US0079 19467B2

(12) United States Patent (10) Patent No.: US 7,919,467 B2 Ramakrishna et al. (45) Date of Patent: Apr. 5, 2011

(54) CYTOTOXICT-LYMPHOCYTE-INDUCING OTHER PUBLICATIONS IMMUNOGENS FOR PREVENTION, Smith et al. (Nature Biotechnology 15:1222-1223 (1997)).* TREATMENT, AND DIAGNOSIS OF CANCER Brenner (Trends in Genetics 15:132-133 (1999)).* Voskoglou-Nomikos (Clin. Can. Res. 9:4227-4239 (2003)).* (75) Inventors: Venky Ramakrishna, Riegelsville, PA Dennis (Nature 442:739-741 (2006)).* (US); Mark M. Ross, Charlottesville, Cespcles et al. (Clin. Transl. Oncol. 8(5):318-329 (2006)).* VA (US); Ramila Philip, Ivyland, PA Talmadge et al. (Am. J. Pathol 170(3):793-804 (2007)).* (US); Lorraine H. Keller, Pipersville, clinicaltrials.gov search (ImmunoVaccine Technologies, Inc., pp. PA (US) 1-3; Jan. 29, 2011).* Adachiet al., 1992, Nucleic Acids Research, vol. 20, pp. 5297-5303. (73) Assignee: Immunotope, Inc., Doylestown, PA Boon, T. et al., Ann. Rev. Immunol. 12:337-365 (1994). Celluzzi, C. M. et al., J. Exp. Med., 183:283-287 (1996). (US) European Search Report, dated Jan. 26, 2005. Gilboa, E., Immunity, 11:263-270 (1999). (*) Notice: Subject to any disclaimer, the term of this Gluzman, Cell, 23:175 (1981). patent is extended or adjusted under 35 Harding, C. H. III, Eur, J. Immunol. 22:1865-1869 (1992). U.S.C. 154(b) by 560 days. Henderson R. A. etal, DirectIdentification of an Endogenous Peptide Reognized by Multiple HLA-A2.1 Specific Cytotoxic T Cells, (21) Appl. No.: 11/426,161 National Academy of Sciences, vol. 90, pp. 10275-10279 (Nov. 1993). (22) Filed: Jun. 23, 2006 Hogan, K. T. et al., The peptide Recognized by HLAA68.2-Re stricted, Squamous Cell Carcinoma of the Lung-Specific Cytotoxic T (65) Prior Publication Data Lymphocytes is Derviced froma Mutated Elongation Factor 2 Gene, American Association for Cancer Research, vol. 58, No. 22, pp. US 2008/O2O7497 A1 Aug. 28, 2008 5144-5150 (Nov. 15, 1998). Hunt, D. F. et al., Proc. Natl. Acad. Sci. USA, 83:6233-6237 (1986). Related U.S. Application Data Hunt, D. F. et al., Science, 255:1261-1263 (1992). Jacob, L. et al., Int. J. Cancer, 71:325-332 (1997). (63) Continuation-in-part of application No. 10/006, 177, Joslyn G. et al., Identification of Deletion Mutations and Three New filed on Dec. 4, 2001, now Pat. No. 7,083,789. Genes at the Familial Polyposis Locus, Cell, vol. 66, No. 3, pp. (60) Provisional application No. 60/251,022, filed on Dec. 604-614 (1991). Kabat et al., J. Biol. Chem. 252:6609-6616 (1977). 4, 2000, provisional application No. 60/256,824, filed Kinzler, K.W. et al., Identification of FAP Locus Genes from Chro on Dec. 20, 2000. mosome 5.Q21, Science, vol. 253, No. 5020, pp. 661-665 (1991). Lauritzsen et al (International Journal of Cancer, 1998, vol. 78, pp. (51) Int. C. 216-222. A6 IK38/6 (2006.01) Ljunggren, H. G. et al., Nature, 346:476-480 (1990). A6IP35/00 (2006.01) Loannides, C. G. et al., J. Immunol. 146:1700-1707 (1991). (52) U.S. Cl...... S14/19.3 Mayordome, J. et al., Nat. Med., 1:1297-1302 (1995). (58) Field of Classification Search ...... 424/185.1 Moore, J. W. et al., Cell, 54.777-785 (1988). See application file for complete search history. North, R. J. et al., Infect. Immun., 67:2010-2012 (1999). Parkhurst, M. R. et al., J. Immunol. 157:2539-2548 (1996). Peiper, M. et al., Eur, J. Immunol. 27: 1115-1123 (1997). (56) References Cited Peoples, G.E. et al., Surgery, 114:227-234 (1993). Perez-Diez, A. et al., Cancer Res., 58:5305-5309 (1998). U.S. PATENT DOCUMENTS Plebanski et al., Eur, J. Immunol., 25:1783 (1995). 4,235,871 A 11/1980 Papahadjopoulos et al. Posneft, D. N. et al., J. Biol. Chem., 263:1719-1725 (1988). 4,501,728 A 2f1985 Geho et al. Reddy, R. et al., J. Immunol Methods, 141: 157-163 (1991). 4,690,915 A 9/1987 Rosenberg Riddell, S. R. et al., J. Immunol. Methods, 128:189-201 (1990). 4,722,848 A 2f1988 Paolette et al. Riddell, S. R. et al., Science, 257:238-241 (1992). 4,837,028 A 6, 1989 Allen 4,844,893 A 7, 1989 Honski et al. Rock, K. L. et al., Ann. Rev. Immunol., 17:739-779 (1999). 5,019,369 A 5, 1991 Presant et al. Rosenberg, S.A. et al., A New Era for Cancer Immunotherapy Based 5,635,363 A 6, 1997 Altman et al. on the Genes that encode cancer antigens, Immunity, Cell Press, US, 5,645,994 A 7/1997 Huang et al. vol. 10, No. 3, pp. 281-287 (Mar. 1999). 5,747,269 A 5/1998 Rammensee et al. 5,763,219 A 6/1998 Keyomarsi (Continued) 5,972,643 A 10, 1999 Lobanenkov 6,140,464 A 10, 2000 Pfreundschuh et al. Primary Examiner — Lynn Bristol 6,168,804 B1* 1/2001 Samuel et al...... 424/450 (74) Attorney, Agent, or Firm — Ballard Spahr LLP 6,548,064 B1 4/2003 Tureci et al. 6,867.283 B2 3/2005 Barnea 7,083,789 B2 8/2006 Ramakrishna et al. (57) ABSTRACT 7,087,712 B1 8, 2006 Brossart et al. The present invention relates to compositions and methods 7,270,819 B2 9, 2007 Tureci et al. for the prevention, treatment, and diagnosis of cancer, espe 2004/0236091 A1* 11, 2004 Chicz et al...... 536,235 cially carcinomas, Such as ovarian carcinoma. The invention FOREIGN PATENT DOCUMENTS discloses peptides, polypeptides, and polynucleotides that WO 93.10814 6, 1993 can be used to stimulate a CTL response against cancer. WO WO O2/46416 6, 2002 WO WO 2007/081680 7/2007 14 Claims, No Drawings US 7,919.467 B2 Page 2

OTHER PUBLICATIONS Huynh Khanh D et al., “BCor, a novel corepressor involved in BCL-6 repression' Genes and Development, vol. 14, No. 14, Jul. 15, 2000, Rosenberg, S. A. et al., N. Engl. J. Med., 319:1676-1680 (1988). pp. 1810-1823. Rosenberg, S. A. et al., Nat. Med., 4:321-327 (1998). Ramakrishna V et al., “Generation and Phenotypic Characterization Sarma et al., Journal of Experimental Medicine, 1999, vol. 189, pp. of New Human Ovarian Cancer Cell Lines With the Identification of 811-820. Antigens Potetially Recognizable by HLA-Restricted Cytotoxic T Cells' Journal of Cancer, John Wiley & Sons, Inc., United States, Schendel, D. J. et al., J. Immunol. 151:4209-4220 (1993). Switzerland, Germany vol. 73, No. 1, Sep. 26, 1997, pp. 143-150. Sherman, LA et al., 1998, Critical Reviews in Immunol., 18(1-2): EPO 019995455.1 Office Action (Feb. 29, 2008). 47-54. EPO 019995455.1 Result of Consultation (Aug. 13, 2009). Slingluff, C. L. Jr. et al., Cancer Res., 54:2731-2737 (1994). 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Medicine, Rockefeller University Press, United States, vol. 181, No. 6, Jun. 1, 1995, pp. 2109-2117. * cited by examiner US 7,919,467 B2 1. 2 CYTOTOXCTLYMPHOCYTE-INDUCING located on the surface of the cancerous cell. MHC (major IMMUNOGENS FOR PREVENTION, histocompatibility-complex)-encoded molecules have been TREATMENT, AND DIAGNOSIS OF CANCER subdivided into two types, and are referred to as class I and class II MHC-encoded molecules. CROSS-REFERENCE TO RELATED In the human immune system, MHC molecules are referred APPLICATIONS to as human leukocyte antigens (HLA). Within the MHC, located on chromosome six, are three different genetic loci This application is a continuation-in-part of U.S. applica that encode for class I MHC molecules. MHC molecules tion Ser. No. 10/006, 177, filed Dec. 4, 2001, and now U.S. encoded at these loci are referred to as HLA-A, HLA-B, and Pat. No. 7,083,789, which claims priority from U.S. provi 10 HLA-C. The genes that can be encoded at each of these loci sional application No. 60/251,022, filed Dec. 4, 2000, and are extremely polymorphic, and thus, different individuals U.S. provisional application No. 60/256,824, filed Dec. 20. within the population express different class I MHC mol 2000, the disclosures of which are all herein incorporated by ecules on the surface of their cells. HLA-A1 HLA-A2, HLA reference in their entireties. A3, HLA-B7, and HLA-B8 are examples of different class I 15 MHC molecules that can be expressed from these loci. The FIELD OF THE INVENTION present disclosure involves peptides that are associated with the HLA-A1 HLA-A2, or HLA-A11 molecules, HLA-A1 The present invention relates generally to the field of Supertypes, HLA-A2 Supertypes, and HLA-A11 Supertypes immunogens whose structures incorporate polypeptides and with the gene and protein that gives rise to these peptides. comprising epitopic peptides derived from proteins A Supertype is a group of HLA molecules that present at least expressed by cancer cells and to uses of said immunogens in one shared epitope. eliciting cytotoxic T lymphocyte (CTL) responses for the The peptides that associate with the MHC molecules can diagnosis, prevention and treatment of cancer, preferably car either be derived from proteins made within the cell, in which cinoma, most preferably ovarian carcinoma. case they typically associate with class I MHC molecules 25 (Rock, K. L. and Golde, U., Ann. Rev. Immunol. 17:739-779, BACKGROUND OF THE INVENTION (1999)) or they can be derived from proteins that are acquired from outside of the cell, in which case they typically associate The mammalian immune system has evolved a variety of with class II MHC molecules (Watts, C., Ann. Rev. Immunol. mechanisms to protect the host from cancerous cells, an 15:821-850, (1997)). Peptides that evoke a cancer-specific important component of this response being mediated by 30 CTL response most typically associate with class I MHC cells referred to as T cells. Cytotoxic T lymphocytes (CTLs) molecules. The peptides that associate with a class I MHC are specialized T cells that function primarily by recognizing molecule are typically nine amino acids in length, but can and killing cancerous cells or infected cells, but also by vary from a minimum length of eight amino acids to a maxi secreting soluble molecules referred to as cytokines that can mum of fourteen amino acids in length. A class I MHC mediate a variety of effects on the immune system. 35 molecule with its bound peptide, or a class II MHC molecule Evidence Suggests that immunotherapy designed to stimu with its bound peptide, is referred to as an MHC-peptide late a tumor-specific CTL response would be effective in complex. controlling cancer. For example, it has been shown that The process by which intact proteins are degraded into human CTLS recognize sarcomas (Slovin, S. F. et al., J. peptides is referred to as antigen processing. Two major path Immunol. 137:3042-3048, (1987)), renal cell carcinomas 40 ways of antigen processing occur within cells (Rock, K. L. (Schendel, D.J. et al., J. Immunol. 151:4209-4220, (1993)), and Golde, U., Ann. Rev. Immunol., 17:739-779, (1999); colorectal carcinomas (Jacob, L. et al., Int. J. Cancer, 71:325 Watts, C., Ann. Rev. Immunol. 15:821-850, (1997)). One 332, (1997)), ovarian carcinomas (Loannides, C. G. et al., J. pathway, which is largely restricted to cells that are antigen Immunol., 146:1700-1707, (1991)) (Peoples, G. E. et al., presenting cells Such as dendritic cells, macrophages, and B Surgery, 114:227-234. (1993)), pancreatic carcinomas 45 cells, degrades proteins that are typically phagocytosed or (Peiper, M. et al., Eur. J. Immunol., 27:1115-1123, (1997); endocytosed into the cell. Peptides derived in this pathway Wolfel, T. et al., Int. J. Cancer. 54:636-644, (1993)), squa typically bind to class II MHC molecules. A second pathway mous tumors of the head and neck (Yasumura, S. et al., of antigen processing is present in essentially all cells of the Cancer Res., 53:1461-1468, (1993)), and squamous carcino body. This second pathway primarily degrades proteins that mas of the lung (Slingluff, C. L. Jr et al., Cancer Res., 50 are made within the cells, and the peptides derived from this 54:2731-2737, (1994): Yoshino, I. et al., Cancer Res., pathway primarily bind to class I MHC molecules. It is the 54:3387-3390, (1994)). The largest number of reports of peptides from this second pathway of antigen processing that human tumor-reactive CTLS have concerned cancers (Boon, are referred to herein. Antigen processing by this latter path T. et al., Ann. Rev. Immunol. 12:337-365, (1994)). The abil way involves polypeptide synthesis and proteolysis in the ity of tumor-specific CTLS to mediate tumor regression, in 55 cytoplasm. The peptides produced are then transported into both human (Rosenberg, S. A. et al., N. Engl. J. Med., 319. the endoplasmic reticulum of the cell, associate with newly 1676-1680, (1988)) and animal models (Celluzzi, C. M. et al., synthesized class I MHC molecules, and the resulting MHC J. Exp. Med., 183:283-287. (1996); Mayordomo, J. I. et al., peptide complexes are then transported to the cell Surface. Nat. Med., 1:1297-1302, (1995); Zitvogel, L. et al., J. Exp. Peptides derived from membrane and secreted proteins have Med., 183:87–97, (1996)), suggests that methods directed at 60 also been identified. In some cases these peptides correspond increasing CTL activity would likely have a beneficial effect to the signal sequence of the proteins that are cleaved from the with respect to tumor treatment. protein by the signal peptidase. In other cases, it is thought In order for CTLs to kill or secrete cytokines in response to that Some fraction of the membrane and secreted proteins are a cancer cell, the CTL must first recognize that cell as being transported from the endoplasmic reticulum into the cyto cancerous. This process involves the interaction of the T cell 65 plasm where processing Subsequently occurs. receptor, located on the surface of the CTL, with what is Once bound to the class I MHC molecule and displayed on generically referred to as an MHC-peptide complex which is the Surface of a cell, the peptides are recognized by antigen US 7,919,467 B2 3 4 specific receptors on CTLs. Mere expression of the class I tumor cell expressing the appropriate MHC molecules. MHC molecule itself is insufficient to trigger the CTL to kill Sequencing is readily performed by tandem mass spectrom the target cell if the antigenic peptide is not bound to the class etry (Henderson, R. A. et al., Proc. Natl. Acad. Sci. U.S.A., I MHC molecule. Several methods have been developed to 90:10275-10279, (1993); Hogan, K. T. et al., Cancer Res., identify the peptides recognized by CTL, each method rely 58:5144-5150, (1998); Hunt, D. F. et al., Science, 255:1261 ing on the ability of a CTL to recognize and kill only those 1263, (1992); Slingluff, C. L.Jret al., J. Immunol., 150:2955 cells expressing the appropriate class I MHC molecule with 2963, (1993)). the peptide bound to it (Rosenberg, S.A., Immunity, 10:281 Immunization with cancer-derived, class I MHC-encoded 287. (1999)). Such peptides can be derived from a non-self molecule-associated peptides, or with a precursor polypep Source. Such as a pathogen (for example, following the infec 10 tide or protein that contains the peptide, or with a gene that tion of a cell by a bacterium or a virus) or from a self-derived encodes a polypeptide or protein containing the peptide, are protein within a cell. Such as a cancerous cell. Examples of forms of immunotherapy that can be employed in the treat sources of self-derived proteins in cancerous cells have been ment of cancer. These forms of immunotherapy require that reviewed (Gilboa, E., Immunity, 11:263-270, (1999); Rosen immunogens be identified so that they can be formulated into berg, S.A., Immunity, 10:281-287. (1999)) and include: (i) 15 an appropriate vaccine. Although a variety of cancer-derived mutated genes; (ii) aberrantly expressed genes Such as an antigens have been identified (Rosenberg, S.A., Immunity, alternative open reading frame or through an intron-exon 10:281-287. (1999)), not all of these are appropriate for boundary; (iii) normal genes that are selectively expressed in broad-based immunotherapy as the expression of some pep only the tumor and the testis; and (iv) normal differentiation tides is limited to the tumor derived from a specific patient. genes that are expressed in the tumor and the normal cellular Furthermore, the number of class I MHC molecules from counterpart. which tumor-derived peptides have been discovered is largely Four different methodologies have typically been used for restricted to HLA-A2. Thus, it would be useful to identify identifying the peptides that are recognized by CTLs. These additional peptides that complex with class I MHC molecules are: (i) the genetic method; (2) motif analysis; (3) SErological other than HLA-A2. Such peptides would be particularly analysis of REcombinant cDNA expression libraries 25 useful in the treatment of cancer patients who do not express (SEREXTM); and (iv) the analytical chemistry approach or the the HLA-A2 molecule, HLA-A1 or HLA-A11 antigens, Direct Identification of Relevant Epitopes for Clinical Thera HLA-A1 supertypes, HLA-A2 supertypes and HLA-A11 peutics (DIRECTTM). Supertypes, for example. It is also particularly useful to iden The genetic method is an approach in which progressively tify antigenic peptides that are derived from different original smaller subsets of cDNA libraries from tumor cells are trans 30 proteins, even if the derived peptides associate with the same fected into cells that express the appropriate MHC molecule class I MHC molecule. Because an active immune response but not the tumor-specific epitope. The molecular clones can result in the outgrowth of tumor cells that have lost the encoding T cell epitopes are identified by their ability to expression of a particular precursor protein for a given anti reconstitute tumor specific T cell recognition of transfected genic peptide, it is advantageous to stimulate an immune cells. The exact T cell epitope is then identified by a combi 35 response against peptides derived from more than one pro nation of molecular subcloning and the use of synthetic pep tein, as the chances of the tumor cell losing the expression of tides based on the predicted amino acid sequence. Such meth both proteins is the multiple of the chances of losing each of ods, however, are susceptible to inadvertent identification of the individual proteins. cross-reacting peptides, and are not capable of identifying important post-translational modifications. 40 SUMMARY OF THE INVENTION Motif analysis involves scanning a protein for peptides containing known class I MHC binding motifs, followed by The present invention relates to Immunogens comprising synthesis and assay of the predicted peptides for their ability polypeptides with amino acid sequences comprising epitopic to be recognized by tumor-specific CTL. This approach sequences selected from the sequences of SEQID NO: 1-791 requires prior knowledge of the protein from which the pep 45 and 1514-1533 and which immunogens facilitate a cytotoxic tides are derived. This approach is also greatly hampered by T lymphocyte (CTL)-mediated immune response against the fact that not all of the predicted peptide epitopes are cancers. The present invention also relates to nucleic acid presented on the surface of a cell (Yewdell, J.W. and Bennink, molecules that encode for the polypeptides and/or the full J. R. Ann. Rev. Immunol., 17:51-88, (1999)), thus additional length proteins from which the polypeptides are derived, of experimentation is required to determine which of the pre 50 Such immunogens, and which can also be used to facilitate an dicted epitopes is useful. immune response against cancer. The SEREXTMapproach relies on using antibodies in the The present invention provides compositions comprising serum of cancer patients to screen cDNA expression libraries the immunogen described herein, and polynucleotides that for a clone that expresses a protein recognized by the anti direct the synthesis of such polypeptides, whereby the oli body. This methodology presumes that an antibody response 55 gopeptides and polypeptides of Such immunogens are will necessarily have developed in the presence of a T cell capable of inducing a CTL response against cells expressing response, and thus, the identified clone is good candidate to a protein comprising an epitopic sequence of at least one of encode a protein that can be recognized by T cells. SEQ ID NO: 1-791 and 1514-1533. The cells are usually DIRECTTM involves a combination of cellular immunol cancer cells, preferably carcinoma cells, most preferably ova ogy and mass spectrometry. This approach involves the actual 60 rian carcinomas expressing such proteins. identification of CTL epitopes by sequencing the naturally The present invention further relates to polynucleotides occurring peptides associated with class I MHC molecules. In comprising the gene coding for a polypeptide of the immu this approach, cells are first lysed in a detergent solution, the nogens disclosed herein. peptides associated with the class I MHC molecules are puri The present invention also provides methods that comprise fied, and the peptides fractionated by high performance liquid 65 contacting a lymphocyte, especially a CTL, with an immu chromatography (HPLC). The peptides are then used to nogen of the invention under conditions that induce a CTL reconstitute recognition by tumor-specific CTLS on a non response against a tumor cell, and more specifically against a US 7,919,467 B2 5 6 cancer cell. The methods may involve contacting the CTL A peptide, oligopeptide, protein, or polynucleotide coding with the immunogenic peptide in Vivo, in which case the for Such a molecule is “immunogenic' (and thus an “immu peptides, polypeptides, and polynucleotides of the invention nogen' within the present invention) if it is capable of induc are used as vaccines, and will be delivered as a pharmaceuti ing an immune response. In the case of the present invention, cal composition comprising a pharmaceutically acceptable immunogenicity is more specifically defined as the ability to carrier and the immunogen, typically along with an adjuvant induce a CTL-mediated response. Thus, an “immunogen or one or more cytokines. would be a molecule that is capable of inducing an immune Alternatively, the immunogens of the present invention can response, and in the case of the present invention, a molecule be used to induce a CTL response in vitro. The generated CTL capable of inducing a CTL response. can then be introduced into a patient with cancer, more spe 10 cifically cancer, colorectal carcinoma, ovarian carcinoma, A T cell "epitope' is a short peptide molecule that binds to breast carcinoma, lung carcinoma, or prostate carcinoma. a class I or II MHC molecule and that is subsequently recog Alternatively, the ability to generate CTL in vitro could serve nized by a T cell. T cell epitopes that bind to class I MHC as a diagnostic for cancer generally, including colorectal car molecules are typically 8-14 amino acids in length, and most cinoma, ovarian carcinoma, breast carcinoma, lung carci 15 typically 9 amino acids in length. T cell epitopes that bind to noma, or prostate carcinoma. class II MHC molecules are typically 12-20 amino acids in length. In the case of epitopes that bind to class II MHC DETAILED DESCRIPTION OF THE INVENTION molecules, the same T cell epitope may share a common core segment, but differ in the length of the carboxy- and amino Definitions terminal flanking sequences due to the fact that ends of the As used herein and except as noted otherwise, all terms are peptide molecule are not buried in the structure of the class II defined as given below. The term "peptide' is used herein to designate a series of MHC molecule peptide-binding cleft as they are in the class amino acid residues, connected one to the other typically by I MHC molecule peptide-binding cleft. peptide bonds between the alpha-amino and carbonyl groups 25 There are three different genetic loci that encode for class of the adjacent amino acids. The peptides are typically 9 I MHC molecules: HLA-A. HLA-B, and HLA-C. HLA-A1, amino acids in length, but can be as short as 8 amino acids in HLA-A2, and HLA-A11 are examples of different class I length, and as long as 14 amino acids in length. MHC molecules that can be expressed from these loci. The The term "oligopeptide' is used hereinto designate a series present invention also involves peptides that are associated of amino acid residues, connected one to the other typically 30 with HLA-A1 supertypes, HLA-A2 supertypes, and HLA by peptide bonds between the alpha-amino and carbonyl All Supertypes. A Supertype is a group of HLA molecules groups of the adjacent amino acids. The length of the oli that present at least one shared epitope. MHC molecule pep gopeptide is not critical to the invention as long as the correct tides that have been found to bind to one member of the MHC epitope or epitopes are maintained therein. The oligopeptides allele supertype family (A1 for example) are thought to be are typically less than about 30 amino acid residues in length, likely to bind to other members of the same supertype family and greater than about 14 amino acids in length. (A32 for example; see Table 1, below. TABLE 1

