly465 470 475 480Arg Thr Arg His Thr Val Arg Cys Gly Gln Ala Val Ser Val His Cys485 490 495Ser Asn Pro Cys Glu Asn Ile Leu Asn Cys Gly Gln His Gln Cys Ala500 505 510Glu Leu Cys His Gly Gly Gln Cys Gln Pro Cys Gln Ile Ile Leu Asn515 520 525Gln Val Cys Tyr Cys Gly Ser Thr Ser Arg Asp Val Leu Cys Gly Thr530 535 540Asp Val Gly Lys Ser Asp Gly Phe Gly Asp Phe Ser Cys Leu Lys Ile545 550 555 560Cys Gly Lys Asp Leu Lys Cys Gly Asn His Thr Cys Ser Gln Val Cys565 570 575His Pro Gln Pro Cys Gln Gln Cys Pro Arg Leu Pro Gln Leu Val Arg580 585 590Cys Cys Pro Cys Gly Gln Thr Pro Leu Ser Gln Leu Leu Glu Leu Gly595 600 605Ser Ser Ser Arg Lys Thr Cys Met Asp Pro Val Pro Ser Cys Gly Lys610 615 620Val Cys Gly Lys Pro Leu Pro Cys Gly Ser Leu Asp Phe Ile His Thr625 630 635 640Cys Glu Lys Leu Cys His Glu Gly Asp Cys Gly Pro Cys Ser Arg Thr645 650 655Ser Val Ile Ser Cys Arg Cys Ser Phe Arg Thr Lys Glu Leu Pro Cys660 665 670Thr Ser Leu Lys Ser Glu Asp Ala Thr Phe Met Cys Asp Lys Arg Cys675 680 685Asn Lys Lys Arg Leu Cys Gly Arg His Lys Cys Asn Glu Ile Cys Cys690 695 700Val Asp Lys Glu His Lys Cys Pro Leu Ile Cys Gly Arg Lys Leu Arg705 710 715 720Cys Gly Leu His Arg Cys Glu Glu Pro Cys His Arg Gly Asn Cys Gln725 730 735Thr Cys Trp Gln Ala Ser Phe Asp Glu Leu Thr Cys His Cys Gly Ala740 745 750Ser Val Ile Tyr Pro Pro Val Pro Cys Gly Thr Arg Pro Pro Glu Cys755 760 765Thr Gln Thr Cys Ala Arg Val His Glu Cys Asp His Pro Val Tyr His770 775 780Ser Cys His Ser Glu Glu Lys Cys Pro Pro Cys Thr Phe Leu Thr Gln785 790 795 800Lys Trp Cys Met Gly Lys His Glu Phe Arg Ser Asn Ile Pro Cys His805 810 815Leu Val Asp Ile Ser Cys Gly Leu Pro Cys Ser Ala Thr Leu Pro Cys820 825 830Gly Met His Lys Cys Gln Arg Leu Cys His Lys Gly Glu Cys Leu Val835 840 845Asp Glu Pro Cys Lys Gln Pro Cys Thr Thr Pro Arg Ala Asp Cys Gly850 855 860His Pro Cys Met Ala Pro Cys His Thr Ser Ser Pro Cys Pro Val Thr865 870 875 880Ala Cys Lys Ala Lys Val Glu Leu Gln Cys Glu Cys Gly Arg Arg Lys885 890 895Glu Met Val Ile Cys Ser Glu Ala Ser Ser Thr Tyr Gln Arg Ile Ala900 905 910Ala Ile Ser Met Ala Ser Lys Ile Thr Asp Met Gln Leu Gly Gly Ser915 920 925Val Glu Ile Ser Lys Leu Ile Thr Lys Lys Glu Val His Gln Ala Arg930 935 940Leu Glu Cys Asp Glu Glu Cys Ser Ala Leu Glu Arg Lys Lys Arg Leu945 950 955 960Ala Glu Ala Phe His Ile Ser Glu Asp Ser Asp Pro Phe Asn Ile Arg965 970 975Ser Ser Gly Ser Lys Phe Ser Asp Ser Leu Lys Glu Asp Ala Arg Lys980 985 990Asp Leu Lys Phe Val Ser Asp Val Glu Lys Glu Met Glu Thr Leu Val995 1000 1005Glu Ala Val Asn Lys Val Glu Val Glu Thr Ser His Trp Thr Phe1010 1015 1020Leu671799DNAHomo sapiensMAD-CT-2 67agtcacatag ctgcgtggta cgtgactgga ggtgtatcct tgtcctcgtc tgaatcacgc 60tgatgtggcc ccaaccccac ctccctcccc accccatgat gtcagaaaaa accagacaga 120acaaattggc tgaggccaag aaaaagttta cagactatcg tcagtggaac attgctggtg 180ttggtaccgg agcaactgac accaaaaaga agaaaataaa tcatggcact aaccctgaga 240caaccacttc ggggggctgc cactcgcctg aggatacaca acagaaccga gcgcagctga 300aagaagaaaa gaagatgcgg gagcaggaag atgtggagac aggaggagag gctgcaggag 360caggagaagc agatgtggga gcaggaggag aagatgcggg atcaggagca gaagatgtgg 420gaccaggagg agaggatgtg ggagcaggac gagaggctgc ggcagaagga ggagagaatg 480cgggagcaga agatgtggca gcaggtggag aagatgcggg aggagaagaa gacgcaggag 540caggagaaga agacatggga ccaggagaag atgcgagagg aggagagcat gcgggagcgg 600gagaagaaga tgcgggagga ggaggagatg