Supertype Motif Genotypes A1 TI(SVLM) A*0101, A*0102, A*2501, A*2601, A*2604, XXXXX WFY A*3201, A*3601, A*4301, A*8001 A2 LIVMATO A*0201, A*0202, A*0203, A*0204, A*0205, XXXXX LIVMAT A*0206, A*0207, A*6802, A*6901 A3 AILMVST A*0301, A*1101, A*3101, A*3301, A*6801 XXXXX RK A24 YF(WIVLMT) A*2301, A*2402, A*2403, A*2404, A*3001, XXXXX A*3002, A*3003 PXXXXXX B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, ALIMVFWY B*3502, B*3503, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, B*7801 B27 XXXXXX B*1401, B*1402, B*1503, B*1509, B*1510, B*1518, B*2701, B*2702, B*2703, B*2704, B*2705, B*2706, B*2707, B*2708, B*3801, B*3802, B*3901, B*3902, B*3903, B*3904, B*4801, B*4802, B*7301 B44 XXXXXX B*18, B*3701, B*4001, B*4006, B*4101, B*4402, B*4403, B*4501, B*4901, B*5001 B58 B*1516, B*1517, B*5701, B*5702, B*58 B*1301, B*1302, B*1501, B*1502, B*1506, B*1512, XXXXXX B*1513, B*1514, B*1519, B*1521, B*4601, B*52

The term “polypeptide' designates a series of amino acid 60 As used herein, reference to a DNA sequence includes both residues, connected one to the other typically by peptide single stranded and double stranded DNA. Thus, the specific bonds between the alpha-amino and carbonyl groups of the sequence, unless the context indicates otherwise, refers to the adjacent amino acids. The length of the polypeptide is not single strand DNA of Such sequence, the duplex of Such critical to the invention as long as the correct epitopes are sequence with its complement (double stranded DNA) and maintained. In contrast to the terms peptide or oligopeptide, 65 the complement of Such sequence. the term polypeptide is meant to refer to protein molecules of The term "coding region” refers to that portion of a gene longer than about 30 residues in length. which either naturally or normally codes for the expression US 7,919,467 B2 7 8 product of that gene in its natural genomic environment, i.e., definition, and can include preparations that are highly puri the region coding in vivo for the native expression product of fied or preparations that are only partially purified, as those the gene. The coding region can be from a normal, mutated or terms are understood by those of skill in the relevant art. For altered gene, or can even be from a DNA sequence, or gene, example, individual clones isolated from a cDNA library have wholly synthesized in the laboratory using methods well been conventionally purified to electrophoretic homogeneity. known to those of skill in the art of DNA synthesis. Purification of starting material or natural material to at least The term “nucleotide sequence” refers to a heteropolymer one order of magnitude, preferably two or three orders, and of deoxyribonucleotides. The nucleotide sequence encoding more preferably four or five orders of magnitude is expressly for a particular peptide, oligopeptide, or polypeptide may be contemplated. Furthermore, the claimed polypeptide which naturally occurring or they may be synthetically constructed. 10 has a purity of preferably 0.001%, or at least 0.01% or 0.1%; Generally, DNA segments encoding the peptides, polypep and even desirably 1% by weight or greater is expressly tides, and proteins of this invention are assembled from contemplated. cDNA fragments and short oligonucleotide linkers, or from a The nucleic acids and polypeptide expression products series of oligonucleotides, to provide a synthetic gene which disclosed according to the present invention, as well as is capable of being expressed in a recombinant transcriptional 15 expression vectors containing Such nucleic acids and/or Such unit comprising regulatory elements derived from a microbial polypeptides, may be in “enriched form.” As used herein, the or viral operon. term "enriched' means that the concentration of the material The term “expression product” means that polypeptide or is at least about 2, 5, 10, 100, or 1000 times its natural protein that is the natural translation product of the gene and concentration (for example), advantageously 0.01%, by any nucleic acid sequence coding equivalents resulting from weight, preferably at least about 0.1% by weight. Enriched genetic code degeneracy and thus coding for the same amino preparations of about 0.5%, 1%. 5%, 10%, and 20% by acid(s). weight are also contemplated. The sequences, constructs, The term "fragment, when referring to a coding sequence, vectors, clones, and other materials comprising the present means a portion of DNA comprising less than the complete invention can advantageously be in enriched or isolated form. coding region whose expression product retains essentially 25 The term “active fragment’ means a fragment that gener the same biological function or activity as the expression ates an immune response (i.e., has immunogenic activity) product of the complete coding region. when administered, alone or optionally with a Suitable adju The term “DNA segment” refers to a DNA polymer, in the vant, to an animal. Such as a mammal, for example, a rabbit or form of a separate fragment or as a component of a larger a mouse, and also including a human, Such immune response DNA construct, which has been derived from DNA isolated at 30 taking the form of stimulating a CTL response within the least once in Substantially pure form, i.e., free of contaminat recipient animal. Such as a human. Alternatively, the “active ing endogenous materials and in a quantity or concentration fragment may also be used to induce a CTL response invitro. enabling identification, manipulation, and recovery of the As used herein, the terms “portion.” “segment, and “frag segment and its component nucleotide sequences by standard ment, when used in relation to polypeptides, refer to a con biochemical methods, for example, by using a cloning vector. 35 tinuous sequence of residues, such as amino acid residues, Such segments are provided in the form of an open reading which sequence forms a Subset of a larger sequence. For frame uninterrupted by internal nontranslated sequences, or example, if a polypeptide were subjected to treatment with introns, which are typically present in eukaryotic genes. any of the common endopeptidases, such as trypsin or chy Sequences of non-translated DNA may be present down motrypsin, the oligopeptides resulting from Such treatment stream from the open reading frame, where the same do not 40 would represent portions, segments or fragments of the start interfere with manipulation or expression of the coding ing polypeptide. This means that any such fragment will regions. necessarily contain as part of its amino acid sequence a seg The term “primer means a short nucleic acid sequence ment, fragment orportion, that is substantially identical, if not that is paired with one strand of DNA and provides a free exactly identical, to a sequence of SEQ ID NO: 792-1513, 3'OH end at which a DNA polymerase starts synthesis of a 45 which correspond to the naturally occurring, or “parent pro deoxyribonucleotide chain. teins of the SEQID NO: 1-791 and 1514-1533. When used in The term “promoter” means a region of DNA involved in relation to polynucleotides, such terms refer to the products binding of RNA polymerase to initiate transcription. produced by treatment of said polynucleotides with any of the The term “open reading frame (ORF) means a series of common endonucleases. triplets coding for amino acids without any termination 50 In accordance with the present invention, the term "percent codons and is a sequence (potentially) translatable into pro identity” or “percent identical, when referring to a sequence, tein. means that a sequence is compared to a claimed or described The term "isolated” means that the material is removed sequence after alignment of the sequence to be compared (the from its original environment (e.g., the natural environmentif “Compared Sequence') with the described or claimed it is naturally occurring). For example, a naturally-occurring 55 sequence (the “Reference Sequence'). The Percent Identity is polynucleotide or polypeptidepresent in a living animal is not then determined according to the following formula: isolated, but the same polynucleotide or polypeptide, sepa rated from Some or all of the coexisting materials in the Percent Identity=1001-(C/R) natural system, is isolated. Such polynucleotides could be wherein C is the number of differences between the Refer part of a vector and/or Such polynucleotides or polypeptides 60 ence Sequence and the Compared Sequence over the length of could be part of a composition, and still be isolated in that alignment between the Reference Sequence and the Com Such vector or composition is not part of its natural environ pared Sequence wherein (i) each base or amino acid in the ment. Reference Sequence that does not have a corresponding The polynucleotides, and recombinant or immunogenic aligned base oramino acid in the Compared Sequence and (ii) polypeptides, disclosed in accordance with the present inven 65 each gap in the Reference Sequence and (iii) each aligned tion may also be in “purified form. The term “purified' does base or amino acid in the Reference Sequence that is different not require absolute purity; rather, it is intended as a relative from an aligned base or amino acid in the Compared US 7,919,467 B2 10 Sequence, constitutes a difference; and R is the number of amino acid sequence, nonapeptide fragments having up to 8 bases or amino acids in the Reference Sequence over the amino acids identical to a sequence of SEQID NO: 1-4 such length of the alignment with the Compared Sequence with that the polypeptide comprises, in a specific embodiment, 2 any gap created in the Reference Sequence also being counted segments with at least 8 residues identical to SEQID NO: 1 as a base or amino acid. 5 and one segment with at least 8 residues identical to SEQID If an alignment exists between the Compared Sequence NO: 3. In other embodiments, other combinations and per and the Reference Sequence for which the percent identity as mutations of the epitopic sequences disclosed herein may be calculated above is about equal to or greater than a specified part of an immunogen of the present invention or of Such a minimum Percent Identity then the Compared Sequence has polypeptide so long as any Such polypeptide comprises at the specified minimum percent identity to the Reference 10 least 2 such epitopes, whether such epitopes are different or Sequence even though alignments may exist in which the the same. Thus, in a specific embodiment, a polypeptide of herein above calculated Percent Identity is less than the speci the present invention may comprise 2 copies of the sequence fied Percent Identity. of SEQID NO: 2 at some point or points within its length. Of The present invention relates generally to immunogens and course, any combinations and permutations of the epitopes immunogenic compositions, and methods of use therefore, 15 disclosed herein, as long as they are present at least two in for the prevention, treatment, and diagnosis of cancer, espe number in Such polypeptides, are expressly contemplated. cially carcinomas, including ovarian carcinomas. Disclosed All of the epitopic peptides of SEQ ID NO: 1-791 and according to the invention are immunogens comprising pro 1514-1533 are derived from proteins expressed by cancer teins or polypeptides whose amino acid sequences comprises cells and sequences and were identified through the method one or more epitopic oligopeptides with sequences selected of Automated High Through-put Sequencing (HTPS). from the group SEQID NO: 1-791 and 1514-1533. In addi Accordingly, SEQ ID NO: 792-1513 are polypeptides that tion, the invention further relates to polynucleotides that can comprise at least one of SEQID NO: 1-791 and 1514-1533. be used to stimulate a CTL response against cancer, and more Oligopeptides as disclosed herein may themselves be pre specifically carcinoma, especially ovarian carcinomas. pared by methods well known to those skilled in the art. In accordance with the present invention there are dis 25 (Grant, G. A., Synthetic Peptides: A User's Guide, 1992, W. closed specific oligopeptide sequences with amino acid H. Freeman and Company, New York; Coligan, J. E. et al. sequences shown in SEQ ID NO: 1-791 and 1514-1533, Current Protocols in Protein Science, 1999, John Wiley & which represent epitopic peptides (i.e. immunogenic oli Sons, Inc., New York). gopeptide sequences) of at least about 8 amino acids in Besides the sequences of SEQ ID NO:1-791 and 1514 length, preferably about 9 amino acids in length (i.e., non 30 1533, the proteins and polypeptides forming the immunogens apeptides), and no longer than about 10amino acids in length of the present invention may also comprise one or more other and present as part of a larger structure, such as a polypeptide immunogenic amino acid stretches known to be associated or full length protein. with cancer, and more specifically with carcinomas and mela The polypeptides forming the immunogens of the present nomas, including colorectal carcinoma, ovarian carcinoma, invention have amino acid sequences that comprise at least 35 breast carcinoma, lung carcinoma, or prostate carcinoma, and one stretch, possibly two, three, four, or more stretches of which may stimulate a CTL response whereby the immuno about 8 to 10 residues in length and which stretches differ in genic peptides associate with HLA-A1 or HLA-A11, or amino acid sequence from the sequences of SEQ ID NO: HLA-A2, or another class I MHC (i.e., MHC-1) molecule. 1-791 and 1514-1533 by no more than about 1 amino acid The immunogens of the present invention can be in the residue, preferably a conservative amino acid residue, espe 40 form of a composition of one or more of the different immu cially amino acids of the same general chemical character, nogens and wherein each immunogen is present in any Such as where they are hydrophobic amino acids. desired relative abundance. Such compositions can be homo Said polypeptides can be of any desired length so long as geneous or heterogeneous with respect to the individual they have immunogenic activity in that they are able, under a immunogenic peptide components present therein, having given set of desirable conditions, to elicit in vitro or in vivo the 45 only one or more than one of Such peptides. activation of cytotoxic T lymphocytes (CTLs) (i.e., a CTL The oligopeptides and polypeptides useful in practicing the response) against a presentation of a cancer specific protein, present invention may be derived by fractionation of naturally especially a carcinoma or sarcoma specific protein, most occurring proteins by methods such as protease treatment, or especially MAGE D, MAGE 4, MFG-E8 or human retino they may be produced by recombinant or synthetic method blastoma-like protein, especially when such proteins are pre 50 ologies that are well known and clear to the skilled artisan sented along with MHC-1 proteins, such as where said pro (Ausubel, F. M. etal, Current Protocols in Molecular Biology, teins are presented in vitro or in vivo by an antigen presenting 1999, John Wiley & Sons, Inc., New York; Coligan, J. E. etal, cell (APC). The proteins and polypeptides forming the immu Current Protocols in Protein Science, 1999, John Wiley & nogens of the present invention can be naturally occurring or Sons, Inc., New York; Molecular Cloning: A Laboratory may be synthesized chemically. According to the present 55 Manual, 1989, Cold Spring Harbor Laboratory Press, Cold invention the polypeptides may comprise at least one of SEQ Spring Harbor). The polypeptide may comprise a recombi ID NO: 792-1513. nant or synthetic polypeptide that comprises at least one of The present invention is also directed to an isolated SEQID NO: 1-791 and 1514-1533 which sequences may also polypeptide, especially one having immunogenic activity, the be present in multiple copies. Thus, oligopeptides and sequence of which comprises within it one or more stretches 60 polypeptides of the present invention may have one, two, comprising any 2 or more of the sequences of SEQ ID NO: three, or more such immunogenic peptides within the amino 1-791 and 1514-1533 and in any relative quantities and acid sequence of said oligopeptides and polypeptides, and wherein said sequences may differ by one amino acid resi said immunogenic peptides, or epitopes, may be the same or dues from the sequences of SEQ ID NO: 1-791 and 1514 may be different, or may have any number of such sequences 1533 in any given stretch of 8 to 10amino acid residues. Thus, 65 wherein some of them are identical to each other in amino within the present invention, by way of a non-limiting acid sequence while others within the same polypeptide example only, such polypeptide may contain as part of its sequence are different from each other and said epitopic US 7,919,467 B2 11 12 sequences may occur in any order within said immunogenic molecule, in which case the goal is to create a peptide that has polypeptide sequence. The location of such sequences within a higher affinity for the class I MHC molecule than does the the sequence of a polypeptide forming an immunogen of the original peptide. The peptides can also be modified at amino invention may affect relative immunogenic activity. In addi acid residues that are predicted to interact with the T cell tion, immunogens of the present invention may comprise 5 receptor on the CTL, in which case the goal is to create a more than one protein comprising the amino acid sequences peptide that has a higher affinity for the T cell receptor than disclosed herein. Such polypeptides may be part of a single does the original peptide. Both of these types of modifications composition or may themselves be covalently or non-co can result in a variant peptide that is related to an original valently linked to each other. peptide, but which is better able to induce a CTL response The immunogenic peptides disclosed herein may also be 10 than is the original peptide. As used herein, the term “original linked directly to, or through a spacer or linker to: an immu peptide' means an oligopeptide with the amino acid sequence nogenic carrier Such as serum albumin, tetanus toxoid, key selected from SEQID NO: 1-791 and 1514-1533. hole limpet hemocyanin, dextran, or a recombinant virus The original peptides disclosed herein can be modified by particle; an immunogenic peptide known to stimulate a T the substitution of one or more residues at different, possibly helper cell type immune response; a cytokine Such as inter 15 selective, sites within the peptide chain. Such substitutions feron gamma or GMCSF; a targeting agent Such as an anti may be of a conservative nature, for example, where one body or receptor ligand; a stabilizing agent Such as a lipid; or amino acid is replaced by an amino acid of similar structure a conjugate of a plurality of epitopes to a branched lysine core and characteristics, such as where a hydrophobic amino acid structure, Such as the so-called “multiple antigenic peptide' is replaced by another hydrophobic amino acid. Even more described in (Posneft, D. N. et al., J. Biol. Chem., 263:1719 conservative would be replacement of amino acids of the 1725, (1988)); a compound such as polyethylene glycol to same or similar size and chemical nature. Such as where increase the halflife of the peptide; or additional amino acids leucine is replaced by isoleucine. In studies of sequence Such as a leader or secretory sequence, or a sequence variations in families of naturally occurring homologous pro employed for the purification of the mature sequence. Spacers teins, certain amino acid Substitutions are more often toler and linkers are typically comprised of relatively small, neutral 25 ated than others, and these are often show correlation with molecules, such as amino acids and which are substantially similarities in size, charge, polarity, and hydrophobicity uncharged under physiological conditions. Such spacers are between the original amino acid and its replacement, and Such typically selected from the group of nonpolar or neutral polar is the basis for defining "conservative substitutions.” amino acids. Such as glycine, alanine, serine and other similar Conservative Substitutions are herein defined as exchanges amino acids. Such optional spacers or linkers need not be 30 within one of the following five groups: Group 1—Small comprised of the same residues and thus may be either homo aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, or hetero-oligomers. When present, such linkers will com Pro, Gly); Group 2 polar, negatively charged residues and monly be of length at least one or two, commonly 3, 4, 5, 6, their amides (Asp, ASn, Glu, Gln); Group 3 polar, positively and possibly as much as 10 or even up to 20 residues (in the charged residues (His, Arg, Lys); Group 4-large, aliphatic, case of amino acids). In addition, such linkers need not be 35 nonpolar residues (Met, Leu, lie, Val, Cys); and Group composed of amino acids but any oligomeric structures will 4—large, aromatic residues (Phe, Tyr, Trp). do as well so long as they provide the correct spacing so as to Less conservative Substitutions might involve the replace optimize the desired level of immunogenic activity of the ment of one amino acid by another that has similar charac immunogens of the present invention. The immunogen may teristics but is somewhat different in size, such as replacement therefore take any form that is capable of eliciting a CTL 40 of an alanine by an isoleucine residue. Highly nonconserva response. tive replacements might involve substituting an acidic amino In addition, the immunogenic peptides of the present acid for one that is polar, or even for one that is basic in invention may be part of an immunogenic structure via character. Such radical Substitutions cannot, however, be dis attachments other than conventional peptide bonds. Thus, any missed as potentially ineffective since chemical effects are manner of attaching the peptides of the invention to an immu 45 not totally predictable and radical substitutions might well nogen of the invention, Such as an immunogenic polypeptide give rise to serendipitous effects not otherwise predictable as disclosed herein, could provide an immunogenic structure from simple chemical principles. as claimed herein. Thus, immunogens, such as proteins of the Of course, such substitutions may involve structures other invention, are structures that contain the peptides disclosed than the common L-amino acids. Thus, D-amino acids might according to the present invention but Such immunogenic 50 be substituted for the L-amino acids commonly found in the peptides may not necessarily be attached thereto by the con antigenic peptides of the invention and yet still be encom ventional means of using ordinary peptide bounds. The passed by the disclosure herein. In addition, amino acids immunogens of the present invention simply contain Such possessing non-standard R groups (i.e., R groups other than peptides as part of their makeup, but how Such peptides are to those found in the common 20 amino acids of natural pro be combined to form the final immunogen is left to the talent 55 teins) may also be used for Substitution purposes to produce and imagination of the user and is in no way restricted or immunogens and immunogenic polypeptides according to limited by the disclosure contained herein. the present invention. The peptides that are naturally processed and bound to a If substitutions at more than one position are found to result class I MHC molecule, and which are recognized by a tumor in a peptide with Substantially equivalent or greater antigenic specific CTL, need not be the optimal peptides for stimulating 60 activity as defined below, then combinations of those substi a CTL response. See, for example, (Parkhurst, M. R. et al., J. tutions will be tested to determine if the combined substitu Immunol., 157:2539-2548, (1996); Rosenberg, S. A. et al., tions result in additive or Syngeneic effects on the antigenicity Nat. Med., 4:321-327, (1998)). Thus, there can be utility in of the peptide. At most, no more than 4 positions within the modifying a peptide. Such that it more readily induces a CTL peptide would simultaneously be substituted. response. Generally, peptides may be modified at two types of 65 Based on cytotoxicity assays, an epitope is considered positions. The peptides may be modified at amino acid resi substantially identical to the reference peptide if it has at least dues that are predicted to interact with the class I MHC 10% of the antigenic activity of the reference peptide as US 7,919,467 B2 13 14 defined by the ability of the substituted peptide to reconstitute pany, New York; Coligan, J. E. et al. Current Protocols in the epitope recognized by a CTL in comparison to the refer Protein Science, 1999, John Wiley & Sons, Inc., New York). ence peptide. Thus, when comparing the lytic activity in the The coding sequences can also be modified Such that a pep linear portion of the effector:target curves with equimolar tide or polypeptide will be produced that incorporates a concentrations of the reference and substituted peptides, the desired amino acid Substitution. The coding sequence can be observed percent specific killing of the target cells incubated provided with appropriate linkers, be ligated into suitable with the substituted peptide should be equal to that of the expression vectors that are commonly available in the art, and reference peptide at an effector:target ratio that is no greater the resulting DNA or RNA molecule can be transformed or than 10-fold above the reference peptide effector:target ratio transfected into suitable hosts to produce the desired fusion at which the comparison is being made. 10 protein. A number of Such vectors and Suitable host systems Preferably, when the CTLs specific for a peptide of SEQID are available, and their selection is left to the skilled artisan. NO: 1-791 and 1514-1533 are tested against the substituted For expression of the fusion proteins, the coding sequence peptides, the peptide concentration at which the substituted will be provided with operably linked start and stop codons, peptides achieve half the maximal increase in lysis relative to promoter and terminator regions, and a replication system to background is no more than about 1 mM, preferably no more 15 provide an expression vector for expression in the desired than about 1 uM, more preferably no more than about 1 nM. host cell. For example, promoter sequences compatible with and still more preferably no more than about 100 pM, and bacterial hosts are provided in plasmids containing conve most preferably no more than about 10pM. It is also preferred nient restriction sites for insertion of the desired coding that the substituted peptide be recognized by CTLs from more sequence. The resulting expression vectors are transformed than one individual, at least two, and more preferably three into Suitable bacterial hosts. Of course, yeast, insect, and individuals. mammalian host cells may also be used, employing Suitable Thus, the epitopes of the present invention may be identical vectors and control sequences. to naturally occurring tumor-associated or tumor-specific Host cells are genetically engineered (transduced or trans epitopes or may include epitopes that differ by no more than formed or transfected) with the vectors of this invention 4 residues from the reference peptide, as long as they have 25 which may be, for example, a cloning vector or an expression Substantially identical antigenic activity. vector. The vector may be, for example, in the form of a It should be appreciated that an immunogen may consist plasmid, a viral particle, a phage, etc. The engineered host only of a peptide of SEQ ID NO:1-791 or 1514-1533, or cells can be cultured in conventional nutrient media modified comprise a peptide of SEQ ID NO:1-791 or 1514-1533, or as appropriate for activating promoters, selecting transfor comprise a plurality of peptides selected from SEQID NO:1- 30 mants or amplifying the genes of the present invention. The 791 and 1514-1533, or comprise a polypeptide that itself culture conditions, such as temperature, pH and the like, are comprises one or more of the epitopic peptides of SEQ ID those previously used with the host cell selected for expres NO: 1-791 and 1514-1533. Sion, and will be apparent to the ordinarily skilled artisan. The immunogenic peptides and polypeptides of the inven More particularly, the present invention also includes tion can be prepared synthetically, by recombinant DNA 35 recombinant constructs comprising one or more of the technology, or they can be isolated from natural Sources Such sequences as broadly described above. The constructs com as tumor cells expressing the original protein product. prise a vector, Such as a plasmid or viral vector, into which a The polypeptides and oligopeptides disclosed herein can sequence of the invention has been inserted, in a forward or be synthesized in solution or on a Solid Support in accordance reverse orientation. In a preferred aspect of this embodiment, with conventional techniques. Various automated peptide 40 the construct further comprises regulatory sequences, includ synthesizers are commercially available and can be used in ing, for example, a promoter, operably linked to the sequence. accordance with known protocols. See, for example, (Grant, Large numbers of Suitable vectors and promoters are known G. A., Synthetic Peptides: A User's Guide, 1992, W. H. Free to those of skill in the art, and are commercially available. man and Company, New York; Coligan, J. E. et al. Current In a further embodiment, the present invention relates to Protocols in Protein Science, 1999, John Wiley & Sons, Inc., 45 host cells containing the above-described constructs. The New York). Fragments of polypeptides of the invention can host cell can be a higher eukaryotic cell. Such as a mammalian also be synthesized as intermediates in the synthesis of a cell, or a lower eukaryotic cell. Such as a yeast cell, or the host larger polypeptide. cell can be a prokaryotic cell. Such as a bacterial cell. Intro Recombinant DNA technology may be employed wherein duction of the construct into the host cell can be effected by a nucleotide sequence which encodes an immunogenic pep 50 calcium phosphate transfection, DEAE-Dextran mediated tide or polypeptide of interest is inserted into an expression transfection, or electroporation (Ausubel, F. M. et al. Current vector, transformed or transfected into an appropriate host Protocols in Molecular Biology, 1999, John Wiley & Sons, cell, and cultivated under conditions Suitable for expression. Inc., New York; Molecular Cloning: A Laboratory Manual, These procedures are well known in the art to the skilled 1989, Cold Spring Harbor Laboratory Press, Cold Spring artisan, as described in (Coligan, J. E. etal, Current Protocols 55 Harbor). Such cells can routinely be utilized for assaying in Immunology, 1999, John Wiley & Sons, Inc., New York; CTL activity by having said genetically engineered, or Ausubel, F. M. et al. Current Protocols in Molecular Biology, recombinant, host cells express the immunogenic peptides of 1999, John Wiley & Sons, Inc., New York; Molecular Clon the present invention. ing: A Laboratory Manual, 1989, Cold Spring Harbor Labo Various mammalian cell culture systems can also be ratory Press, Cold Spring Harbor). Thus, recombinantly pro 60 employed to express recombinant protein. Examples of mam duced peptides or polypeptides can be used as the malian expression systems include the COS-7 lines of mon immunogens of the invention. key kidney fibroblasts, described by Gluzman, Cell, 23:175 The coding sequences for peptides of the length contem (1981), and other cell lines capable of expressing a compat plated herein can be synthesized on commercially available ible vector, for example, the C127, 3T3, CHO, HeLa and automated DNA synthesizers using protocols that are well 65 BHK cell lines. Mammalian expression vectors will comprise know in the art. See for example, (Grant, G. A. Synthetic an origin of replication, a Suitable promoter and enhancer, and Peptides: A User's Guide, 1992, W. H. Freeman and Com also any necessary ribosome binding sites, polyadenylation US 7,919,467 B2 15 16 site, splice donor and acceptor sites, transcriptional termina MHC molecules as they are not efficiently processed into an tion sequences, and 5' flanking non-transcribed sequences. appropriately sized peptide in the extracellular milieu. There DNA sequences derived from the SV40 splice, and polyade a variety of approaches that are known in the art, however, that nylation sites may be used to provide the required nontran allow oligopeptides and polypeptides to be exogenously scribed genetic elements. acquired by a cell, which then allows for their subsequent The polypeptide can be recovered and purified from processing and presentation by a class I MHC molecule. recombinant cell cultures by methods including ammonium Representative, but non-limiting examples of Such Sulfate or ethanol precipitation, acid extraction, anion or cat approaches include electroporation of the molecules into the ion exchange chromatography, phosphocellulose chromatog cell (Harding, C. H. III, Eur. J. Immunol., 22:1865-1869, raphy, hydrophobic interaction chromatography, affinity 10 (1992)), encapsulation of the molecules in liposomes which chromatography, hydroxylapatite chromatography and lectin are fused to the cells of interest (Reddy, R. et al., J. Immunol. chromatography. Protein refolding steps can be used, as nec Methods, 141:157-163, (1991)), or osmotic shock in which essary, in completing configuration of the mature protein. the molecules are taken up via pinocytosis (Moore, M. W. et High performance liquid chromatography (HPLC) can be al., Cell, 54:777-785, (1988)). Thus, oligopeptides and employed for final purification steps. 15 polypeptides that comprise one or more of the peptides of the The immunogenic peptides of the present invention may be invention can be provided to antigen presenting cells in Such used to elicit CTLs ex vivo from either healthy individuals or a fashion that they are delivered to the cytoplasm of the cell, from cancer patients with cancer, Such as colorectal carci and are Subsequently processed to allow presentation of the noma, lung carcinoma, ovarian carcinoma, breast carcinoma, peptides. or prostate carcinoma. Such responses are induced by incu Antigen presenting cells Suitable for stimulating an in vitro bating in tissue culture the individual’s CTL precursor lym CTL response that is specific for one or more of the peptides phocytes together with a source of antigen presenting cells of the invention can also be prepared by introducing poly and the appropriate immunogenic peptide. Examples of Suit nucleotide vectors encoding the sequences into the cells. able antigen presenting cells include dendritic cells, mac These polynucleotides can be designed Such that they express rophages, and activated B cells. Typically, the peptide at 25 only a single peptide of the invention, multiple peptides of the concentrations between 10 and 40 ug/ml, would be pre-incu invention, or even a plurality of peptides of the invention. bated with the antigen presenting cells for periods ranging There are a variety of approaches that are known in the art, from 1 to 18 hrs. B-microglobulin (4 g/ml) can be added that allow polynucleotides to be introduced and expressed in during this time period to enhance binding. The antigen pre a cell, thus providing one or more peptides of the invention to senting cells may also be held at room temperature during the 30 the class I MHC molecule binding pathway. Representative, incubation period (Ljunggren, H.-G. et al., Nature, 346:476 but non-limiting examples of Such approaches include the 480, (1990)) or pretreated with acid (Zeh, H. J., III et al., introduction of plasmid DNA through particle-mediated gene Hum. Immunol. 39:79-86, (1994)) to promote the generation transfer or electroporation (Tuting, T. et al., J. Immunol. of denatured class I MHC molecules which can then bind the 160:1139-1147, (1998)), or the transduction of cells with an peptide. The precursor CTLs (responders) are then added to 35 adenovirus expressing the polynucleotide of interest (Perez the antigen presenting cells to which the immunogenic pep Diez, A. et al., Cancer Res., 58:5305-5309, (1998)). Thus, tide has bound (stimulators) at responder to stimulator ratios oligonucleotides that code for one or more of the peptides of of between 5:1 and 50:1, and most typically between 10:1 and the invention can be provided to antigen presenting cells in 20:1. The co-cultivation of the cells is carried out at 37°C. in such a fashion that the peptides associate with class I MHC RPMI 1640, 10% fetal bovine serum, 2 mM L-glutamine, and 40 molecules and are presented on the Surface of the antigen IL-2 (5-20 Units/ml). Other cytokines, such as IL-1, IL-7, and presenting cell, and consequently are available to stimulate a IL-12 may also be added to the culture. Fresh IL-2-containing CTL response. media is added to the cultures every 2-4 days, typically by By preparing the stimulator cells used to generate an in removing one-half the old media and replenishing it with an vitro CTL response in different ways, it is possible to control equal volume of fresh media. After 7-10 days, and every 7-10 45 the peptide specificity of CTL response. For example, the days thereafter, the CTL are re-stimulated with antigen pre CTLs generated with a particular peptide will necessarily be senting cells to which immunogenic peptide has been bound specific for that peptide. Likewise, CTLs that are generated as described above. Fresh IL-2-containing media is added to with a polypeptide or polynucleotide expressing or coding for the cells throughout their culture as described above. Three to particular peptides will be limited to specificities that recog four rounds of stimulation, and sometimes as many five to 50 nize those peptides. More broadly, stimulator cells, and more eight rounds of stimulation, are required to generate a CTL specifically dendritic cells, can be incubated in the presence response that can then be measured in vitro. The above of the whole protein. As a further alternative, stimulator cells, described protocol is illustrative only and should not be con and more specifically dendritic cells, can be transduced or sidered limiting. Many in vitro CTL stimulation protocols transfected with RNA or DNA comprising the polynucleotide have been described and the choice of which one to use is well 55 sequence encoding the protein. Under these alternative con within the knowledge of the skilled artisan. The peptide ditions, peptide epitopes that are naturally cleaved out of the specific CTL can be further expanded to large numbers by protein, and which are generated in addition to peptide treatment with anti-CD3 antibody. For example, see (Riddell, epitopes of SEQID NO:1-791 and 1514-1533 can associate S. R. and Greenberg, P. D., J. Immunol. Methods, 128:189 with an appropriate class I MHC molecule, which may or may 201, (1990); Walter, E. A. et al., N. Engl. J. Med., 333:1038 60 not include HLA-A1, A2, or -A3. The selection of antigen 1044, (1995)). presenting cells and the type of antigen with which to stimu Antigen presenting cells that are to be used to stimulate a late the CTL, is left to the ordinary skilled artisan. CTL response are typically incubated with peptide of an In certain embodiments, the methods of the present inven optimal length, most commonly a nonapeptide, that allows tion include a method for inducing a CTL response in vitro for direct binding of the peptide to the class I MHC molecule 65 that is specific for a tumor cell expressing a molecule from without additional processing. Larger oligopeptides and A1, A2, or A3 supertypes (A11 is a member of the A3 super polypeptides are generally ineffective in binding to class I type), whereby the method comprises contacting a CTL pre US 7,919,467 B2 17 18 cursor lymphocyte with an antigen presenting cell that has however, about 1x10° to about 1x10", more preferably about bound an immunogen comprising one or more of the peptides 1x10 to about 1x10', and even more preferably, about disclosed according to the invention. 