atgcgggagc aggaggagaa gatgcaggag 660caggaagaaa agatgcagga gcaggaggag gagatgtggg agcaggagga gaagatgtgg 720gagcaggaag agaagatgtg ggagcagcag aggctaccgg aacagaagga gaggctgtgg 780gaacacgaga agatgcagga gcaggagaag atatgggagc aggaggagaa gatgcgggac 840caggaggaga agatgcgggg ccaggaggag aagatgcggg ggcaggagga gaagatgcgg 900gggcaggagg agaagatgtg ggggcaggag gagaagatgt gggggcagga ggagaagatg 960tggggccagg aggagaagat gtgggggcag gaggagaaga tgtggggcca ggaggagaag 1020atgcgggggg caggaggaga agatgcgggg gcaggaggag aagatgcggg gccaggagga 1080gaagatgcgg gggcaggagg aggagatgcg gggggcagga ggagaagatg cggggggcag 1140gaggagaaga tgcgggggac aggaggagaa gatgcggggg ccaggaggag aagatgcggg 1200agcaggagga gaagatgcgg gagcaggagg agaagatgca gggccaggag gagaagatgc 1260gggagcagga ggagaagatg cggggccagg aggagaagat gcgggagcag gaggagaaga 1320tgcggggcca ggaggagaag atgtggggcc aggaggagaa gatgtggggg caggaggaga 1380agatgtgggg gcaggaggag atgatgcgag agaaggagga gaggatatga gatcagaaag 1440agaagatgca ggagaggctg ccagagcacg aggagcggtg ctcagagccc tgcctccctc 1500cctccaaagt tctttgtaat atgagccaca ctggcagtgt ggagcctgca ggaggagagg 1560ctggggaggg ttctccgcag gacaacccca ctgcacagga gatcatgcag ctgttttgtg 1620ggatgaagaa cgcccagcag tgcccaggat taggcagtac ctcctgcatc ccattcttct 1680accgaggaga caagagaaag atgaagatca tcaatatcta aaagttggca ctgtcaacaa 1740ggcctacaga agcataagcc gccatgtcac tgtgtgaata tagtctgagc acaaacttg 179968102PRTHomo sapiensMAD-CT-1 68Met Val Arg Tyr Arg Val Arg Ser Leu Ser Glu Arg Ser His Glu Val1 5 10 15Tyr Arg Gln Gln Leu His Gly Gln Glu Gln Gly His His Gly Gln Glu20 25 30Glu Gln Gly Leu Ser Pro Glu His Val Glu Val Tyr Glu Arg Thr His35 40 45Gly Gln Ser His Tyr Arg Arg Arg His Cys Ser Arg Arg Arg Leu His50 55 60Arg Ile His Arg Arg Gln His Arg Ser Cys Arg Arg Arg Lys Arg Arg65 70 75 80Ser Cys Arg His Arg Arg Arg His Arg Arg Gly Cys Arg Thr Arg Lys85 90 95Arg Thr Cys Arg Arg His10069146PRTHomo sapiensPAGE-1 69Met Gly Phe Leu Arg Arg Leu Ile Tyr Arg Arg Arg Pro Met Ile Tyr1 5 10 15Val Glu Ser Ser Glu Glu Ser Ser Asp Glu Gln Pro Asp Glu Val Glu20 25 30Ser Pro Thr Gln Ser Gln Asp Ser Thr Pro Ala Glu Glu Arg Glu Asp35 40 45Glu Gly Ala Ser Ala Ala Gln Gly Gln Glu Pro Glu Ala Asp Ser Gln50 55 60Glu Leu Val Gln Pro Lys Thr Gly Cys Glu Pro Gly Asp Gly Pro Asp65 70 75 80Thr Lys Arg Val Cys Leu Arg Asn Glu Glu Gln Met Lys Leu Pro Ala85 90 95Glu Gly Pro Glu Pro Glu Ala Asp Ser Gln Glu Gln Val His Pro Lys100 105 110Thr Gly Cys Glu Arg Gly Asp Gly Pro Asp Val Gln Glu Leu Gly Leu115 120 125Pro Asn Pro Glu Glu Val Lys Thr Pro Glu Glu Asp Glu Gly Gln Ser130 135 140Gln Pro14570180PRTHomo sapiensNY-ESO-1 70Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp1 5 10 15Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly20 25 30Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala35 40 45Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro50 55 60His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala65 70 75 80Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe85 90 95Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp100 105 110Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val115 120 125Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln130 135 140Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met145 150 155 160Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser165 170 175Gly Gln Arg Arg180