1x10 to about 1x10" peptide-specific CTL are infused. In specific embodiments, the methods of the present inven Methodology for reinfusing the T cells into a patient are well tion include a method for inducing a CTL response in vitro 5 known and exemplified in U.S. Pat. No. 4,844,893 to Honski, that is specific for a tumor cell expressing a molecule from et al., and U.S. Pat. No. 4,690,915 to Rosenberg. A1, A2, or A3 supertypes, whereby the method comprises The peptide-specific CTL can be purified from the stimu contacting a CTL precursor lymphocyte with an antigen pre senting cell that has exogenously acquired an immunogenic lator cells prior to infusion into the patient. For example, oligopeptide or polypeptide that comprises one or more of the 10 monoclonal antibodies directed towards the cell surface pro peptides disclosed according to the invention. tein CD8, present on CTL, can be used in conjunction with a A yet additional embodiment of the present invention is variety of isolation techniques such as antibody panning, flow directed to a process for inducing a CTL response in vitro that cytometric sorting, and magnetic bead separation to purify is specific for a tumor cell expressing a molecule from A1, A2, the peptide-specific CTL away from any remaining non-pep or A3 Supertypes, comprising contacting a CTL precursor 15 tide specific lymphocytes or from the stimulator cells. These lymphocyte with an antigen presenting cell that is expressing methods are well known in the art, and are their selection is a polynucleotide coding for a polypeptide of the invention left to the skilled artisan. It should be appreciated that gen and wherein said polynucleotide is operably linked to a pro eration of peptide-specific CTL in this manner, obviates the moter. need for stimulating the CTL in the presence of tumor. Thus, In specific embodiments, the methods of the present inven- 20 there is no chance of inadvertently reintroducing tumor cells tion include a method for inducing a CTL response in vitro into the patient. that is specific for a tumor cell expressing HLA-A1 HLA-A2, Thus, one embodiment of the present invention relates to a or HLA-A11, whereby the method comprises contacting a process for treating a subject with cancer characterized by CTL precursor lymphocyte with an antigen presenting cell tumor cells expressing complexes of a molecule from A1, A2, that has bound an immunogen comprising one or more of the 25 or A3 supertypes, for example, HLA-A1 HLA-A2, or HLA peptides disclosed according to the invention. A11, whereby CTLS produced in vitro according to the In specific embodiments, the methods of the present inven present invention are administered in an amount Sufficient to tion include a method for inducing a CTL response in vitro destroy the tumor cells through direct lysis or to effect the that is specific for a tumor cell expressing HLA-A1 HLA-A2, or HLA-A11, whereby the method comprises contacting a 30 destruction of the tumor cells indirectly through the elabora CTL precursor lymphocyte with an antigen presenting cell tion of cytokines. that has exogenously acquired an immunogenic oligopeptide Another embodiment of the present invention is directed to or polypeptide that comprises one or more of the peptides a process for treating a subject with cancer characterized by disclosed according to the invention. tumor cells expressing any class I MHC molecule and an A yet additional embodiment of the present invention is 35 epitope of SEQ ID NO: 1-791 and 1514-1533, whereby the directed to a process for inducing a CTL response in vitro that CTLs are produced in vitro and are specific for the epitope or is specific for a tumor cell expressing HLA-A1 HLA-A2, or original protein and are administered in an amount Sufficient HLA-A11, comprising contacting a CTL precursor lympho to destroy the tumor cells through direct lysis or to effect the cyte with an antigen presenting cell that is expressing a poly destruction of the tumor cells indirectly through the elabora nucleotide coding for a polypeptide of the invention and 40 tion of cytokines. wherein said polynucleotide is operably linked to a promoter. In the foregoing embodiments the cancer to be treated may A variety of techniques exist for assaying the activity of include a colorectal carcinoma, an ovarian carcinoma, a CTL. These techniques include the labeling of target cells breast carcinoma, a lung carcinoma, and prostate carcinoma, with radionuclides such as NaCrO, or H-thymidine, and but especially ovarian carcinoma. measuring the release or retention of the radionuclides from 45 The ex vivo generated CTL can be used to identify and the target cells as an index of cell death. Such assays are isolate the T cell receptor molecules specific for the peptide. well-known in the art and their selection is left to the skilled The genes encoding the alpha and beta chains of the T cell artisan. Alternatively, CTL are known to release a variety of receptor can be cloned into an expression vector system and cytokines when they are stimulated by an appropriate target transferred and expressed in naive T cells from peripheral cell, such as a tumor cell expressing the relevant class I MHC 50 blood, T cells from lymph nodes, or T lymphocyte progenitor molecule and the corresponding peptide. Non-limiting cells from bone marrow. These T cells, which would then be examples of such cytokines include IFN-y, TNFC., and GM expressing a peptide-specific T cell receptor, would then have CSF. Assays for these cytokines are well known in the art, and anti-tumor reactivity and could be used in adoptive therapy of their selection is left to the skilled artisan. Methodology for cancer, and more specifically cancer, colorectal carcinoma, measuring both target cell death and cytokine release as a 55 ovarian carcinoma, breast carcinoma, lung carcinoma, and measure of CTL reactivity are given in (Coligan, J. E. et al. prostate carcinoma. Current Protocols in Immunology, 1999, John Wiley & Sons, In addition to their use for therapeutic or prophylactic Inc., New York). purposes, the immunogenic peptides of the present invention After expansion of the antigen-specific CTLS, the latter are are useful as screening and diagnostic agents. Thus, the then adoptively transferred back into the patient, where they 60 immunogenic peptides of the present invention, together with will destroy their specific target cell. The utility of such adop modern techniques of gene Screening, make it possible to tive transfer is demonstrated in North, R. J. et al. (Infect. screen patients for the presence of genes encoding such pep Immun., 67:2010-2012, (1999)) and Riddell, S. R. et al. (Sci tides on cells obtained by biopsy of tumors detected in such ence, 257:238-241, (1992)). In determining the amount of patients. The results of Such screening may help determine cells to reinfuse, the skilled physician will be guided by the 65 the efficacy of proceeding with the regimen of treatment total number of cells available, the activity of the CTL as disclosed herein using the immunogens of the present inven measured in vitro, and the condition of the patient. Preferably, tion. US 7,919,467 B2 19 20 Alternatively, the immunogenic peptides disclosed herein, case, the term prophylaxis can be applied to encompass both as well as functionally similar homologs thereof, may be used prevention and suppression. Conversely, the term “treatment to screen a sample for the presence of CTLs that specifically is often utilized to mean the clinical application of agents to recognize the corresponding epitopes. The lymphocytes to be combat an already existing condition whose clinical presen screened in this assay will normally be obtained from the tation has already been realized in a patient. This would occur peripheral blood, but lymphocytes can be obtained from other where an individual has already been diagnosed as having a Sources, including lymph nodes, spleen, tumors, and pleural tumor. fluid. The peptides of the present invention may then be used It is understood that the Suitable dosage of an immunogen as a diagnostic tool to evaluate the efficacy of the immuno of the present invention will depend upon the age, sex, health, therapeutic treatments disclosed herein. Thus, the in vitro 10 generation of CTL as described above would be used to and weight of the recipient, the kind of concurrent treatment, determine if patients are likely to respond to the peptide in if any, the frequency of treatment, and the nature of the effect vivo. Similarly, the in vitrogeneration of CTL could be done desired. However, the most preferred dosage can be tailored with samples of lymphocytes obtained from the patient to the individual subject, as determined by the researcher or before and after treatment with the peptides. Successful gen 15 clinician. The total dose required for any given treatment will eration of CTL in vivo should then be recognized by a corre commonly be determined with respect to a standard reference spondingly easier ability to generate peptide-specific CTL in dose as set by a manufacturer, such as is commonly done with vitro from lymphocytes obtained following treatment incom vaccines, such dose being administered either in a single parison to those obtained before treatment. treatment or in a series of doses, the success of which will The oligopeptides of the invention, such as SEQID NO: depend on the production of a desired immunological result 1-791 and 1514-1533, can also be used to prepare class I (i.e., Successful production of a CTL-mediated response to MHC tetramers which can be used in conjunction with flow the antigen, which response gives rise to the prevention and/ cytometry to quantitate the frequency of peptide-specific or treatment desired). Thus, the overall administration sched CTL that are present in a sample of lymphocytes from an ule must be considered in determining the Success of a course individual. Specifically, for example, class I MHC molecules 25 of treatment and not whether a single dose, given in isolation, comprising peptides of SEQ ID NO: 1-791 and 1514-1533, would or would not produce the desired immunologically would be combined to form tetramers as exemplified in U.S. therapeutic result or effect. Pat. No. 5,635,363. Said tetramers would find use in moni The therapeutically effective amount of a composition con toring the frequency of CTLs in the peripheral blood, lymph taining one or more of the immunogens of this invention, is an nodes, or tumor mass of an individual undergoing immuno 30 therapy with the peptides, proteins, or polynucleotides of the amount sufficient to induce an effective CTL response to the invention, and it would be expected that successful immuni antigen and to cure or arrest disease progression. Thus, this Zation would lead to an increase in the frequency of the dose will depend, among other things, on the identity of the peptide-specific CTL. immunogens used, the nature of the disease condition, the As stated above, a vaccine in accordance with the present 35 severity of the disease condition, the extent of any need to invention may include one or more of the hereinabove prevent Such a condition where it has not already been described polypeptides or active fragments thereof, or a com detected, the manner of administration dictated by the situa position, or pool, of immunogenic peptides disclosed herein. tion requiring Such administration, the weight and state of When employing more than one polypeptide or active frag health of the individual receiving Such administration, and the ment, two or more polypeptides and/or active fragments may 40 Sound judgment of the clinician or researcher. Thus, for pur be used as a physical mixture or as a fusion of two or more poses of prophylactic ortherapeutic administration, effective polypeptides or active fragments. The fusion fragment or amounts would generally lie within the range of from 1.0 ug fusion polypeptide may be produced, for example, by recom to about 5,000 ug of peptide for a 70 kg patient, followed by binant techniques or by the use of appropriate linkers for boosting dosages of from about 1.0 g to about 1,000 ug of fusing previously prepared polypeptides or active fragments. 45 peptide pursuant to a boosting regimen over days, weeks or The immunogenic molecules of the invention, including even months, depending on the recipient's response and as vaccine compositions, may be utilized according to the necessitated by Subsequent monitoring of CTL-mediated present invention for purposes of preventing, Suppressing or activity within the bloodstream. Of course, such dosages are treating diseases causing the expression of the immunogenic to be considered only a general guide and, in a given situation, peptides disclosed herein, such as where the antigen is being 50 may greatly exceed such suggested dosage regimens where expressed by tumor cells. As used in accordance with the the clinician believes that the recipient’s condition warrants present invention, the term “prevention” relates to a process more a aggressive administration schedule. Needless to say, of prophylaxis in which an animal, especially a mammal, and the efficacy of administering additional doses, and of increas most especially a human, is exposed to an immunogen of the ing or decreasing the interval, may be re-evaluated on a con present invention prior to the induction or onset of the disease 55 tinuing basis, in view of the recipient’s immunocompetence process. This could be done where an individual has a genetic (for example, the level of CTL activity with respect to tumor pedigree indicating a predisposition toward occurrence of the associated or tumor-specific antigens). disease condition to be prevented. For example, this might be For Such purposes, the immunogenic compositions accord true of an individual whose ancestors show a predisposition ing to the present invention may be used against a disease toward certain types of cancer. Alternatively, the immunogen 60 condition Such as cancer by administration to an individual by could be administered to the general population as is fre a variety of routes. The composition may be administered quently done for infectious diseases. Alternatively, the term parenterally or orally, and, if parenterally, either systemically “suppression' is often used to describe a condition wherein or topically. Parenteral routes include Subcutaneous, intrave the disease process has already begun but obvious symptoms nous, intradermal, intramuscular, intraperitoneal, intranasal, of said condition have yet to be realized. Thus, the cells of an 65 transdermal, or buccal routes. One or more such routes may individual may have become cancerous but no outside signs be employed. Parenteral administration can be, for example, of the disease have yet been clinically recognized. In either by bolus injection or by gradual perfusion over time. US 7,919,467 B2 21 22 Generally, vaccines are prepared as injectables, in the form Aerosol administration is also an alternative, requiring of aqueous Solutions or Suspensions. Vaccines in an oil base only that the immunogens be properly dispersed within the are also well known such as for inhaling. Solid forms which aerosol propellant. Typical percentages of the peptides or are dissolved or Suspended prior to use may also be formu polypeptides of the invention are 0.01%-20% by weight, pref lated. Pharmaceutical carriers, diluents and excipients are erably 1%-10%. The use of a surfactant to properly disperse generally added that are compatible with the active ingredi the immunogen may be required. Representative surfactants ents and acceptable for pharmaceutical use. Examples of such include the esters or partial esters of fatty acids containing carriers include, but are not limited to, water, saline Solutions, from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, dextrose, or glycerol. Combinations of carriers may also be palmitic, Stearic, linoleic, linolenic, olesteric and oleic acids used. These compositions may be sterilized by conventional, 10 with an aliphatic polyhydric alcohol or its cyclic anhydride. well known sterilization techniques including sterile filtra Mixed esters, such as mixed or natural glycerides may be tion. The resulting Solutions may be packaged for use as is, or employed. The surfactant may constitute 0.1-20% by weight the aqueous Solutions may be lyophilized, the lyophilized of the composition, preferably 0.25-5%. Typical propellants preparation being combined with sterile water before admin 15 for Such administration may include esters and similar chemi istration. Vaccine compositions may further incorporate addi cals but are by no means limited to these. A carrier, such as tional Substances to stabilize pH, or to function as adjuvants, lecithin for intranasal delivery, may also be included. wetting agents, or emulsifying agents, which can serve to The peptides and polypeptides of the invention may also be improve the effectiveness of the vaccine. delivered with an adjuvant. Adjuvants include, but are not The concentration of the CTL stimulatory peptides of the limited to complete or incomplete Freund's adjuvant, Mon invention in pharmaceutical formulations are subject to wide tanide ISA-51, Lymphocyte Activation Gene-3 (LAG-)3, alu variation, including anywhere from less than 0.01% by minum phosphate, aluminum hydroxide, alum, and saponin. weight to as much as 50% or more. Factors such as volume Adjuvant effects can also be obtained by injecting a variety of and Viscosity of the resulting composition must also be con cytokines along with the immunogens of the invention. These sidered. The solvents, or diluents, used for Such compositions 25 cytokines include, but are not limited to IL-1, IL-2, IL-7, include water, possibly PBS (phosphate buffered saline), or IL-12, and GM-CSF. saline itself, or other possible carriers or excipients. The peptides and polypeptides of the invention can also be The immunogens of the present invention may also be added to professional antigen presenting cells Such as den contained in artificially created structures such as liposomes, dritic cells that have been prepared ex vivo. For example, the ISCOMS, slow-releasing particles, and other vehicles which 30 dendritic cells could be prepared from CD34 positive stem increase the immunogenicity and/or half-life of the peptides cells from the bone marrow, or they could be prepared from or polypeptides in serum. Liposomes include emulsions, CD14 positive monocytes obtained from the peripheral foams, micelies, insoluble monolayers, liquid crystals, phos blood. The dendritic cells are generated ex vivo using cytok pholipid dispersions, lamellar layers and the like. Liposomes ines such as GM-CSF, IL-3, IL-4, TNF, and SCF. The cultured for use in the invention are formed from standard vesicle 35 DC are then pulsed with peptides at various concentrations forming lipids which generally include neutral and negatively using standard methods that are well known in the art. The charged phospholipids and a sterol, Such as cholesterol. The peptide-pulsed dendritic cells can then be administered either selection of lipids is generally determined by considerations intraveneously, Subcutaneously, or intradermally, and the such as liposome size and stability in the blood. A variety of immunization may also include cytokines such as IL-2 or methods are available for preparing liposomes as reviewed, 40 IL-12. for example, by (Coligan, J. E. et al. Current Protocols in The present invention is also directed to a vaccine in which Protein Science, 1999, John Wiley & Sons, Inc., New York) an immunogen of the present invention is delivered or admin and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4.837,028, istered in the form of a polynucleotide encoding the a and 5,019,369. polypeptide or active fragment as disclosed herein, whereby Liposomes containing the peptides or polypeptides of the 45 the peptide or polypeptide or active fragment is produced in invention can be directed to the site of lymphoid cells where vivo. The polynucleotide may be included in a suitable the liposomes then deliver the selected immunogens directly expression vector and combined with a pharmaceutically to antigen presenting cells. Targeting can be achieved by acceptable carrier. For example, the peptides or polypeptides incorporating additional molecules such as proteins or could be expressed in plasmid DNA and nonreplicative viral polysaccharides into the outer membranes of said structures, 50 vectors such as vaccinia, fowlpox, Venezuelan equine thus resulting in the delivery of the structures to particular encephalitis virus, adenovirus, or other RNA or DNA viruses. areas of the body, or to particular cells within a given organ or These examples are meant to be illustrative only and should tissue. Such targeting molecules may a molecule that binds to not be viewed as self-limiting A wide variety of other vectors receptor on antigen presenting cells. For example an antibody are available and are apparent to those skilled in the art from that binds to CD80 could be used to direct liposomes to 55 the description given herein. In this approach, a portion of the dendritic cells. nucleotide sequence of the viral vector is engineered to The immunogens of the present invention may also be express the peptides or polypeptides of the invention. Vac administered as solid compositions. Conventional nontoxic cinia Vectors and methods useful in immunization protocols Solid carriers including pharmaceutical grades of mannitol, are described in U.S. Pat. No. 4,722,848, the disclosure of lactose, starch, magnesium, cellulose, glucose. Sucrose, 60 which is incorporated herein by reference in its entirety. Sodium saccharin, and the like. Such solid compositions will Regardless of the nature of the composition given, addi often be administered orally, whereby a pharmaceutically tional therapeutic agents may also accompany the immuno acceptable nontoxic composition is formed by incorporating gens of the present invention. Thus, for purposes of treating the peptides and polypeptides of the invention with any of the tumors, compositions containing the immunogens disclosed carriers listed above. Generally, Such compositions will con 65 herein may, in addition, contain other antitumor pharmaceu tain 10-95% active ingredient, and more preferably 25-75% ticals. The use of Such compositions with multiple active active ingredient. ingredients is left to the discretion of the clinician. US 7,919,467 B2 23 24 In addition, the immunogens of the present invention can the antibodies are synthesized and, possibly, optimized for be used to stimulate the production of antibodies for use in binding to the polypeptides disclosed herein as being recep passive immunotherapy, for use as diagnostic reagents, and tors. Such antibodies may be chimeric or humanized antibod for use as reagents in other processes such as affinity chro ies and may be fully tetrameric instructure, or may be dimeric matography. and comprise only a single heavy and a single light chain. The present invention also relates to antibodies that react Such antibodies may also include fragments, such as Fab and with immunogens, such as a polypeptide comprising one or F(ab)' fragments, capable of reacting with and binding to any more of the epitopic peptides of SEQ ID NO: 1-791 and of the polypeptides disclosed herein as being receptors. 1514-1533 as disclosed herein. Active fragments of such A further embodiment of the present invention relates to a antibodies are also specifically contemplated. Such antibod 10 method for inducing a CTL response in a Subject comprising ies, and active fragments of such antibodies, for example, and administering to Subjects that express HLA-A1 antigens an Fab structure, may react with, including where it is highly effective (i.e., CTL-stimulating amount) of an immunogen of selective or specific for, an immunogenic structure compris the invention that does not comprise the entire protein ing 2, 3, 4 or more of the epitopic peptides of the invention. expressing the epitopic peptides disclosed herein (i.e., one With the advent of methods of molecular biology and that comprises less than the entire protein where the protein is recombinant technology, it is now possible to produce anti 15 a naturally occurring polypeptide) in an amount Sufficient to body molecules by recombinant means and thereby generate induce a CTL response to tumor cells expressing at least gene sequences that code for specific amino acid sequences HLA-A1 or HLA-A2, as the case may be, thereby eliciting a found in the polypeptide structure of the antibodies. Such cellular response against said tumor cells. antibodies can be produced by either cloning the gene A still further embodiment of the present invention relates sequences encoding the polypeptide chains of said antibodies to a method for inducing a CTL response in a subject, wherein or by direct synthesis of said polypeptide chains, with in vitro the immunogen is in the form of a polynucleotide. In one assembly of the synthesized chains to form active tetrameric non-limiting example, the method comprises administering (HL) structures with affinity for specific epitopes and anti to subjects that express HLA-A1 at least one CTL epitope, genic determinants. This has permitted the ready production wherein said epitope or epitopes are selected from a group of antibodies having sequences characteristic of neutralizing 25 comprising the peptides disclosed according to the invention, antibodies from different species and Sources. and are coded within a polynucleotide sequence that does not Regardless of the source of the antibodies, or how they are comprise the entire protein coding region, in an amount Suf recombinantly constructed, or how they are synthesized, in ficient to induce a CTL response to tumor cells expressing vitro or in Vivo, using transgenic animals. Such as cows, goats HLA-A1 or HLA-A2. and sheep, using large cell cultures of laboratory or commer 30 While the below examples are provided to illustrate the cial size, in bioreactors or by direct chemical synthesis invention, it is to be understood that these methods and employing no living organisms at any stage of the process, all examples in no way limit the invention to the embodiments antibodies have a similar overall 3 dimensional structure. described herein and that other embodiments and uses will no This structure is often given as H2L2 and refers to the fact that doubt suggest themselves to those skilled in the art. All pub antibodies commonly comprise 2 light (L) amino acid chains 35 lications, patents, and patent applications cited herein are and 2 heavy (H)amino acid chains. Both chains have regions hereby incorporated by reference, as are the references cited capable of interacting with a structurally complementary therein. It is also to be understood that throughout this dis antigenic target. The regions interacting with the target are closure where the singular is used, the plural may be inferred referred to as “variable' or “V” regions and are characterized and vice versa and use of either is not to be considered by differences in amino acid sequence from antibodies of limiting. different antigenic specificity. 40 The variable regions of either H or L chains contains the EXAMPLE 1. amino acid sequences capable of specifically binding to anti genic targets. Within these sequences are Smaller sequences Cell Lines dubbed “hypervariable” because of their extreme variability For HLA-A1 and HLA-A11 studies, ARGOV57, a HLA between antibodies of differing specificity. Such hypervari 45 A1/11 positive ovarian cell line, was established by culturing able regions are also referred to as “complementarity deter tumor cells from an asciitic fluid from an ovarian patient. mining regions' or “CDR' regions. These CDR regions For HLA-A2 studies, OVCAR3, a HLA-A2 positive ova account for the basic specificity of the antibody for a particu rian carcinoma cell line, was established by culturing tumor lar antigenic determinant structure. cells from an asciitic fluid from an ovarian patient. The CDRS represent non-contiguous stretches of amino 50 SKOV3-A2, a HLA-A2 stably expressing ovarian carci acids within the variable regions but, regardless of species, noma cell line, was established by culturing tumor cells from the positional locations of these critical amino acid sequences an ascitic fluid from an ovarian patient and transduced with within the variable heavy and light chain regions have been HLA-A2 gene. found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light EXAMPLE 2 chains of all antibodies each have 3 CDR regions, each non 55 contiguous with the others (termed L1, L2, L3, H1, H2,H3) Immunoaffinity Purification for the respective light (L) and heavy (H) chains. The ARGOV57 cells were grown in 10-chamber Nunc cell accepted CDR regions have been described in the text and factories (Fisher, Pittsburgh, Pa.). The cells were harvested by figures of Kabatetal. (J. Biol. Chem. 252:6609-6616 (1977)). treatment with 0.45% trypsin and 0.32 mM EDTA, washed In all mammalian species, antibody polypeptides contain 60 two times in phosphate-buffered saline solution (pH 7.4), and constant (i.e., highly conserved) and variable regions, and, stored as cell pellets at -80° C. Aliquots of 6-8x10" cells within the latter, there are the CDRs and the so-called “frame were solubilized at 5-10x10 cells/ml in 20 mM Tris, pH 8.0, work regions’ made up of amino acid sequences within the 150 mM. NaCl, 1% CHAPS, 18.5 ug/ml iodoacetamide, 5 variable region of the heavy or light chain but outside the ug/ml aprotonin, 10ug/ml leupeptin, 10 g/ml pepstatin A, 5 CDRS. 65 mM EDTA, 0.2% sodium azide, and 17.4 ug/ml phenylmeth The antibodies disclosed according to the invention may ylsulfonyl fluoride for 1 h. This and all subsequent steps were also be wholly synthetic, wherein the polypeptide chains of performed with ice-cold solutions and at 4°C. The lysates US 7,919,467 B2 25 26 were then centrifuged at 100,000xg, the pellets discarded, tion assays, whereas the remaining four-fifths of the effluent and the Supernatants passed through a 0.22 um filter. The was directed into the mass spectrometer. Ions were formed by Supernatants were then passed over a series of columns with electrospray ionization, and mass spectra were recorded by the first containing Sepharose, and the second containing the scanning between mass to charge ratios (m/z) 300 and 1400 HLA-A1-specific monoclonal antibody, GAP-A1, bound to a every 1.5 seconds. Peptide sequences were determined by protein A-Sepharose matrix. The second column was then CAD (collision-activated dissociation) tandem mass spec sequentially washed with 20 column volumes of 20 mM Tris, trometry as described in the literature (Hunt, D. F. et al., Proc. pH 8.0, 150mMNaCl, 20 column volumes of 20 mM Tris, pH Natl. Acad. Sci. U.S.A, 83:6233-6237, (1986)). 8.0, 1.0 MNaCl, and 20 column volumes of 20 mM Tris, pH 8.0. The peptides were eluted from the column with 5 column 10 EXAMPLE 5 Volumes of 10% acetic acid. The isolated HLA-A1 molecules were then boiled for 5 min to further dissociate any bound Homology Searches of Identified Peptide Sequences peptide from the heavy chains. The peptides were then sepa Proteins containing peptides corresponding to the masses rated from the co-purifying class I heavy chain and B-micro identified by MS were analyzed with the search algorithm, globulin by centrifugation on a Ultrafree-CL membrane with 15 SEQUEST. Searches were also carried out on the GenBank a nominal molecular weight cut-off of 5,000 Daltons (Milli non-redundant sequence database (http:/ncbi.nlm.nih.gov/ pore, Bedford, Mass.). For a separate study, OVCAR3 or SKOV3 cells were suc Entrez/) as well as on our own unique database of 2943 cessfully prepared using the same procedure as just described specific sequences compiled from GenBank and EST data except that HLA-A2 molecules were prepared using HLA base entries. Upon experimental confirmation of the peptide Sequence, a tRLASTn search of the GenBank non redundant A2 specific antibodies. databaxe was performed to identify any genes containing the EXAMPLE 3 DNA sequence encoding the peptice. Peptide Fractionation EXAMPLE 6 The peptide extracts were fractionated by RP-HPLC (Re 25 versed Phase-High Performance Liquid Chromatography) Peptide Synthesis using an Applied Biosystems (ABI) model 140B system. The Peptides were synthesized using a Gilson (Madison, Wis.) extracts were concentrated by vacuum centrifugation from AMS 422 multiple peptide synthesizer. Quantities of 10uMol about 20 ml down to 250 ul and injected into either a Brown were synthesized using conventional FMOC amino acids, lee (Norwalk, Conn.) C. Aquapore column (2.1 mmx3 cm; 30 resins, and chemical techniques. Peptides were purified by 300 A; 7um) or a Higgins (Mountain View, Calif.) C18Haisil RP-HPLC using a 4.6 mmx100mm POROS (Perseptive Bio column (2.1 mmx4 cm; 300 A; 5um). The peptides were systems, Cambridge, Mass.) column and a 10 min, 0-60% eluted by first using a gradient of acetonitrile/0.085% TFA acetonitrile in 0.1% TFA gradient. (trifluoroacetic acid) in 0.1%TFA/water, with the concentra tion of acetonitrile increasing from 0-9% (0-5 minutes), 35 EXAMPLE 7 9-36% (5-55 minutes), and 36-60% (55-62 minutes). A sec ond dimension fractionation of combined fractions 17 and 18 Generation of Monocyte-Derived DC and Peptide Loading from the first dimension (TFA) fraction was accomplished using the same gradient but with the substitution of HFBA PBMC were purified from HLA-A2" normal donor blood (heptafluorobutyric acid) for TFA. The flow rate was 200 using lymphocyte separation media (Cappel ICN Biomedi ul/min, and fractions were collected at 1 min (Brownlee col 40 cal, Aurora, Ohio). PBMC (5.3x10) were added to individual umn) or 40 second (Higgins column) intervals. A third dimen wells of a 24-well cluster plate (Costar, Corning, N.Y.) in 1.0 sion of RP-HPLC was achieved using an Eldex (Napa, Calif.) ml of serum-free AIM-V medium (Life Technologies) and MicroPro Pump, a homemade Cs microcapillary column, allowed to adhere for 60 min at 37° C. Non-adherent cells and an ABI model 785A UV absorbance detector. The col were removed and saved as a source of effector T cells. umn was prepared by packing a 27 cm bed of 10 Lim Cs 45 Adherent PBMC (-8.3x10/well) were then pulsed with 50 particles in a section of 285 um o.d./75 um i.d. fused silica mg/ml synthetic peptides in serum-free AIM-V medium con (Polymicro Technologies, Phoenix, Ariz.). Peptides in com taining 1.5 mg/ml f-microglobulin (Calbiochem-Novabio bined fractions 26 and 27 of the second dimension fraction chem, San Diego, Calif.) and incubated for 2 h at 37° C. were loaded onto this column and eluted with a gradient of Unbound peptides were aspirated and the wells washed with acetonitrile/0.67% triethylamine acetate/water in 0.1% tri 50 media. ethylamine acetate?water, with the concentration of acetoni Monocyte-derived DC were generated as follows. PBMC trile increasing from 0-60% in 40 minutes. The flow rate was (5.3x107) were allowed to adhere in T-75 flasks (Corning) in about 300 nl/min, and fractions were collected into 25 ul of 10 ml of Serum-free AIM-V medium for 60 min at 37° C. water every 30s. In all RP-HPLC experiments, peptides were Non-adherent cells were collected as a source of effector T detected by monitoring UV absorbance at 214 nm. 55 cells and pooled with the previous collection above. Adherent monocytes inflasks were then exposed to recombinant human EXAMPLE 4 granulocyte macrophage colony stimulating factor (GM Mass Spectrometric Analysis CSF, 25 ng/ml. Peprotech) and recombinant human IL-4 (100 The second dimension HPLC fraction was analyzed using ng/ml. Peprotech) in 10 ml of AIM-V medium containing an affluent splitter on the microcapillary HPLC column. In 60 10% heat-inactivated FBS. DC obtained by this methodim this experiment, the column (360 umo.d.x100 um i.d. with a mature DC (iDC) are characterized by expression of low 25 cm Cs bed) was butt connected with a zero dead volume levels of CD83, CD80, CD86, and HLA class I and class II tee (Valco, Houston, Tex.) to two pieces of fused silica of molecules (data not shown). different lengths (25um and 40 umi.d.). Peptides were eluted Mature DC (mDC) were obtained by exposing day 5 DC with a 34 min gradient of 0-60% acetonitrile. The 25 m 65 cultures to recombinant soluble CD40 ligand (SCD40L: Pep capillary deposited one-fifth of the HPLC effluent into the rotech) at 1.5 mg/ml for 24 h in the presence of 25 ng/ml wells of a microtiter plate for use in CTL epitope reconstitu GM-CSF and are characterized by expression of high levels US 7,919,467 B2 27 28 of CD80, CD86, and HLA class I and class II molecules. EXAMPLE 11 mDC were harvested, washed, pulsed with 5 mg/ml peptide in serum-free AIM-V medium and irradiated (5000 rad) prior to 'Cr-Release Cytotoxicity Assay use as stimulators. The standard 4-h Cr-release assay was performed in 5 96-well V-bottomed microplates. Target cells in suspension (T2, C1RA2, B-LCL and K562) were labeled with 100 mCi EXAMPLE 8 NaCrO4 (NEN Life Science, Boston, Mass.) per 1.3x10 cells either overnight (-6+18 h) in 5 ml RPMI 1640 media Generation of Peptide-Specific CTL containing 2+5% FBS or for 60+90 min at 37° C. directly The protocol used here is a modification of the method 10 with the cell pellet in the case of adherent cells (tumor cell described by Plebanski et al. (Eur. J. Immunol. 25:1783, lines and control lines). Labeling was terminated by washing (1995)). CTL to peptide were generated by 3+4 cycles of the targets with cold media containing 5% FBS for a total of stimulation with peptide-loaded APC. For the first round of three washes. Target cells were resuspended at a concentra stimulation (day 0), T cells or non-adherent PBMC from 15 tion of 2-3x10"/ml. About 2-3x10 targets in 100 ml were above (2.3x10°/ml or 4.3x10° per well) were added in bulk delivered to each well containing CTL (effectors) seeded at (CD4", CD8", NK, etc.) to adherent PBMC-loaded peptides different E:T ratios. Spontaneous release wells contained tar in serum-free medium (50 mg/ml), f-microglobulin (1.5 gets in media alone, while maximal release wells contained mg/ml) (Calbiochem-Novabiochem), recombinant human targets in 2% NP-40 detergent (Igepal CA-630; Sigma). HLA IL-7(5 ng/ml) (Peprotech) and keyhole limpet hemocyanin (5 restriction of CTL-mediated killing was achieved by prein mg/ml) (Sigma, St Louis, Mo.). Cultures were re-stimulated cubation of targets with HLA-specific antibodies prior to with iDC every 7 days, pulsed with varying amounts of pep incubation with CTL. tide (second round 25 mg/ml, third round 10 mg/ml) and The plate was gently spun for 1+2 min and incubated at 37° irradiated (5000 rad) on day 8. At each re-stimulation, the T C. for 4h. For harvesting assay plates, 100 ml of supernatants cells were transferred to new plates by first aspirating 70% of 25 from the wells was transferred to counting tubes (USA Sci spent media in wells and then transferring the pooled contents entific) and g counts were determined in a g counter (ICN to a new plate. Fresh IL-7 was added at each re-stimulation. Micromedic Systems, Huntsville, Ala.). Cytolytic activity of The responder:stimulator (T cell:DC) ratio was set at 20 for T cells was expressed in percent specific lysis as follows: each stimulation. Recombinant human IL-2 (10 U/ml) was specific lysis-experimental release (c.p.m.)tspontaneous added on day 5 after each re-stimulation. 30 release (c.p.m.)/maximal release (c.p.m.)tspontaneous Prior to 'Cr-release assay, the T cells were harvested and release (c.p.m.). CD8" T cells were purified by positive selection using CD8" microbeads immunomagnetic cell separation with MACS kit EXAMPLE 12 (Miltenyi Biotec, Auburn, Calif.). If a fourth round of stimu 35 Competitive Inhibition Assay lation was necessary following CTL analysis, the CTL were Peptide-stimulated CTL were reacted with 'Cr-labeled pulsed as before, except with 5-10 mg/ml of peptide. Ov2 tumor cells (E:T ratio of 40) in the presence of excess of cold targets in a 4-hCr-release assay. Cold targets were either EXAMPLE 9 empty T2 cells, T2 cells pulsed with 1 mg/ml relevant peptide 40 (used to stimulate CTL) or irrelevant (control) peptides Generation of Allospecific CTL (HER-2/neu 369+377 or MART 127+35), or IFN-y pre treated tumor cells (SKOV3.A2 and OVCAR3) with the cold HLA-A2-allospecific CTL were obtained in a mixed lym target in 5-fold excess of the hot target. Results indicate that phocyte reaction by repeated stimulation of HLA-A3 PBMC (i) CTL show specific interaction with the peptide to which (responders) with irradiated HLA-A2" stimulator PBMC at a 45 they are sensitized to, and (ii) CTL compete for similar ratio of 10:1 in the presence of 10U/ml IL-2. Stimulation was epitopes presented on Ov2 as were found in SKOV3.A2 and repeated weekly with PBMC from different HLA-A2" OVCAR3 cell lines by MS. donors so as to minimize alloresponse to non-HLA-A2 anti gens. T cells were assessed for lysis on several HLA-A2" TABL E 2 targets including tumor cells, EBV-B cells and HLA-A3" 50 targets every week after the third round of stimulation. Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 EXAMPLE 10 Parent 55 Swiss CTL Expansion Prot SEQ Identi Expansion of large numbers of peptide-specific or HLA ID Parent Sequence fication A2-allospecific CTL was achieved by culturing NO: Fragment Identification No. 5.3x10+1.3x10T cells around day 6 or 7 post peptide- or 1. AEAEFYROV BCL - 6 corepressor long Q6W2J9 allostimulation in the presence of 2.5-3.0x10 irradiated 60 (5000 rad) allogeneic normal donor PBMC coated with anti 2 IYNGDMEKI isoform E1B 19K/Bcl-2- Q12983 CD3 antibody at 10 ng/ml (BD PharMingen, San Diego, interacting protein Calif.) and 25 U/ml of recombinant human IL-2 (Peprotech) Nip3 in a final volume of 30 ml RPMI medium. Media changes 3 KEFDGKSLW Similar to Heat shock PO8238 with IL-2 addition (50 U/ml) were effected on days 5 and 8. 65 protein HSP 90-beta Cells were harvested for cytotoxicity assays on days 10+12 (HSP 84) (HSP 90) and re-stimulated or frozen for later use. US 7,919,467 B2 29 30 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. HIPAGTLVOV Cytochrome P450 11B2, P19099 23 GNGAPDVFOT Adaptor-related protein mitochondrial precursor NFO1 O1953.7