Claims

1. A method for identifying a human subject as a candidate for further prostate cancer examination comprising the step of: determining whether the human subject has developed an immune reaction to a prostate cancer antigen selected from SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, MAD-Pro-30 (SEQ ID NO: 58), MAD-Pro-42 (SEQ ID NO: 61), transgelin encoded by SEQ ID NO: 62 or a conservatively modified variant thereof, ZCWCC3 (SEQ ID NO: 63), ACAA1 (SEQ ID NO: 64), actinin (SEQ ID NO: 65), and NFX2 (SEQ ID NO: 66) wherein the presence of an immune reaction indicates that the human subject is a candidate for further prostate cancer examination.

2. The method of claim 1, wherein the antigen is selected from SSX-2 encoded by a nucleic acid comprising SEQ ID NO: 60 or a conservatively modified variant thereof, MAD-Pro-30 (SEQ ID NO: 58), MAD-Pro-42 (SEQ ID NO: 61), transgelin (SEQ ID NO: 62), ZCWCC3 (SEQ ID NO: 63), and ACAA1 (SEQ ID NO: 64).

3. The method of claim 1, wherein the antigen is MAD-Pro-30 (SEQ ID NO: 58).

4. The method of claim 3, wherein whether the human subject developed an immune reaction to antigens in an antigen panel is determined, and wherein the panel comprises MAD-Pro-30 (SEQ ID NO: 58), SSX-2 encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 60 or a conservatively modified variant thereof, androgen receptor (AR) ligand binding domain (SEQ ID NO: 59), and MAD-Pro-22 (PSA) (SEQ ID NO: 57).

5. The method of claim 1, wherein the development of an immune reaction is determined by testing whether a blood sample from the human subject contains an antibody to an antigen.

6. A method for determining whether an immune therapy has elicited a tumor-specific immune response in a prostate cancer patient, the method comprising the steps of: providing an immune therapy to a prostate cancer patient; and determining whether the patient developed an immune reaction to a prostate cancer antigen selected from SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, MAD-Pro-30 (SEQ ID NO: 58), MAD-Pro-42 (SEQ ID NO: 61), transgelin encoded by the nucleotide sequence of SEQ ID NO: 62 or a conservatively modified variant thereof, ZCWCC3(SEQ ID NO: 63), ACAA1 (SEQ ID NO: 64), actinin (SEQ ID NO: 65), and NFX2 (SEQ NO: 66), wherein the presence of an immune reaction indicates that the therapy has elicited a tumor-specific immune response.