SLAEGLRTW 2'-5' oligoadenylate 24 IDAIRIPWL Lung alpha/beta hydro synthetase 3 15 lase protein 1 YLGDGPKLW 26S protease regulatory P62191 25 FIASKGWKLW Alpha-actinin-3 Q08043 subunit 4 (P26 S4) 26 HRPDLIDY Alpha-actinin-3 Q08043 YLASLIRSW 26S proteasome non P51665 ATPase regulatory 27 SPOGLELALPS Ankyrin-2 (Brain Q01484 subunit 7 ankyrin) (Ankyrin-B)

FWDDYTVRV 26S proteasome non OOO487 28 KIWKRPSLOFL Ankyrin repeat and SOCS ATPase regulatory box protein 17 subunit 14 29 TLWTVSAAKT Anti-colorectal carcin KLLEPWILL 40S ribosomal protein P62249 25 oma heavy chain S16 3 O KVLDGSPIEW APOBEC1 complementation KLIEWDDERKL 40S ribosomal protein P62753 factor (APOBEC1 S6 (Phosphoprotein stimulating protein) NP33) 30 31 FLAEHPNWTL Probable DNA dC->dU RLFEGINALL 40S ribosomal protein P46781 editing enzyme APOBEC S9 3D (EC 3.5. 4.- )

TLYEAWREW 60S ribosomal protein 32 NLVODSLDL Apollipoprotein-L4 pre L10a (CSA-19) cursor (Apollipoprotein 35 L-IV) NMWAKWDEW 60S ribosomal protein L10a (CSA-19) 33 ISENEKLOK Apoptosis stimulating of p53 protein 1 SLIKQIPRI 60S ribosomal protein L10a (CSA-19) 34 WLAARNPAKW Nucleoporin 188 kDa (arachin) FLSEEGGHWAW 6-phosphofructo-2- O16877 40 kinase/fructose-2, 6– 35 RYFDGNLEKL Protein ariadne-1 homo biphosphatase 4 (6PF-2- log (ARI-1) (Ubiquitin K/Fru-2, 6-P2ASE testis conjugating enzyme E2 type isozyme) binding protein 1)

16 IETINFHEW Cleavage and polyadeny 45 36 TLADWLYHW Set1/Ash2 histone lation specificity methyltransferase com factor, 73 kDa subunit plex subunit ASH2 (CPSF 73 kDa subunit) (ASH2-like protein)

17 YLN DLIHSW A kinase anchor protein O43572 37 ATP synthase FO subunit 10, mitochondrial 50 8 Splice isoform 2 of precursor Q9H7FO

18 RWA PEERHPWL Actin, cytoplasmic 1. P60709 38 ISSMLWLFF ATPase family homo (Beta-actin) log up-regulated in Senescence cells 19 KIPVOLV Activated T-cell marker 55 Probable phospholipid CD109 transporting 39 SPDEGALWRA ATPase 1A (EC 3.. 6.3.1) LSD FLKANW Activin receptor type P27 O37 (Chromaffin granule 2A precursor (EC ATPase II) 2.7.11.30) 60 4 O ILLITLIPY ATP-binding cassette 21 DLC FEKVNV ADAM19 protein A1O

22 DINAQL AP-1 complex subunit Q10567 41 NLEQQETEP ATP-binding cassette beta-1 (Adapter-related sub-family A member 2 protein complex 1 beta (ATP-binding cassette 1 subunit) (Beta 65 transporter 2) (ATP adapt in 1) binding cassette 2) US 7,919,467 B2 31 32 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 42 RKWLYWMEL Autoantigen RCD8 6 O MLNEHDFEW Breast cancer 1 early Onset 43 EAIPARKLK Xonemal dynein heavy chain 8 61 WNTDFSPYL Breast cancer 1 early 15 Onset 44 SLRLENITW Butyrophilin-like pro tein 8 precursor 62 EFMLWYKFAR Breast and ovarian cancer susceptibility 45 SYWL KKAOW Ubiquitin carboxyl protein terminal hydrolase 20 (EC 3.1.2.15) 63 TLWWDPYEW BTG2 protein (NGF P78543 inducible anti-proli 46 KLIH PKLEY Bardet-Biedl syndrome 7 ferative protein PC3) protein (BBS2-like protein 1) 64 FLDHIIASW Nuclear protein 5gNCA

47 EFDO LDOEN Large proline-rich pro P4 8634 65 TLNDREYOL CAD protein Includes: tein BAT2 (HLA-B-assoc 25 Glutamine-dependent iated transcript 2) carbamoyl-phosphate synthase (EC 6.3. 5.5); 48 TWLL RIGDEL Bcl-2 related ovarian Aspartate carbamoyl killer transferase (EC 2.1.3.2) ; Dihydrooro - 49 LFEI Lipopolysaccharide PSO851 30 tase (EC 3.5.2.3) responsive and beige like anchor protein 66 WEWMVNDVN Cadherin EGF LAG seven (CDC4-like protein) pass G-type receptor 3 precursor (Flamingo SO KLELDETGOE Splice isoform 3 of P356.12-3 homolog 1) (hEmi 1) P35 612 35 (Multiple epidermal growth factor-like do 51 LAIGAFTIII UDP-GlcNAc : beta Gal mains 2) (Epidermal beta-1,3-N-acetylgluco growth factor-like 1) saminyltransferase 3 (EC 2.4.1. - ) 67 LSIYLSIYL Cadherin FIB3 40 52 QILLDETLK Cell growth inhibiting O2 TTR2 68 SLSMWNHRL Integrin alpha-3 pre P26 OO6 protein 39 cursor (Galactoprotein B3) 53 DECITNLLW BH3-interacting domain Pssssf. 69 Calcineurin B homolo O43745 death agonist (BID) gous protein 2 (Hepato 45 cellular carcinoma-as 54 TWWSGSNWILN CD48 antigen precursor POS326 sociated antigen 52O) (B-lymphocyte activa tion marker BLAST-1 70 MTDKAPPGV Calcium/calmodulin dependent protein ki 55 SLDERPWAW Bone morphogenetic pro Q13873 nase II inhibitor alpha tein receptor type-2 50 (CaMKIINalpha) precursor (EC 2.7.11.30) 71. WTNPOFKI Calpain-11 (EC 3. 4. 22. - ) 56 MVDSOOKSP Bullous pemphigoid an tigen 1, isoform 7 72 IMAOLPOEOKA Alpha-1 catenin (Cad P35221 55 herin-associated pro SLLLLPEKN BRCA1 associated RING tein) (Alpha E-catenin) domain 1 variant 73 KIDPLEWEE Neural cell adhesion 58 WLCWSDIISL Breast cancer type 2 PS1587 molecule variant susceptibility protein (Fanconi anemia group 60 74 KLPEKWESW Ribosomal L1 domain Of 6021 D1 protein) containing protein 1 (Cellular senescence 59 FLPDPSALONL Protein BRE (Brain and inhibited gene protein) reproductive organ expressed protein) LIEKEKWLN CENP-F kinetochore pro P494.54 (BRCA1/BRCA2 - containing 65 tein (Centromere pro complex subunit 45) tein F) (Mitosin) US 7,919,467 B2 33 34 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 76 FEVKEDOVK Centaurin-delta 1 (Cnt 96 TWLDFGWLASI Cyclin M3, isoform 1 d1) (Arf-GAP, Rho- GAP, ankyrin repeat and 97 MPSETPOAE Cystathionine beta pleckstrin homology do synthase human homolog main-containing protein 15 of Cynomolgus monkey 2) gene product

77 DTEAEKSOV Centrosomal protein 2 98 FILEALRKT Cytochrome P450 2E1 (EC POS181 (Centrosomal Nek2-as 1. 14.14.1) sociated protein. 1) (C- NAP1) 99 KMLETKWSL Keratin, type II cyto POsf87 skeletal 8 78 FLKEHMDEW Pericentriol material. 1 Q15154 1 OO QPLLKOSPW CPEB2 protein 79 KLIGELHTL Pericentriol material. 1 Q15154 YLLPAIWHI Probable ATP-dependent P17844 TLWEAFPTL Cervical cancer sup RNA helicase DDX5 (EC pressor gene 5 25 3. 6.1. - )

81 OSNKGFVVIN T-complex protein 1 Q92526 1 O2 KLLPGDIHOI Dedicator of cytokine Q14.185 subunit zeta-2 sis protein 1

82 LADGALIYR Chemokine-like factor 103 Development and differ O4315 O (C32) 30 entiation-enhancing factor 2 83 GLGAEIEIR Vacuolar protein sorting 13A 104 NAEWLLWSEI Probable ubiquitin OOOSOf carboxyl-terminal hy 84 GKLILLDKL Chromodomain-helicase O14647 drolase FAF-Y (EC DNA-binding protein 2 35 3.1.2.15) (EC 3.. 6.1. - ) 105 RLWGEPWNL Probable ubiquitin car OOOSOf 85 POTICRKP FERM domain-containing boxyl-terminal hy protein 6 drolase FAF-Y (EC 3.1.2.15) 86 RSYYLNEI Putative protein C21orf56 40 106 OLIDLSSPLI G2 and S phase ex pressed protein 1 87 TTITWSPFY Adiponutrin (iPLA2 epsilon) 1. Of YIDYTGAAYA HUMAN CDNA FLJ3O829 fis, clone 88 RLPDDDPTAW Coatomer subunit gamma FEBRA2001790, highly 2 45 similar to Xenopus laevis RRM-containing 89 LWAISTWSFSI Sodium/potassium/ protein SEB-4 mRNA calcium exchanger 2 precursor 108 WIENKSDEKWI KIAA1799 protein

9 O WLIDYORNV Exportin-1 (Chromosome O1498O 50 109 PSPOLWTV Peroxisomal prolifera region maintenance 1 tor-activated receptor protein homolog) A-interacting complex 285 kDa protein (EC 91 SILNEGGIK CUB and Sushi domain 3. 6.1. -) (ATP-dependent containing protein 3 helicase PRIC285) precursor 55 11O EGRGGIPAGLPW HUMAN KIAA1922 92 YMADRILLGW Cullin-7 (CUL-7) Q14999 111 Transcription elonga OOO267 93 YLKDLIEEW Cyclic AMP-dependent P18848 tion factor SPT5 (DLC transcription factor 1) (deleted in liver ATF-4 60 cancer-1)

94 YLDIKCLLD S-phase kinase-associ 112 DNA damage-binding pro Q16531 ated protein 1A (Cyclin tein 1 (Damage-specific A/CDK2-associated DNA-binding protein 1) protein p19) 65 113 ONFVDSKEV DNA excision repair 95 PCLSELHKA Cyclin-A1 P78396 protein ERCC-6 US 7,919,467 B2 35 36 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 114 ALIEKLWEL DNA polymerase alpha Q14181 31 KLIEKLDIKL Elongation factor 2 P13639 subunit B (DNA poly (EF-2) merase alpha 70 kDa subunit) 32 YLNEIKDSW Elongation factor 2 P13639 15 (EF-2) 115 WIEDDWNMAIR DNA replication licens P49736 ing factor MCM2 (Mini 33 YLAEKYEWDW Elongation factor 2 P13639 chromosome maintenance (EF-2) protein 2 homolog) 34 WFEESOVAGT Elongation factor 2 P13639 116 SODEIKOEW DNA2-like homolog (EC P51530 (EF-2) 3. 6.1. - ) (DNA replica tion ATP-dependent hel 35 DAQKEIWRAOK J domain protein i case-like homolog) C21orf55

117 HLNGSCHILLI Estrogen response ele Q77798 36 DLEETWFTAS J domain protein ment binding protein C21orf55 (cotton-top Tarmarin), 25 DNA2-like homolog 37 AMLEGGWDGLL EMILIN-3 precursor (human) (EMILIN-5) (Elastin mi crofibrill interface 118 ALIDRMWNL DNA damage-inducible P35638 located protein 5) transcript 3 (DDIT-3) (Growth arrest and DNA 30 138 RKADEKRIR Synaptotagmin-like pro damage-inducible pro tein 4 (Exophilin-2) tein GADD153) 139 ALQEMWHOV Enhancer of filamenta Q14511 119 SOKIOEAVKA DNA-directed RNA poly tion 1 (HEF1) merase I largest sub unit (EC 2.7.7.6) 35 14 O ILAINKPONK Enhancer of filamenta Q14511 tion 1 (HEF1) 12O LFDLVEEVO DnaJ homolog subfamily C member 1 141 SMYGWDLHHA Band 4.1-like protein 3 (4.1B) (Differentially 121 LLAALL LDP Splice isoform 2 of P354 62-2 expressed in adenocar P354.62 cinoma of the lung pro 40 tein. 1) (DAL-1) 122 FLDESRSTOYM RuvB-like 2 (EC 3. 6.1. - ) (48-kDa TATA 142 SEDITRYYL Band 4.1-like protein 3 box-binding protein (4.1B) (Differentially interacting protein) expressed in adenocar cinoma of the lung pro 23 DRE1 protein 45 tein. 1) (DAL-1)

24 TIDELKE.OV Dynactin-1 (150 kDa 143 NOOEQEDLE Epidermal growth factor dynein-associated receptor substrate 15 polypeptide) 144 SKEEDPENW Epidermal growth factor 25 NLAYENWKE Dynein heavy chain, Q14204 50 receptor substrate 15 cytosolic (DYHC) 145 FLDKOGFYV Epidermal growth factor 26 SEVEOYWKY Dynein heavy chain, Q14204 receptor substrate 15 cytosolic (DYHC) (Protein Eps15) (AF-1p protein) 27 ETOLTYRR Echinoderm microtubule 55 associated protein-like 146 TGALIYAIHA. Epithelial membrane PS4852 5 protein 3 (EMP-3) (YMP protein) 28 IKDDLEDLI ECT2 protein (Epithel ial cell-transforming sequence 2 oncogene) 147 AWOWLMWLSL Epithelial membrane PS4852 60 protein 3 (EMP-3) (YMP 29 QWLGKIERA Endothelial differenti protein) ation-related factor 1 (EDF-1) 148 TLKEWEELEQL ZyXin (Zyxin-2) Q15942

IOINLORKM Developmentally-regul O43854 149 WLMITEDIKL Eukaryotic translation lated endothelial cell 65 initiation factor 4 locus 1 protein) gamma 1 US 7,919,467 B2 37 38 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 150 EEKKOKEMD Eukaryotic translation 166 OCPVEGDPPPL Fibroblast growth fac initiation factor 4 tor receptor-like 1 gamma 1 precursor (FGF recep tor-like protein 1) 151 ELQALYALQAL Eukaryotic translation 15 initiation factor 4 167 TEDNWMKIA Fibroblast growth fac P224.55 gamma 1 tor receptor 4 precur sor (EC 2.7.10.1) 152 WSNKYDPPL F-actin capping protein beta subunit 168 YLLDWLERS Fibroblast growth fac P224.55 tor receptor 4 precur 153 NLSDLIDLW F-actin capping protein sor (EC 2.7.10.1) beta subunit 169 TASPDYLEI Fibroblast growth fac 154 FISHKLDIK Protocadherin Fat 2 tor receptor 2 precur precursor (hPat2) (Mul sor (EC 2.7.10.1) tiple epidermal growth (FGFR-2) factor-like domains 1) 25 17O TENNWMKIA Fibroblast growth fac 155 WEPALRKPP Protocadherin Fat 2 tor receptor 2 precur precursor (hPat2) (Mul sor (EC 2.7.10.1) tiple epidermal growth (FGFR-2) factor-like domains 1) 30 171 ETFKOIDMDND FK506-binding protein 7 156 OVWYSLPDSA Protocadherin Fat 2 precursor (EC 5.2.1. 8) precursor (hPat2) (Mul tiple epidermal growth 172 GLLELIEEP Glomulin (FKBP-associa Q9299 O factor-like domains 1) ted protein) (FK506 binding protein-assoc 157 EKISSYOLK Protocadherin Fat 2 35 iated protein) precursor (hPat2) (Mul tiple epidermal growth 173 FWEEWIDNK Glomulin (FKBP-associa Q9299 O factor-like domains 1) ted protein) (FK506 binding protein-assoc 158 EMDPOKMPYL KIAA1752 protein iated protein) 40 1.59 WTNRARASKD Fo alpha/mu receptor 174 LOLYINKLD Glomulin (FKBP-associa Q9299 O ted protein) (FK506 160 SMNLTISAGP Fo alpha/mu receptor binding protein-assoc iated protein) 161 WTYLONGKGR Low affinity immuno PO8637 17s EOSLETTKV globulin gamma Fc re 45 gion receptor III-A 176 WFNDELPASI Flavin containing mono precursor (IgG Fc re oxygenase 3 isoform 2 ceptor III-2) variant

162 ELLKTARSSK FYVE, RhoGEF and PH do 177 SLFPGKLEW Protein flightless -1 Q13045 main-containing protein 50 homolog 2 (Zinc finger FYVE do main-containing protein 178 OKKLVDTIE Guanylate-binding pro 4) tein 4

163 FYVE, RhoGEF and PH do 179 DVGKDOEFTV Filamin-A (Alpha P21333 main-containing protein 55 filamin) (Filamin-1) 2 (Zinc finger FYVE do (Endothelial actin main-containing protein binding protein) 4) 18O YLLKDKGEYTL Filamin-A (Alpha P21333 filamin) (Filamin-1) 164 YLNKLLITR Fibroblast growth fac (Endothelial actin tor receptor-like 1 60 binding protein) precursor (FGF recep tor-like protein 1) 181 FLJ10101 protein

1.65 IARPWGSSWR Fibroblast growth fac 182 IEQERLER CDNA FLJ14503 fis, tor receptor-like 1 clone NT2RM1 OOO252, precursor (FGF recep 65 weakly similar to H. tor-like protein 1) sapiens E- MAP-115 mRNA US 7,919,467 B2 39 40 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 83 KINSAPSSPIK E2F8 protein 2 OO MINLAWFDL Probable G-protein coupled receptor 55 84 NNDICLDEW Human Hypothetical protein EASALAWAPSAK Probable G-protein 15 coupled receptor 35 85 WFAEWGCSPC HUMAN CDNA FLJ34154 fis, clone FCBBF3 O13058 TFWLTIILV G-protein coupled re ceptor family C group 5 86 NIWETWLDL Hypothetical protein member C precursor FLJ43654 (Hypothetical (Retinoic acid-induced protein C3orf62) gene 3 protein)

87 IYIDGVOEVF HUMAN CDNA FLJ4618O FILDFEEDL Leucine-rich repeat O7473 fis, clone TESTI4 OO4.031 containing G-protein coupled receptor 5 pre 88 KIMTEKELLAW Flotillin-2 (Epidermal Q14254 cursor (Orphan G-pro surface antigen) (ESA) tein coupled receptor 25 HG38) (G-protein 89 WEAQEIILR Flotillin-2 (Epidermal Q14254 coupled receptor 49) surface antigen) (ESA) (G-protein coupled re ceptor 67) 90 MLLDFIOHI Serine/threonine-pro Q13535 tein kinase ATR (EC 2O4. FAMDSYGTSN Probable G-protein 2.7.11.1) (Ataxia tel 30 coupled receptor 133 angiectasia and Rad3 precursor (G-protein related protein) (FRAP coupled receptor PGR25 related protein 1) 2O5 MELSEPIWEN G1 to S phase transi P1517 O 191 SLLESWOKL Serine/threonine-pro Q13535 tion protein 1 homolog tein kinase ATR (EC 35 (GTP-binding protein 2.7.11.1) (Ataxia tel GST1 - HS) angiectasia and Rad3 related protein) (FRAP WLENALGKL Gamma-aminobutyric-acid Q16445 related protein 1) receptor alpha- 6 sub unit precursor (GABA(A) 192 YLOPKLLGI Serine/threonine-pro Q13535 receptor) tein kinase ATR (EC 40 2.7.11.1) (Ataxia tel KILEHDDWSYL Ganglioside-induced angiectasia and Rad3 differentiation-assoc related protein) (FRAP iated protein 1-like 1 related protein 1) (GDAP1-L1)

93 YLLWGTIFIL Frizzled 5 precursor Q13467 45 SOONTDNLV Gap junction alpha-5 P3 6.382 (Frizzled-5) protein (Connexin- 4 O (CX40) 94 MAAGDYPEA Frizzled 5 precursor Q13467 (Frizzled-5) 209 SKILCEETPI GEM-interacting protein (GMIP) 95 LYLLWGTIFLH Frizzled 5 precursor Q13467 50 (Frizzled-5) QLWWELKDI Golgin subfamily B Q14789 member 1 (Giantin) 96 ALSDHHWYL Fructose-bisphosphate POS 972 aldolase C (EC WFDIFOFAK UDP-N-acetylhexosamine Q16222 4.1.2. 13) pyrophosphorylase 55 (Antigen X) 97 YLAPHWRTL G protein pathway sup NIANHFFTW UDP-N-acetylhexosamine Q16222 pressor 1 isoform 1 pyrophosphorylase variant (Antigen X)

98 YLONWSHVL G protein pathway sup HLIHEWTKW Neutral alpha-glucosi Q14697 pressor 1 isoform 1 60 dase AB precursor (EC variant 3.2.1. 84)

99 FAALMLLGLW KiSS-1 receptor (KiSS FLDPNNIPKA Probable dolichyl pyro 1R) (Kisspeptins recep phosphate tor) (Metastin recep Glc1ManeGlcNAc2 alpha tor) (G-protein coupled 65 1,3-glucosyltransferase receptor 54) (EC 2. 4.1. - ) US 7,919,467 B2 41 42 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 215 KINEAWECLISL Bifunctional aminoacyl PO7814 229 EDFHRKWYNI GTP-binding protein tRNA synthetase In Rhes (Ras homolog en cludes: Glutamyl-tRNA riched in striatum) synthetase (EC (Tumor endothelial 6.1.1.17) (Glutamate-- marker 2) tRNA ligase); Prolyl 15 tRNA synthetase (EC 23 O YIDDWFHAL GTP-binding protein Q92963 6.1.1.15) (Proline-- Rit1 (Ras-like protein tRNA ligase) expressed in many tissues) 216 LLOTPKLLL Glycoprotein nmb-like protein 231 EOLAELROEF

217 WLLYSWWWW Prolactin-releasing P49683 232 GLLERWKEL Hypothetical protein peptide receptor (PrRP HDLBP receptor) (PrRPR) (G- protein coupled recep 233 DAILRIWGE Hypothetical protein tor 10) 25 HDLBP

218 KFKOCKLLQ G protein-coupled re 234 RHKLVSDGO Heat shock protein 75 Q12931 ceptor 112 kDa, mitochondrial pre cursor (HSP 75) (Tumor 219 DWLSTSSAISL G protein-coupled re necrosis factor type 1 ceptor 112 30 receptor-associated protein) 22O YIDDHSWTL Growth factor receptor Q14449 bound protein 14 (GRB14 adapter protein) 235 IOLVMKVIE Heat shock protein apg-1 221 SLYEENNKL GRIP and coiled-coil 35 domain-containing pro 236 MITREELWKN Tumor rejection antigen tein 2 (Golgi coiled (Gpg. 6) 1 coil protein GCC185 (CTCL tumor antigen 237 ALKDKIEKA. Tumor rejection antigen Se1-1) (Gpg. 6) 1 40 222 KLLEVOILE GRIP and coiled-coil 238 KIILRHILIE Heat-shock protein Q12988 domain-containing pro beta-3 (HspB3) (Heat tein 2 (Golgi coiled shock 17 kDa protein) coil protein GCC185 (CTCL tumor antigen 239 Low-density lipoprotein O71.97 Se1-1) 45 receptor-related pro tein S precursor 223 KPLLEOKEL GR P and coiled-coil domain-containing pro 24 O KGOGGAGGQFL Regulator of telomere tein 2 (Golgi coiled elongation helicase 1 coil protein GCC185 (EC 3.. 6.1. -) (Helicase (CTCL tumor antigen 50 like protein NHL) Se1-1) 241 KEFLWWASW Hematopoietic protein 1 224 FPWELDPDWS GROS1-L protein 242 KIAQKALDL Heme oxygenase 1 (EC POS 6O1 225 YLSAAINPIL Growth hormone secreta Q92847 1.14.99.3) (HO-1) gogue receptor type 1 55 (GHS-R) 243 ITEPLPELOL Heparan sulfate gluco samine 3-O-sulfotrans 226 QLSLADWILL Glutathione S-transfer O1521.7 ferase 5 (EC 2.8.2. 23) ase A4 - 4 (EC 2.5.1.18) 244 KLRKEKEEF Hepatocellular carcin 227 QSFLVGNOL Glutathione S-transfer O1521.7 60 oma-associated antigen ase A4 - 4 (EC 2.5.1.18) 66

228 LKNKTKEAAE GTP-binding protein 245 EDWFPNILN Melanoma-associated an Rhes (Ras homolog en tigen E2 (MAGE-E2 an riched in striatum) tigen) (Hepatocellular (Tumor endothelial 65 carcinoma-associated marker 2) protein 3) US 7,919,467 B2 43 44 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No.

246 IAWMLLEGGAN 26S proteasome non O75832 262 AMFDHIPWGW Hypothetical protein ATPase regulatory sub (Novel protein HSPC117) unit 10 (26S proteasome (DJ149A16. 6 protein) regulatory subunit p28) (Hypothetical protein 15 HSPC117) 247 WDLFPGTFEW Hepatocellular carcin oma-associated protein 263 WSFCLACW Claudin domain-contain p28-II Hephaestin ing protein 1 (Membrane protein GENX-3745) 248 MWCGSPDIPL HECT domain and RCC1 O9571.4 Q9NY35 like domain-containing protein 2 (HERC2) 264 NLLFPIIYL Large neutral amino acids transporter small 249 DAPHSEGDMHLL HECT domain and RCC1 O9571.4 subunit 2 (L-type amino like domain-containing acid transporter 2) protein 2 (HERC2) (hLAT2)

250 DTIEITDR Heterogeneous nuclear P22 626 25 265 SLLENLEKI Heterogeneous nuclear ribonucleoproteins A2/ ribonucleoprotein C B1 (hnRNP A2/hnRNP B1) like 1 (hnRNP core protein C-like 1) 251 RLFWGSIPK Heterogeneous nuclear O43390 ribonucleoprotein R 266 ILDOKINEV Ornithine decarboxylase P11926 (hnRNP R) 30 (EC 4.1.1.17) (ODC)

252 FLSEYOHOP HEXIM1 protein (HMBA O94992 267 DOINIETKN Regulator of nonsense inducible) transcripts 2 (Nonsense mRNA reducing factor 2) 253 ALMISMISAD Histatin-1 precursor His (Up-frameshift suppres (Histidine-rich protein tatin-1 35 sor 2 homolog) (hUpf2) 1) precur SC) 268 PFONLLKEY Regulator of nonsense (Histi transcripts 2 (Nonsense dine mRNA reducing factor 2) rich (Up-frameshift suppres protein sor 2 homolog) (hUpf2) 1) 40 269 LELELENLEI Regulator of nonsense 254 RMLPHAPGW Histone deacetylase 1 Q13547 transcripts 2 (Nonsense (HD1) mRNA reducing factor 2) (Up-frameshift suppres 255 THNLLLNYGL Histone deacetylase 1 Q13547 sor 2 homolog) (hUpf2) (HD1) 45 27 O GLADASILKKW ATX10 HUMAN Ataxin-10 256 SPNMNAWISL Histone deacetylase 9 (HD9) (HD7) (HD7) 271 GOILEAAWSW KIAA1833 protein

257 EFIDLLKKM Homeodomain-interacting 272 RV WSWSFRV HUMAN UDP-GalNAc : protein kinase 2 (EC 50 betaClcNAc beta 1,3- 2.7.11.1) galactosaminyltransfer ase, polypeptide 2 258 KMINHDSEKED Cullin-2 (CUL-2) Q13617 (Beta 1, 3-N-acetylgal actosaminyltransferase 259 AWDEDRKMYL Cullin-2 (CUL-2) Q13617 II) (MGC39558) 55 26 O LFELLEKEI SWI/SNF-related matrix 273 TOKRLDVYL Hypothetical protein associated actin-de KIAA1033 pendent regulator of chromatin subfamily A 274 AMLTWILHEI Activating signal coin member 5 (EC 3.. 6.1. - ) tegrator 1 complex sub 60 unit 3 (EC 3.. 6.1. - ) 261 FISEFEHRW HUMAN HSPCO27 26S pro teasome non-ATPase ARLAALVQR Delta-interacting pro Q15834 regulatory subunit 13 tein A (Hepatitis delta Synonyms 26S proteasome antigen-interacting regulatory subunit S11 protein A) (Coiled-coil 26S proteasome regula 65 domain-containing pro tory subunit p 40.5 tein 85B) US 7,919,467 B2 45 46 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No.

276 FAWHFYRS Hypothetical protein 296 ONNNLOTOI Hypothetical protein FLJ14466 DKFZp686DO 630

277 FNITYLDID Interferon-inducible Of 5.569 297 FLDDWWHSL Jumonji domain-contain Q15652 double stranded RNA 15 ing protein 1C (Thyroid dependent protein receptor-interacting kinase activator A protein 8) (TRIP-8) 278 GLAKRWWSL Hypothetical protein C9 orf142 298 NMWDLNDY Coatomer subunit beta PS3618 (Beta-coat protein) 279 HLIDATKLLL Tetratricopeptide re (Beta- COP) peat protein 17 299 YLLKEDMAGI FLJ10462 fis, clone 28O IGSFHGWISL CDNA FLJ14 O58 fis, NT2RP1 OO1494, weakly clone HEMBB1 OOO554 similar to MALE STERILITY PROTEIN 2 281 ILDLIDDAW Anaphase promoting com 25 plex subunit 13 3 OO KLFEKWKEW FLJ10462 fis, clone NT2RP1 OO1494, weakly 282 KILLEMWRED Hypothetical protein similar to MALE CCDC6 O STERILITY PROTEIN 2 283 LSYLPATWEP Sphingosine kinase 2 30 TVMDEIHTV Cell-cycle and apopto (EC 2.7.1. - ) sis regulatory 284 OLAOFWHEV Probable ATP-dependent protein 1 RNA helicase DDX11 (EC 3. 6.1. - ) (DEADAH box KLISELOKL Telomere-associated protein 11) (CHL1 homo protein RIF1 (Rap1-in log) (Keratinocyte 35 teracting factor 1 hom growth factor-regulated olog) gene 2 protein) (KRG-2) KVIDEIYRV F-box only protein 28 285 SYDESDEEE Protein KIAAO182 Q14687 40 SSLSDGLLLE CDNA FLJ10901 fis, 286 SYSDEFGPS Ras GTPase-activating clone NT2RPSOO3524 protein SyngAP (Synap tic Ras - GTPase-activat 3. OS Acetoacetyl-CoA synthe ing protein. 1) (Synap tase (EC 6.2.1.16) tic Ras - GAP 1) (Neuro nal RasGAP) 45 ELLENIIKN Putative cell cycle 287 TWERADSSHLSI Fibrinogen C domain control protein (DEP containing 1 domain containing 1)

288 WTENELAWIT MGC39581 protein 3. Of ELLSLWONL Synaptopodin 2-like

289 WTYLEDYSA Bcl-2-like 13 protein 50 POOERDFY CDNA FLJ3656O fis, (Mill protein) clone TRACH2OO934 O (Bcl-rambo) GRGGKDPPLEP CDNA FLJ1333 O fis, 290 YLLEKTRWA Myos in head domain con clone OVARC1 OO18O2 taining 1 55 310 LADISLHDPW ATP-dependent RNA heli 291 TLKILDLME WD-repeat protein 51A case DDX31 (EC 3.. 6.1. - ) (DEAD box protein 31) 292 EDLIKELIK KIF27A (Helicain) (OTTHUMPOOOOOO 21559) 311 PSNMGIAIPL Protein C14orf161 293 LSLENLEKI Inositol polyphosphate 60 5-phosphatase F, 312 FMMPOSLGV Cysteine protease ATG4B isoform 1 (EC 3.4.22. - ) (Auto phagy-related protein 294 FLNKAADFIE Myopalladin 4 homolog B)

295 GLDIDGIYRW Rho GTPase activating 65 313 IMWATAWWAI CDNA FLJ14526 fis, protein 12 clone NT2RM1 OO1139 US 7,919,467 B2 47 48 TABLE 2- Continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 - Number for Peptides 1791 and 1514-1533 Parent Parent Swiss- g d SE Giti SEQ Identi Q aen i- ID Parent Sequence fication ID Parent Sequence fication 10 No. Fragment Identification No. NO: Fragment Identification No. 329 SEIEKNKKW CDNA FLJ31846 fis, O96MVO 314 MTKRYEALE Hypothetical protein O9BR77 clone NT2RP7 OOO425, CCDC77 (CDNA FLJ14732 weakly similar to fis, clone MYOSIN HEAVY CHAIN, NT2RP3 OO1969, weakly 15 NONMUSCLE TYPE B similar to TRICHOHYALIN) 33O SLVOIVTTL FLJ32833 fis, clone Q96M43 TESTI2OO3228 315 SLDAKEIYL CDNA FLJ14790 fis, Q96K38 clone NT2RP4 OOO973, 33 1 KILDIRKNW Guanine nucleotide- P384 OS weakly similar to 2O binding protein PROBABLE PROTEIN DI- p.a.it SULFIDE ISOMERASE P5 initing & alpha (EC 5.3. 4.1.) protein, olfactory type) 316 OLLDIKTRL Keratin 24 Q2M215 25 332 QSLELLLLPV CDNA FLJ33811 fis, O8N279 317 FLTDYLNDL BCoR protein (BCL-6 Q6W2J9 clone CTONG2 OO2 O95 corepressor) 333 ALLNNIIEI Transmembrane protein O9BYT9 318 ANOGGFENGE Hypothetical protein Q61921 16C FLJ2O582 30 334 FNOSSSLIIH Zinc finger protein 31 P1704 O 319 ILGLILL HLE Hypothetical protein O9BTO4 (Zinc finger protein FLT22688 KOX29) (Zinc finger and SCAN domain-containing 32O WYOKEGVLAS Hypothetical protein O9H5W3 protein 20) (Zinc FLJ 22.944 35 finger protein 360)

321, YLNDFTHEI Zinc finger protein, O8TBE5 335 LSLSALPWSY Transmembrane 6 Q9BZW4 subfamily 1 A, 5- superfamily member 2

322 SPPLOGEIS Leucine-rich repeats O8IW35 336 YLDLTPNOE CDNA FLJ90251 fis, O8NCH3 and IQ motif containing 40 clone NT2RM4 OOO115 2 337 YLFERIKEL CDNA FLJ90251 fis, O8NCH3 323 LEFEPWTTP Hypothetical protein Q8TEAO clone NT2RM4 OOO115 FLJ23749 http:// WWW e 338 FILDWLLPEA CDNA FLJ9 O76 O fis, Q8N2I4 *pay as clone THYRO1 OOOO61 org/spr 3.39 EFIPEFEK Tubulin--tyrosine Q1416.6 324 WISWPVVT Hypothetical protein ot/user ligase-like protein 12 FLJ25336 man.ht mil-AC 340 DVFPATPGSQN KIAAO3O3 protein O15021 lineQ96L 50 P1 341 FIFDVHVHEV Plexin-B2 precursor O15031 (MM1) 325 NMEIMPEGSL Hypothetical protein O8N 7G6 FLJ25 660 342. ILEWTNNLE Zinc finger and BTB O15062 domain-containing 326 QDQLSALQL CDNA FLJ3 OO58 fis, O96NU6 55 protein 5 clone ADRGL2 OOOO74, weakly similar to 343 ILSKKDLPL Centrosome-associated O8WY2O RHO-GIPASE- ACTIWATING protein 350 PROTEIN 6 344. HEPPKAWDK piccolo (Aczonin) O9Y6VO 327 MEADPDLSR CDNA FLJ3 O106 fis, Q96A82 60 clone BNGH41OOO190, 345 ILDDSHLLW KIAAO560 protein O6 O3O 6 weakly similar to Rattus norvegicus 346 YLDNVVNKO KIAAO676 protein Q96H49 Schlafen- 4 (SLFN 4) mRNA. 347 KLLPYWGLLO Human homolog of Mus Q81OB7 65 SLIT and NTRK-like 328 LYLPATTPY Whirlin Q9P2O2 protein 5 precursor US 7,919,467 B2 49 50 TABLE 2- Continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 - Number for Peptides 1791 and 1514-1533 Parent Parent Swiss Swiss Prot Prot SE Identi SEQ Identi Q aen i- ID Parent Sequence fication ID Parent Sequence fication 10 No. Fragment Identification No. NO: Fragment Identification No. 366 YQDPLDPTRSV InaD-like protein O8NI35 348 QLKSLIQID Human homolog of Mus Q810B7 (Inadil protein) SLIT and NTRK-like (hINADL) (Pals 1-assoc protein 5 precursor iated tight junction 15 protein) (Protein as 349 SLLNNPLSI Nischarin Q6PIB4 sociated to tight junctions) 350 SSLSDALWLE FERM domain-containing Q9P2O2 protein 4A 367 HEFLTPRL InaD-like protein O8NI35 (Inadil protein) 351 DELOOLFNL Leucine-rich repeats Q6UXK5 20 iated(hINADL) tight (Pals junction 1-assoc neuronal protein 1 pre- protein) (Protein as cursor (Neuronal lieu sociated to tight cine-rich repeat pro- junctions) tein. 1) (NLRR-1) 3.68 GLFPWTPKL InaD-like protein O8NI35 352 QILSGRKPEL KIAA1512 protein Q9P216 25 (Inadil protein) (hINADL) (Pals 1-assoc 353 KLWEWIEEW KIAA1598 protein Q9HCH4 iated tight junction protein) (Protein as 354 QTLLKNPLY hosphatidylinositol-3 Q96QU2 sociated to tight phosphate 3-phosphatase junctions) adaptor subunit 30 369 CDVORYNI Nitric oxide synthase, P35228 355 SLLDDLHSA KIAA1730 protein O9COD3 inducible (EC 1.14.13.39) 356 HILDSSIYS KIAA1786 protein Q96 JN9 37O NMYGKVVTV Transcription elonga- OOO267 35 tion factor SPTs 357 OSSPPPPPPS Hypothetical protein Q96EK3 (hSPT5) MGC2O470 371. ONVOVNOKW Inositol-trisphosphate P27987 358 LMCYAIMWT OACT1 protein Q86XC2 3-kinase B (EC 2.7.1.127) (Inositol 359 FLSEEGGHVAV 6-phosphofructo-2-ki- Q16877 40 1,4,5-trisphosphate 3 nase/fructose-2,6-bi- kinase B) phosphatase 4 (6PF-2-K/ 372 SLINOMTOW T I inositol-3, 4 96PE3 Fru-2, 6-P2ASE testis- QMTO ype 1 inositol-3, Q type isozyme) bisphosphate 4-phospha yo y tase (EC 3.1.3.66) 45 (Inositol polyphosphate 36O SPDQELVLL IkappaB kinase complex- O951.63 4-phosphatase type I) associated protein (IKK complex-associated pro- 373 NWTVAWPTV Insulin receptor beta O9UCB7 tein) (p15O) subunit