7. The method of claim 6, wherein the antigen is selected from SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, MAD-Pro-30 (SEQ ID NO: 58), MAD-Pro-42 (SEQ ID NO: 61), transgelin encoded by SEQ ID NO: 62 or a conservatively modified variant thereof, ZCWCC3 (SEQ ID NO: 63), and ACAA1 (SEQ ID NO: 64).

8. The method of claim 6, wherein the antigen is MAD-Pro-30 (SEQ ID NO: 58).

9. The method of claim 8, wherein whether the prostate cancer patient developed an immune reaction to antigens in an antigen panel is determined, and wherein the panel comprises MAD-Pro-30 (SEQ ID NO: 58), SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, androgen receptor (AR) ligand binding domain (SEQ ID NO: 59), and MAD-Pro-22 (PSA) (SEQ ID NO: 57).

10. The method of claim 6, wherein the immune therapy is a non-antigen-specific immune therapy.

11. The method of claim 6, wherein the development of an immune reaction is determined by testing whether a blood sample from the prostate cancer patient contains an antibody to an antigen.

12. (canceled)

13. A method for identifying a human subject as a candidate for further melanoma examination comprising the step of: determining whether the human subject has developed an immune reaction to a melanoma antigen selected from MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof, MAD-CT-1 (SEQ ID NO: 68), and PAGE-1 (SEQ ID NO: 69), wherein the presence of an immune reaction indicates that the human subject is a candidate for further melanoma examination.

14. The method of claim 13, wherein the antigen is MAD-CT-2.

15. The method of claim 14, wherein whether the human subject developed an immune reaction to antigens in an antigen panel is determined, and wherein the panel comprises MAD-CT-1 (SEQ ID NO: 68), PAGE-1 (SEQ ID NO: 69), SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, and NY-ESO-1 (SEQ ID NO: 70).

16. The method of claim 13, wherein the development of an immune reaction is determined by testing whether a blood sample from the human subject contains an antibody to an antigen.

17. A method for determining whether an immune therapy has elicited a tumor-specific immune response in a melanoma patient, the method comprising the steps of: providing an immune therapy to a melanoma patient; and determining whether the patient developed an immune reaction to a melanoma antigen selected from MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof, MAD-CT-1 (SEQ ID NO: 68), and PAGE-1 (SEQ ID NO: 69), wherein the presence of an immune reaction indicates that the therapy has elicited a tumor-specific immune response.

18. The method of claim 17, wherein the antigen is MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof.

19. The method of claim 18, wherein whether the melanoma patient developed an immune reaction to antigens in an antigen panel is determined, and wherein the panel comprises MAD-CT-1 (SEQ ID NO: 68), PAGE-1 (SEQ ID NO: 69), SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof, and NY-ESO-1 (SEQ ID NO: 70).

20. The method of claim 17, wherein the immune therapy is a non-antigen-specific immune therapy.

21. The method of claim 17, wherein the development of an immune reaction is determined by testing whether a blood sample from the melanoma patient contains an antibody to an antigen.

22. A method for identifying a human subject as a candidate for further melanoma examination comprising the step of: determining whether the cells in a region of the subject's skin suspected of being malignant express MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof, wherein the expression of MAD-CT-2 indicates that the subject is a candidate for further melanoma examination.

23. A method for identifying candidate compounds for further testing as preventive or therapeutic agents for melanoma, the method comprising the steps of: providing cells that express MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof; exposing the cells to a test compound; determining the expression of level of MAD-CT-2 in the exposed cells; and comparing the expression level in the exposed cells to that of corresponding control cells that are not exposed to the test compound, wherein a lower expression level than that in the control cells indicates that the compound is a candidate for further testing as a preventive or therapeutic agent for melanoma.

24. A kit comprising: a first polypeptide that comprises MAD-CT-2 encoded by SEQ ID NO: 67 or a conservatively modified variant thereof; a second polypeptide that comprises SSX-2 encoded by SEQ ID NO: 60 or a conservatively modified variant thereof; and a third polypeptide that comprises NY-ESO-1 (SEQ ID NO: 70).