361 FILWWLLKL Immune receptor ex- Q7Z7I3 SO 374 LGLENLCHL Insulin-like growth O8TAYO pressed on myeloid factor binding protein, cells 2 acid labile subunit

362 QIIEANYHS High-affinity cAMP- O60658 375 YYEKLHTYF Integrin beta- 4 precur- P16144 specific and IBMX-in- sor (GP1.50) (CD104 sensitive 3, 5-cyclic 55 antigen) phosphodiesterase 8A (EC 3.1. 4.17) 376 LLIAALL LDP Splice isoform 2 of P354 62-2 P35462 363 ILIDKSGKLEL Bone specific CMF608 Q6WRIO 377 RRDFGFPO Interferon alpha 2 Q16055 60 protein 364 TVMDSKIVOV Importin alpha-7 sub- O6 O684 unit (Karyopherin 378 SLLGFWYKL Interferon-induced pro- POS914 alpha-6) tein with tetratrico peptide repeats 1 365 VMDSKIVOV Importin alpha-7 sub- O6 O684 (IFIT-1) ) (Interferon unit (Karyopherin 65 induced 56 kDa protein) alpha-6) (IFI-56K) US 7,919,467 B2 51 52 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 379 Interleukin-2O precur 397 RTKLTDIOI HUMAN CTCL tumor anti sor (IL-2O) (Four alpha gen HD-CL-04 helix cytokine Zcyto1 O 398 RECKYDLPP Importin-13 (Imp13) O94829 Steroid receptor RNA 15 (Ran-binding protein activator isoform 1 13)

381 KMDOOEFSI Intersectin-2 (SH3 do - 399 OLTKIOTEL KIAAO769 protein O94.868 main-containing protein 1B) (SH3P18) (SH3P18 4 OO Hypothetical protein like WASP-associated KIAAO863 protein) WKAEDKARW Zinc finger protein 382 SLLLLPEEL ITI-like protein (In KIAA1196 - ter-alpha (Globulin) inhibitor H5-like) WLHDRIWSW CRSP complex subunit 3 (Cofactor required for 383 SOONTDNLV Gap junction alpha-5 P3 6.382 25 Sp1 transcriptional protein (Connexin- 40 activation subunit 3) (Transcriptional co 384 WLDETLAQV Kelch-like protein 8 activator CRSP130) (Vitamin D3 receptor 385 WNLGGSKSISIS Keratin, type II cyto PO4264 interacting protein skeletal 1 (Cytokera 30 complex tin-1) RNSIATLQGGR 130 kDa component 386 ANYLDSMYI ADAM 9 precursor (EC Q13443 Pyruvate dehydrogenase 3. 4. 24. – ) (A disinte lipoamide-phospha grin and metallopro tase 2 mitochondrial teinase domain 9) (Met precursor (EC 3.1.3.43) alloprotease/disinte 35 grin/cysteine-rich pro TVNILIVDON Protocadherin-10 tein 9) (Myeloma cell precursor metalloproteinase) 4 OS Leucine-rich repeats 387 HLWNSIHGL Next to BRCA1 gene 1 Q14596 and callponin homology protein (Neighbor of 40 (CH) domain containing BRCA1 gene 1 protein) 2 (Membrane component, chromosome 17, surface NLAKDNEWL Ankyrin repeat domain marker 2) (1A1-3B) 18B 45 4. Of SGDKLKLDOT Kin17 protein (HsKin17 388 SLADLMPRW Hypothetical protein protein) (KIN, antigen DKFZp686 K2O75 ic determinant of recA protein homolog) 389 IDLSASLWLN KIAAO 100 protein Q14667 408 KLTDYOVTL Kines in-like protein 390 HLTYLNWYL Pre-mRNA-splicing fac 50 KIF13A (Kinesin-like tor ATP-dependent RNA protein RBKIN) helicase PRP16 (EC 3. 6.1. -) (ATP-dependent KIOEILTOW Putative RNA binding OOO425 RNA helicase DHX38) protein KOC (DEAH box protein 38 55 410 YLDEOIKKV HUMAN Kinesin-like pro 391 QLVACIESKL KIAAO251 protein tein KIF13A (Kinesin like protein RBKIN) 392 EGKLVVODIE HUMAN KIAA0342 protein 411 SSIWEWDSLH HUMAN Kinesin-like pro tein KIF13A (Kinesin 393 QALEAGAWWLI KIAAO357 protein O15064 like protein RBKIN) 60 394 WLSCSQALKI Hypothetical protein 412 RLASYLDRW Keratin, type I cyto POS783 KIAAO372 skeletal 18 (Cytokeratin-18) 395 LSIEGEOEL KIAAO377 splice variant 2 413 ALLNIKWKL Keratin, type I cyto POS783 65 skeletal 18 396 EFODLNOEW KIAAO386 protein (Cytokeratin-18) US 7,919,467 B2 53 54 TABLE 2 - continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden- Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 Number for peotides 1-791 and 1514-1533 Parent Parent Swiss- g d SE Giti SEQ Identi Q aen i- ID Parent Sequence fication ID Parent Sequence fication 10 No. Fragment Identification No. NO: Fragment Identification No. 429 RDTPHSDFRG RNA-binding protein 6 P78332 414 FNIWKNKTE Kv3.2d voltage-gated Q86WO9 (RNA-binding motif pro potassium channel tein 6) (RNA-binding protein DEF-3) (Lung 415 KAITAPWSL Lethal (3) malignant Q9Y468 15 cancer antigen NY-LU brain tumor-like pro- 12) tein (L (3) mbt-like) (L (3) mbt protein 43 O. HRWLLHLF Lung cancer oncogene 5 O725Q7 homolog) 431 LLFDRPMHW Heterogeneous nuclear PS2272 416 HEYLKAFKV Lactadherin precursor Q08431 20 iroprotein M (Milk fat globule-EGF (hnRNP M) factE. 8s ( MFG-E8t th 432 FLSELTOOL Macrophage migration P14174. Breast epitne- inhibitory factor (MIF) lial antigen BA46) (Phenylpyruvate tauto (MFGM) merase) (EC 5.3.2.1) 25 417 LKAFKWAYS Lactadherin precursor QO8431 433 SLLSHVEOL Mitotic spindle assem- O9UI95 (Milk fat globule-EGF bly checkpoint protein factor 8) (MFG-E8) MAD2B (MAD2-like 2) (HMFG) (Breast epithe- (hREV7) lial antigen BA46) (MFGM) 434 KLILRLHKL Mitogen-activated pro- Q9Y6R4 tein kinase kinase ki 418 RLAWYIDRW Lamin-A/C (70 kDa PO25.45 nase 4 (EC 2.7.11.25) lamin) (MAPK/ERK kinase kinase 4) 419 YLLGNSSPRT Lamin-A/C (70 kDa PO25.45 lamin) 435 RLTHHPvy I Serine/threonine/tyro - Q9Y6J8 Sine-interacting-like 42O EMKWSDLDR Laminin gamma-1 chain P1104.7 protein 1 (Dual-speci precursor (Laminin B2 ficity protein phospha chain) tase 24) (Map kinase 40 phosphatase-like pro 42.1 WRLWDAGGWKL Low-density lipoprotein O751.97 tein MK-STYX) receptor-related pro ein 5 precursor 436 QDNLEKLLO Microtubule-associated Q6POQ8 serine/threonine-pro 422 KPETFEHLF Leptin receptor precur- P48.357 tein kinase 2 (EC sor (LEP-R) (OB receptor) 45 2.7.11.1) 437 MKRLLLLF Matrix metalloprotease Q9H3O6 423. EITDDGNLK Leptin receptor precur- P43357 MMP-27 sor (LEP-R) (OB receptor) 438 DPODILEVK MCM10 protein O7L59 O 50 424 ECHHRYAEL Leptin receptor precur- P43357 439 FLFGEWHKA MCM10 protein O7L59 O sor (LEP-R) (OB receptor) 440 KWIWLWNKWLL Interferon-induced hel - Q9BYX4 icase C domain-contain 425 PSTCPDGFKI Mitogen-activated pro- O43283 ing protein 1 (EC tein kinase kinase ki- 55 3.6.1.) (Melanoma dif nase 13 (EC 2.7.11.25) proteinferentiation-associated 5) 426 RKGIIDWNL Leukemia virus receptor Q08357 2 441 QILSLEEKI Melanoma ubiquitous Q2TAK8 mutated protein 60 427 LIOERDWKK Leukemia-associated O8NFU7 4 42 MLKDIIKEY Melanoma antigen family Q5BJF3 protein with a CXXC D 2 domain 443 KTWGOYWOW Melanocyte protein Pmel P40967 428 LTLEQVVAIE Leukemia-associated O8NFU7 17 precursor (Melano protein with a CXXC 65 cyte lineage - specific domain antigen GP100 US 7,919,467 B2 55 56 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 444 LLDGTATLRL Melanocyte protein Pmel 47 DTNADKOLS Calgranulin B (Migra PO 6702 17 precursor (Melano tion inhibitory factor cyte lineage - specific related protein 14) antigen GP100) (MRP-14) (P14) 15 445 WLKEIWERW GPI-anchored protein Q14444 458 GRWWCKDLPCP UC2 HUMAN Mucin-2 pre QO2817 p137 (p137GPI) (Mem cursor (Intestinal brane component chromo mucin 2) some 11 surface marker 1) Cytoplasmic activa 459 FGNMOKINO UC2 HUMAN Mucin-2 pre QO2817 tion/proliferation cursor (Intestinal associated protein 1 mucin 2)

446 SLLDEFYKL GPI-anchored protein Q14444 460 FPNWTLAOV ucin-5B precursor p137 (p137GPI) (Mem (Mucin 5 subtype B, brane component chromo tracheobronchial) some 11 surface marker 1) Cytoplasmic activa 25 461 ATPSSTPETV ucin-5B precursor tion/proliferation (Mucin 5 subtype B, associated protein 1 tracheobronchial)

447 TLNONGYTLV Hepatocyte growth fac PO 8581 462 FWNDWNLEN ultiple PDZ domain O797 O tor receptor precursor protein (Multi PDZ do (EC 2.7.10.1) (HGF re 30 main protein. 1) (Multi ceptor) (Scatter factor PDZ-domain protein 1) receptor) (SF receptor) (HGF/SF receptor) (Met 463 SENKLILMK RUFY2 (Run and FYVE do proto-oncogene tyrosine main-containing protein kinase) Rabip4 35 4 48 OMPKMNFAN Mitogen-activated pro Q16539 464 TFCVOPGEKV Multidrug resistance tein kinase 14 (EC associated protein 7 2.7.11.24) 465 YLNDGLWHM Multiple copies in a 449 KLADFGWSGE Mitogen-activated pro Q12851 T-cell malignancies (Malignant T cell am tein kinase kinase ki 40 nase kinase 2 (EC plified sequence 1) 2.7.11.1) (MAPK/ERK (MCT1) kinase kinase kinase 2) 466 GTTLRNLEI DNA mismatch repair P2O585 protein Msh;3 450 SIKDYEOAN Mitotic kinesin-related protein 45 467 SPPTLNGAPSP Protein CFA2T2 (MTG8 O43439 like protein) (MTG8 451 EDLMEDEDL Mitotic kinesin-related related protein 1) protein (Myeloid translocation related protein 1) 452 WLISKELISL Sperm-associated anti gen 5 (Astrin) (Mitotic 50 468 NEAAIKNWYL Myomesin-1 (190 kDa P521.79 spindle-associated pro titlin-associated pro tein p126) tein) (190 kDa connec tin-associated protein 453 LIEKVOEAR Myeloid/lymphoid or mixed-lineage leukemia 469 FIDFGMDLO Myos in heavy chain, P.12883 protein 4 (Trithorax 55 cardiac muscle beta homolog 2) isoform (MyHC-beta) 470 LLEAKWKEL Myosin-13 (Myosin heavy 454 Myeloid/lymphoid or chain, skeletal muscle, mixed-lineage leukemia extraocular) (MyHC-eo) protein 4 (Trithorax homolog 2) 60 471 LLAEKVEQL Tumor suppressor candi Q13454 date 3 (N33 protein) 45.5 GLDDIKDLKW Putative helicase MOW 10 (EC 3.. 6.1. - ) (Molo 472 LANARGILGLQ Nebulin-related anchor O8TCHO ney leukemia virus 10 ing protein protein) 65 473 WNRIGOESLE Neural cell adhesion P13592 456 WLAETLTOW MOZ/CBP protein molecule 1, 1 US 7,919,467 B2 57 58 TABLE 2 - continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden- Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 5 Number for peptides 1-791 and 1514-1533

Parent Parent Swiss- Swiss Prot Prot SEQ Identi- SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO: Fragment Identification No. 10 NO: Fragment Identification No. 474 YLEIOGITR Neurotrimin precursor Q9P121 487 ALLDQLYLA Nuclear receptor co- Q15596 activator 2 (NCoA-2) 475 EALENNKEL Ninein Q8N4C6 (Transcriptional in termediary factor 2) 476 NSMVVEROOL Ninein Q8N4C6 15 488 TLFDYEWRL Ubiquitin-like PHD and O96T88 477 HLLERVDOV Ninein Q8N4C6 RING finger domain-con taining protein 1 (EC 478 PERTOLLYL Notch homolog 2 OSVTDO 6.3.2. - )

479 NGGTCEDGIN Neurogenic locus notch P4 6531 489 SILKWWINN Nucleic acid helicase Q8IWW2 homolog protein 1 pre- 2O DDXX cursor (Notch 1) (hN1) (Translocation-associa- 490 LLYGGDLHSA Nucleic acid helicase Q8IWW2 ted notch protein TAN- DDXX 1) Contains: Notch 1 extracellular trunca- 491 KLAENIDAQL Nucleoporin 62 kDa Q6GTM2 tion; Notch 1 intracel- 25 (NUP62 protein) lular domain 492 SLLTDEEDWD Nuclear pore complex P52948 480 QSAADYL.GAL Neurogenic locus notch Q9UM47 protein Nup98-Nup96 homolog protein 3 pre precursor Contains: Nuclear pore complex cursor (Notch 3) Con 30 protein Nup98 (Nucleo tains: Notch 3 extra porin Nup98) (98 kDa cellular truncation; nucleoporin) ; Notch 3 intracellular domain 493 WDITOEPVL Nuclear pore complex P52948 protein Nup98-Nup96 481 ALLVVLSPPAL Neurogenic locus notch Q994.66 35 precursor Contains: homolog protein 4 pre- Nuclear pore complex cursor (Notch. 4) protein Nup98 (Nucleo (hNotch4) Contains: porin Nup98) (98 kDa Notch 4 extracellular nucleoporin) ; truncation; Notch 4 intracellular domain- 40 494. OLEKKLME Nucleoprotein TPR P1227 O

482 LRLDXLFKL Plexin-A1 precursor O9UIW2 495 GLDPLGYEIO Nuclear pore complex Pff 4 O (Semaphorin receptor protein Nup107 NOV) 496 ALLDRIWSW Nuclear pore complex Q92621 483 WLIEDGKWW HUMAN NPDO 11 O9H2R7 45 protein Nup2O5

484 SQPQEPENK Nuclear autoantigen Sp- P23497 497 KILDLETOL ODF2 protein Q6PJQ8 100 (Speckled 100 kDa) (Nuclear dot-associated 498 WTWLKETEW Trophoblast glycopro- Q6PJQ8 Sp1 OO protein) tein precursor (5T4 50 onco fetal trophoblast 485 LILREKWEFL Nuclear factor eryth- Q14494 glycoprotein) roid 2-related factor 1 (NF-E2-related factor 499 WDLPGWINTW Dynamin-like 120 kDa O6 O313 1) (NFE2-related factor protein, mitochondrial 1) (Nuclear factor, precursor (Optic erythroid derived 2, 55 atrophy 1 gene protein) like 1) (Transcription factor 11) (Transcrip- 5 OO TITCLPATLV Orexin receptor type 2 O43 614 tion factor HBZ17) (Ox2r) (Hypocretin (Transcription factor receptor type 2) LCR-F1) (Locus control region-factor 1) 60 501 LLGPRLVLA Transmembrane emp24 do - P49755 main-containing protein 486 YLDDWNEII Nuclear factor of acti- O95644 10 precursor (Transmem vated T-cells, cyto- brane protein Tmp21) plasmic 1 (NFAT trans cription complex cyto- 502 LTTPDAAGVNQ Orphan nuclear receptor P13056 solic component) (NF- 65 TR2 (Testicular ATc1) receptor 2) US 7,919,467 B2 59 60 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. SO3 FLDGHDLOL MKL/myocardin-like pro 519 ELKKINYOV Protein patched homolog Q13635 tein 1 (Myocardin-re 1 (PTC1) (PTC) lated transcription factor A) (MRTF-A) KLFODLODL Rap guanine nucleotide (Megakaryoblastic 15 exchange factor 2 leukemia 1 protein) (Neural RAP guanine nu (Megacaryocytic acute cleotide exchange pro leukemia protein) tein) (nRap GEP) (PDZ domain-containing gua 504 KTTEWLDASA Ovarian cancer related nine nucleotide ex tumor marker CA125 change factor 1) (PDZ GEF1) 505 TSPTWPWTTSIF Ovarian cancer related tumor marker CA125 521 EAIWSHEKN Pecanex-like protein 1 (Pecanex homolog) 506 WTITDTTEH Ovarian cancer related tumor marker CA125 522 GLLPOVNTFW Pecanex-like protein 1 25 (Pecanex homolog) - TITNLOYGE Ovarian cancer related tumor marker CA125 523 KAYDWEREL GC-1-related estrogen receptor alpha coacti 508 ARLTFLNRG Oxysterol-binding pro vator short isoform tein-related protein 8 (OSBP-related protein 30 524 DWLESWLDF PHD finger 8) 525 TMLWLWIRG Hypothetical protein 509 KIDALSSEKL Centrosomal protein of DKFZp686 CO7187 70 kDa (Cep70 protein) (p10-binding protein) 526 DVAQLQALLQ Phosphatidylinositol P42338 35 4,5-bisphosphate 3-ki 510 LLAEAWLTYL Leucine carboxyl nase catalytic subunit methyltransferase 2 (EC beta isoform (EC 2.1.1. -) (p21WAF1/CIP1 2.7.1.153) (P13-kinase promoter-interacting p110 subunit beta) protein) (PtdIns-3-kinase p110) 40 511 F-box/LRR-repeat pro 27 QIIEANYHS Phosphodiesterase 8A, tein 5 (F-box and leu isoform 1 cine-rich repeat pro tein 5) (F-box protein 528 Serine/threonine-pro FBL4/FBL5 tein kinase SMG1 (EC 512 LDTPSOPVNN Inhibitor of growth 45 2.7.11.1) (SMG-1) protein 3 (hSMG-1) (Lambda Aiota protein kinase C-in 513 P53 inducible protein teracting protein) (Lambda-interacting 514 PODYPDKKSLP DNA polymerase alpha POS884 protein) ( catalytic subunit (EC 50 27.7.7) 529 FLDDEWIEL PiggyBac transposable element derived 3 515 Chloride intracellular channel protein 4 (In 53 O WICILPNDDK PIWIL3 protein tracellular chloride ion channel protein 55 531 IQNSQLQLQ Homeobox protein PKNOX1 P55347 p. 64H1 (PBX/knotted homeobox 1) 516 LAAAGGPGQGWA Paired mesoderm homeo - Q994.53 box protein 2B (Paired 532 FAYLLTYMA Transmembrane protein Q12893 like homeobox. 2B) 115 (Protein PL6 (PHOX2B homeodomain 60 protein) (Neuroblastoma 533 GLIDSLWHYW Plakophilin-2 O99959 Phox) 534 REDHPARP Plectin 6 17 GTPPPPGKPE PRB3 protein P81489 535 FILDPWKGERL Plectin 1 (PLTN) (PCN) Q15149 518 SQGAVGLAGV Protein patched homolog Q13635 65 (Hemides mosomal protein 1 (PTC1) (PTC) 1) (HD1) US 7,919,467 B2 61 62 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No.

536 RGONLDVWO Plexin B1; plexin 5; O43157 550 ILDAGGHNWTI 26S proteasome non Q994 6 O semaphorin receptor ATPase regulatory sub unit 1 (26S proteasome s37 SLTGHISTW Pleiotropic regulator 1 O43660 regulatory subunit 15 RPN2) (26S proteasome 538 EPLRWPPDL Blood vessel epicardial regulatory subunit S1) substance (hBVES) (Pop (26S proteasome subunit eye domain-containing p1112) protein. 1) (Popeye 551 YMNLEKPDFI 26S proteasome non Q994 6 O protein 1) ATPase regulatory sub unit 1 (26S proteasome 539 EIPWLNELPW Carboxypeptidase-like regulatory subunit protein X2 precursor RPN2) (26S proteasome regulatory subunit S1) 54 O LYIPAMAFI YIF1B protein YIF1B (26S proteasome subunit protein p11.2) 25 541 SLLOHLIGL Melanoma antigen pre P78395 552 SLADIAOKL 26S proteasome non O43242 ferentially expressed ATPase regulatory sub in tumors (Pr4eferen unit 3 (26S proteasome tially expressed anti regulatory subunit gen of melanoma) (OPA S3) (Proteasome subunit interacting protein 4) 30 p58) 553 OLVDIIEKV Proteasome activator P61289 542 ISSMLWLFF Splice isoform 2 of complex subunit 3 (Pro Q9H7FO tea some activator 28 gamma subunit) 543 P2Y purinoceptor 13 ENHSSOTDNI 35 (PA28gamma) (PA28g) (P2Y13) (G-protein (Activator of multi coupled receptor 86 catalytic protease sub (G-protein coupled unit 3) (11S regulator receptor 94) complex gamma subunit) (REG-gamma) (Ki nuclear autoantigen) 544 ILMGWLKEW Putative pre-mRNA O43143 40 splicing factor ATP dependent RNA helicase 554 SLLKVDOEV Proteasome activator P61289 DHX15 (EC 3.. 6.1. - ) complex subunit 3 (Pro tea some activator 28 (DEAH box protein 15 gamma subunit) (ATP-dependent RNA (PA28gamma) (PA28g) helicase #46) 45 (Activator of multi catalytic protease sub 545 WLFENTDSWHL HUMAN RNA-binding pro P42696 unit 3) (11S regulator tein 34 (RNA-binding complex gamma subunit) motif protein 34) (REG-gamma) (Ki nuclear autoantigen) 546 INMRIODL Prolyl 4-hydroxylase P13674 50 alpha-1 subunit precur 555 QILRLLHIE Protein C14orf166 sor (EC 1.14.11.2) (4- PH alpha-1) (Procolla 556 EMIGGGENNLK Protein KIAA1219 gen-proline, 2-oxoglu tarate-4-dioxygenase 557 NLAEKLIGW Protein KIAA1219 alpha-1 subunit) 55 558 EKSVSVOTNL Protein KIAA1688 547 Profilin-1 PO7737 559 GLLDSLTGILN Protein Plunc precursor 548 GLIEILKKW Programmed cell death O14.737 (Palate lung and nasal protein 5 (TFAR19 pro epithelium clone pro tein) (IF-1 cell apop 60 tein) (Lung-specific tosis-related gene 19 protein X) (Nasopharyn protein) geal carcinoma-related protein) (Tracheal epi 549 Propionyl-CoA carboxy POS166 thelium-enriched pro lase beta chain, mito tein) (Secretory pro chondrial precursor (EC 65 tein in upper respira 6.4.1.3) tory tracts) US 7,919,467 B2 63 64 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 560 SLLPPDALWGL Protein transport pro Q15437 576 LLSNNNOAL Ras-GTPase-activating Q13283 tein Sec23B protein-binding protein 1 (EC 3.. 6.1. - ) (ATP 561 LEEKNTLIQEL Liprin-alpha-2 (Protein Of 5334 dependent DNA helicase tyrosine phosphatase VIII) (GAP SH3-domain receptor type f poly 15 binding protein 1) peptide-interacting (G3BP-1) (HDH-WIII) protein alpha-2) (PTPRF-interacting 577 WLCGNKSDLE Ras-related protein P51159 protein alpha-2) Rab-27A (Rab-27) (GTP binding protein Ram) 562 LLSESNERL Liprin-alpha-2 (Protein Of 5334 tyrosine phosphatase LLMYDIAN Ras-related protein O95716 receptor type f poly Rao-3D peptide-interacting protein alpha-2) sfe SOVNILSKIVSR Nuclear pore complex Pff 4 O (PTPRF-interacting protein Nup107 (Nucleo protein alpha-2) 25 porin Nup107) (107 kDa nucleoporin) 563 LADLGSIESP Protocadherin gamma A12 precursor (PCDH-gamma Receptor-interacting A12) (Cadherin-21) factor 1 (Fibroblast cadherin 3) 30 581 LEWEWIEAR Regulating synaptic 564 OLLKFOLNK Protocadherin gamma A10 membrane exocytosis precursor (PCDH-gamma protein 3 (Nim3) (Rab-3 A10) interacting molecule 3) (RIM 3) (RIM3 gamma) 565 LLAEAWLTYL Leucine carboxyl methyltransferase 2 582 TLLRGIEW Regulator of G protein (EC 2.1. 1. -) (p21WAF1/ 35 signaling protein (Reg CIP1 promoter-interact ulator of G-protein ing protein) signalling like 1)

566 OLLREPHLQ KIAA1636 protein 583 PDFTELDLO MHC class II regulatory P22 670 40 factor RFX1 (RFX) (En 567 TIPNLEOIE Probable G-protein hancer factor C) (EF- C) coupled receptor 160 584 DWLFALFSKL Retinoblastoma-associa PO 64 OO 568 KLWEAESKL Protein C21orf 45 ted protein (PP110 (P105-R) 569 IFHLHELPE Periodic tryptophan Q15269 protein 2 homolog 45 585 RSGERKAVOA Roundabout homolog 3 st O KLFNDAIRL Rab-like protein 2B precursor (Roundabout like protein 3) FENOEWOAI Cell cycle checkpoint O75943 protein RAD17 (hRad17) 586 GLNEEIARW Retinoblastoma-associa O14777 (RF-C/activator 1 50 ted protein HEC (Kine homolog) tochore associated 2)

EY WEKFYRI DNA repair protein O928.78 587 FLFOEPRSI Retinoblastoma-associa RAD50 (EC 3.. 6. - ... - ) ted protein RAP140 (hRAD5O) 55 588 FLFOEPRSIVT Retinoblastoma-associa QIDEIRDK DNA repair protein O928.78 ted protein RAP140 RAD50 (EC 3.. 6. - ... - ) (hRAD5O) 589 KEWDILNLP AT-rich interactive do P29374 main-containing protein FLHEKLESL Ras GTPase-activating P20936 4A (ARID domain-con protein 1 (GTPase-acti 60 taining protein 4A) vating protein) (GAP) (Retinoblastoma-binding (Ras p21 protein acti protein 1) vator) (p120GAP) (RasGAP) 590 YKLPMEDLK Jumonji/ARID domain P2.9375 containing protein 1A sts FELNNELKM Ras guanine nucleotide 65 (Retinoblastoma-binding exchange factor 2 protein 2) (RBBP-2) US 7,919,467 B2 65 66 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No.

591 TMVDRIEEV Jumonji/ARID domain P2.9375 608 WLWDRTFSL Signal transducer and P42224 containing protein 1A activator of transcrip (Retinoblastoma-binding tion 1 - alpha/beta protein 2) (RBBP-2) (Transcription factor 15 ISGF-3 components 592 WEGLITLSDFDL RhoGTPase regulating p91/p84) STAT1 protein variant 609 NWNFFTKPP Signal transducer and ARHGAP2O - lad activator of transcrip tion 3 (Acute-phase 593 405 ribosomal protein response factor) S4, Y isoform 2 ETFSGVYKK 40S ribosomal protein 594 LIKHLLILL RNA binding motif Q13380 S7

595 ALLSRLEQI RNA binding protein QLDDLKVEL 60S ribosomal protein P42766 (Autoantigenic, hnRNP L35 associated with lethal 25 yellow), long isoform MEDLIHEI 60S ribosomal protein P18124 L7 596 DWYEDELWP RNA-binding protein OTDVDNDLV Thrombospondin-2 P354.42 precursor 597 WMLGGRNIKW Ro ribonucleoprotein binding protein 1 30 LLIDPPRYI C3 and PZP-like alpha (SIAHBP1 protein) 2-macroglobulin domain containing 8 598 RLDELGGWYL HUMAN OTTHUMPOOOOOO3 O9 O2 PSIPTSAOHV C3 and PZP-like alpha 35 2-macroglobulin domain 5.99 FEDKLIEDL Ryanodine receptor 2 Q92736 containing 8 (Cardiac muscle-type ryanodine receptor) FLDEPTNHL ATP-binding cassette (RyR2) (RYR-2) (Cardiac sub-family F member 2 muscle ryanodine recep (Iron-inhibited ABC tor- calcium release 40 transporter 2) channel) (hrYR-2) 617 KMDDPDYWRTW Ribosome biogenesis Q14137 OLIDKWWOL SEC14-like protein 1 protein BOP1 (Block of proliferation 1 FLLEPOMKV Secreted and transmem protein) brane protein 1 pre 45 cursor (Protein K12) 618 LANVQQVOI CDNA FLJ13765 fis, clone PLACE4 OOO128, ILNEDGSPNL Neudes in precursor weakly similar, to Mus (Neuron-derived neuro musculus putative trophic factor) transcription factor 50 mRNA 603 LLAILILAL P-selectin glycoprotein Q14242 ligand 1 precursor 619 SLFWWILWT GD2OO cell surface (PSGL-1) (Selectin P glycoprotein receptor ligand) (CD162 antigen) isoform 2 variant 2

604 SMNRGGYMP Semaphorin - 6D precursor 55 ARTIKIRNI LRRC58 protein

605 EFIDGSLOM Serine/threonine/tyro 621 Claudin- 6 (Skullin 2) Ps 6747 Sine-interacting pro tein (Protein tyrosine 622 WISFDKLKL T-box transcription O95935 phosphatase-like factor TBX18 (T-box protein) 60 protein 18)

606 ILWWYWIGL Olfactory receptor 8G5 623 DLMELYKW INTS7 protein (Olfactory receptor OR11-298) 624 LORRKPTGAF FRAS 1-related extracel lular matrix protein 2 TLSERLWLG Shb-like adapter 65 precursor (ECM3 protein, Shf homolog) US 7,919,467 B2 67 68 TABLE 2- Continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 - Number for Peptides 1791 and 1514-1533 Parent Parent Swiss Swiss- Prot SE Giti SEQ Identi Q aen i- ID Parent Sequence fication ID Parent Sequence fication 10 No. Fragment Identification No. NO: Fragment Identification No. 643 SYWNLPTIAL 40S ribosomal protein PO8865 625 KWNNEKFRT Zinc finger protein 318 Q5VUA4 SA (p40) (34/67 kDa (Endocrine regulatory laminin receptor) protein) (Colon carcinoma 15 laminin-binding pro 626 SLDOPTOTV Eukaryotic translation Q996.13 tein) (NEMA1CHD4) (Mull initiation factor 3 tidrug resistance-as subunit 8 (eIF3 p110) sociated protein MGr1 (eIF3 c) Ag)

627 SWTSEGIKAW HUMAN LOC196394 protein Q81Y45 2O 644. SNLEHLGHE N-acetylglucosamine-1- O9 UJJ9 phosphotransferase sub 628 ISLSEPAKPG Hypothetical protein Q8NDZ2 unit gamma precursor FLJ44216 6.45 LKLKLTAWEK Liprin-beta-1 (Protein Q86W92 629 ILDKKWEKW Heat shock protein HSP PO8238 tyrosine phosphatase 90-beta (HSP 84) 25 receptor type f poly (HSP 90) peptide-interacting protein-binding protein 63 O KLSAEWESLK Sarcoma antigen NY-SAR- Q5T9S5 1) 41 (NY-SAR-41) 646 GLKGRWFEW 40S ribosomal protein P61247 631 WTWDAALYI Protein FAM86A Q96GO4 30 S3a

632 YLLPKDIKL Ras-like family 11 Q6T310 647 SLADLONDEV 40S ribosomal protein P61247 member A. S3a (OTTHUMPOOOOOO18162) 648 NNLPHLOVW LOC124512 protein Q86XAO 633 RLLEDGEDFNL Keratin, type I cyto- POS783 35 (Fragment) skeletal 18 (Cytokera tin-18) (CK-18) 649 ISFGGSWOL Hypothetical protein Q96KX1 MGC2 6744 634 RWLPYPFTH U3 small nucleolar RNA- Q9BWJ6 associated protein 14 a 650 SILDOILQ Hypothetical protein O96KW.9 homolog A (Antigen NY- LOC122258 CO-16) 651 TLSDLRWYL Sulfiredoxin-1 (EC O9BYNO 635 QNOERLER Hypothetical protein Q68DMO 1.8.98.2) DKFZp 781D1722 45 652 EAFWNSKN Basalin Q5QJ38 636 ODNIKELEL Chromosome-associated O95239 kines in KIF4A 653 WTWDAALYL Protein FAM86A Q96GO4 (Chromokinesin) 654 WLDDKLVFW Transmembrane protein Q4KMO2 637 ILKORDNEI Kines in-like protein Q62MV9 16F KIF6 50 655 YLLDLHSYL TEB4 protein O14 670 638 ONELDNVSTL Myosin-10 (Myosin heavy P35580 chain, nonmuscle IIb) 656 FLALAVIQL SLC1 OA5 O5PT55 (Nonmuscle myosin heavy chain IIb) 657 TLAEWSTRL Serine/threonine-pro- Psi Oss 55 tein kinase SNF1-like 639 NIDLLDDGSN Hypothetical protein O8IY85 kinase 1 (EC 2.7.11.1) C17orf7 658 VIEWYOEOI LOC391257 protein Q6PO94 64O WLOSNIOHV N- O9BVR8 659 RLWEEAWKA Zinc finger protein 161 Q14119 9 ycanage lomo og 60 (Putative transcription (S. cerevisiae) factor DB1)

641 VFFDIAVDGEPL Peptidyl-prolyl cis- P62937 660 SLKTLMLR Slit homolog 2 protein O94813 trans isomerase A (EC precursor (Slit-2) 5.2.1. 8) 65 661 EIKKKFKL FYN-binding protein O15117 642 DFHFPKFSI Serpin A13 precursor Q6UXR4 (FYN-T-binding protein) US 7,919,467 B2 69 70 TABLE 2 - continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden- Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 - Number for Peptides 1791 and 1514-1533 Parent Parent Swiss- g Prot SEQ Identi SEQ Identi- ID Parent Sequence fication ID Parent Sequence fication 10 No. Fragment Identification No. NO: Fragment Identification No. 67 NMYGKVVTV Transcription elonga- OOO267 662 WHKEMFIMW Jumonji/ARID domain- P4 1229 tion factor SPTs containing protein 1C (hSPT5) (DRB Sensitiv (SmcX protein) (Xe169 ity-inducing factor protein) 15 large subunit) (DSIF large subunit) (DSIF 66.3 WHKEMFIMW Jumonji/ARID domain- Q9BY66 p160) (Tat- cotransacti containing protein 1D vator 1 protein) (Tat (SmcY protein) (Histo- CT1 protein) - citility Y 2O 676 SLFATEQL Synaptogyrin-3 O43761 677 RLQEGDKILSV Synaptojanin-2-binding P571 OS 664 LAGSEVALAGV Monocarboxylate trans- O959. Of protein (Mitochondrial porter 3 (MCT 3) outer membrane protein 25) 665 IPHDLFTEL Solute carrier family 4 Q6U841 sodium bicarbonate 25 678 AMFDKKVOL Synemin Q8TE61 cotransporter-like member 1 O- 679 ALNELLOHV Talin-1 O9Y49 O 666 FLADPDTVNHLL Sorting nexin 14, iso- Q6NUI7 680 RWWSMAALAM TAR RNA loop binding w form a protein (TAR (HIV) RNA 30 binding protein 1) 667 RWADRLYGW Sorting nexin- 4 O95219 681 GIIMOIIDV Taste receptor type 2 O9NYW6 668 HRPDLLDY Spectrin beta chain, O9NRC6 member 3 (T2R3) brain 4 (Spectrin, non erythroid beta chain 4) 682 IFNAIALFL Taste receptor type 2 P595.35 35 member 40 (T2R40) (T2R58) (G-protein 669 TLDENHPSI Spermatogenesis-assoc- O9POW8 coupled receptor 6O) iated protein 7 (Sperm atogenesis-associated 683 LEOGLFSKW Oxidoreductase HTATIP2 Q9BUP3 protein HSD3) (EC 1.1.1. -) (HIV-1 TAT-interactive protein 670 TLAEIAKWEL Non-POU domain-contain- Q15233 40 2) ing octamer-binding protein (Nono protein) 684 KFMHMGKROK Transcription initia- P49848 (54 kDa nuclear RNA- tion factor TFIID sub and DNA-binding pro- unit 6 (Transcription tein) (p54 (nrb.) ) initiation factor TFIID (p54nrb.) (55 kDa 45 70 kDa subunit) nuclear protein) (TAF (II) 7O) (TAFII-70) p (TAFII-80) (TAFII80) 671 DWAWEAIRL Cohesin subunit SA-1 O8WVM'7 685 SNFGNEKL TRAG protein O6PIP7 (Stromal antigen 1) (SCC3 homolog 1) 50 686 FLLDKKIGV T-complex protein 1 P78371 subunit beta (TCP-1- 672 LMWDHWTEW Steroid receptor RNA Q9HD15 beta) (CCT-beta) activator isoform 1 687 RSLAASNPIL Telomerase-binding O86U58 673. SLYEMWSRW Structure-specific QO8945 protelntei EST1A (Ever(E 55 shorter telomeres 1A) recognition protein 1 (Telomerase subunit (SSRP1) (Recombination EST1A) (EST1-like pro signal sequence recog- tein A) (hSmg 5/7a) nition protein) (T16O) (Chromatin-specific 688 EMESLTGHQ Tumor endothelial mar- Q96PEO transcription elonga- 60 ker 6. (Hypothetical tion factor 80 kDa protein TEM6) subunit)bunit 689 LDFQEELEV Ras GTPase-activating - Q13576 like protein IQGAP2 674 SINPKRAKL Suppressor of hairy Q86YH2 wing homolog 2 690 SPNSEGDAGDL Tetratricopeptide re- O8WWT3 (5'OY11.1) (Zinc finger 65 peat protein 15 (TPR protein 632) repeat protein 15) US 7,919,467 B2 71 72 TABLE 2- Continued TABLE 2- Continued

Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514 - 1533 5 Number for peptides 1-791 and 1514-1533

Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication 10 ID fication NO : Fragment Identification No. Parent Sequence NO: Fragment Identification No. 691 LWYLNESSWLH Myosin-18A (Myos in Q92614 XVIIIa) (Myosin con 705 KLEEEQEKNOL Transcriptional repres taining PDZ domain) sor CTCFL (CCCTC-bind (Molecule associated 15 ing factor) (Brother of With JAK3 N-terminus) the regulator of im (MAJN) printed sites) (Zinc finger protein CTCF-T) 692 WAGIKVNOVK Polycystic kidney and (CTCF paralog hepatic disease 1 pre cursor (Fibrocystin) 706 LNWDTPFPL Transducer of regulated CREB protein 3 693 ILYELOVEL TMC4 protein 7 Of ILYELKWEL Transmembrane channel Q72404 694 EWLDELYRW MDC-3.13 isoform 1 like protein 4 (TNFAIP8 protein) 25 Transcription initia P49848 695 TNIEDGWFET Toll-like receptor 8 Toll precursor like tion factor TFIID sub receptor unit 6 (Transcription 8 pre initiation factor TFIID CSC) 70 kDa subunit) 30 (TAF (II) 7O) (TAFII-70) 696 EIRKNEGOI Tolloid-like protein 1 O43897 (TAFII-80) (TAFII80) precursor (EC 3.4. 24. -) 709 HSDEGGWASL Trophinin-associated Q12815 697 IAAKILSYN DNA topoisomerase I, protein (Tastin) (Tro mitochondrial precursor 35 phinin-assisting (EC 5.99.1.2) (TOP1mt) protein) 698 LYGRHFNYL PAP associated domain containing protein 5 710 AMLTGELKKA Tryptophanyl-tRNA syn P23381 (EC 2.77. - ) thetase (EC 6.1.1.2) (Topoisomerase-related (Tryptophan--tRNA function protein 4-2) 40 ligase) (TrpRS) (IFP53) (TRF4-2) (hWRS) 699 NLFNKYPAL Plastin-3 (T-plastin) P13797 711 WFPTHVFPT Tubulin, gamma complex 7 OO YLDEIWKEW Translocated promoter associated protein 3 region (To activated 45 MET oncogene) 712 KELAELRESTS Tumor necrosis factor ligand superfamily ENHSSOTDNI P2Y purinoceptor 13 member 6 (Fas antigen (P2Y13) (G-protein ligand) (Fas ligand) coupled receptor 86 50 (CD178 antigen) (CD95L (G-protein coupled re protein) (Apoptosis ceptor 94) antigen ligand) (APTL) RTHMLSSL Transcript Y 5 Contains: Tumor necro sis factor ligand OATIAPVTV Transcription factor Q024.46 55 superfamily member 6, Sp4 (SPR-1) membrane form

704 NLFRAPIYL Transcription initia P21675 713 LTDKEGWIL Tumor necrosis factor, Q13829 tion factor TFIID sub alpha-induced protein unit 1 (EC 2.7.11.1) 1, endothelial (B12 (Transcription initia 60 tion factor TFIID 25 O protein) kDa subunit) (TAF (II) 25O) (TAFII 714. WWTYKNENI Netrin receptor DCC P431.46 25O) (TAFII25O) (TP precursor (Tumor sup associated factor 250 pressor protein DCC) kDa) (p.25O) (Cell cycle 65 (Colorectal cancer gene 1 protein) suppressor) US 7,919,467 B2 73 74 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No.

TWAEGLIED Adipocyte-derived lieu 728 CKDGEDSIIR Beta-defensin 120 Q8N689 cine aminopeptidase precursor precursor (EC 3.4.11. - ) (A-LAP) (ARTS-1) 729 DNTKKSDKT Alpha-catulin (Catenin Q9 UBT 7 (Aminopeptidase PILS) 15 alpha-like protein 1) (Puromycin-insensitive (Alpha - catenin-related leucyl-specific amino protein) peptidase) (PILS-AP) (Type 1 tumor necrosis 73 O EFLGDSIMQ Ribonuclease III (EC O9NRR4 factor receptor 3.1. 26.3) (RNase III) shedding aminopeptidase 731 EFLQEGLEK Seizure related 6 Q53EL9 71.6 NEKIKKDEL U1 Small nuclear ribo POSO12 homolog nucleoprotein A (U1 snRNP protein A) (U1A 732 FLLKCLEOW Granulocyte colony- POS 919 protein) (U1-A stimulating factor precursor (G-CSF) 717 ILDESHERW U6 snRNA-associated O95777 25 (Pluripoietin) (Fil Sm-like protein LSm8 grastim) (Lenograstim)

718 NLYSDYILN Ubiquitin-protein Q05086 733 FLTDSNNIKEW Lysyl-tRNA synthetase Q9HB23 ligase E3A (EC 6.3.2. - ) (E6AP ubiquitin-protein 734 GGWQELLNQQ Protein C6orf130 O9Y53 O ligase) (Oncogenic pro 30 tein-associated protein 73 GKPRRKSNL Melanophilin (Exo- Q9BV36 E6-AP) (Human papillo philin-3) (Synaptotag mavirus E6-associated min-like protein 2a) protein) (Slp homolog lacking C2 domains a) 719 RYWNGHAK Ubiquitin carboxyl 35 terminal hydrolase 3 736 HIKEELMHG Novel protein (Possible Q5VTR6 (EC 3.1.2. 15) (Ubiqui ortholog of mouse phos tin thioesterase 3) phoinositide-3-kinase (Ubiquitin-specific adaptor protein 1 processing protease 3) (Pik3ap1) (Deubiquitinating en zyme 3) 40 737 HIPFFLHN AER61 glycosyltrans- O6P985 ferase KLLDIELAPS UBX domain-containing O92575 protein 2 738 ILEKKWEKW Heat shock protein HSP PO79 OO 9 O-alpha (HSP 86) 721 YLYDLNHTL UNC93 homolog B1 (UNC 93B protein) (hUNC93B1) 45 739 ILMEHIHKL 60S ribosomal protein P84 O98 L.19 722 FFFWLMEL Splice isoform 5 of Q9H171 740 KASOLGIFISKW PDZ domain-containing Q5EBL8 protein 11 723 ELSSLKETHI CDNA FLJ46282 fis, clone TESTI4 O31 O 66 50 74.1 KILEWMHTK Dedicator of cytokine- Q5 JSL3 sis 11 – ; Cdc42-associa 724 CCDC73 protein ted guanine nucleotide exchange factor 725 ALWERIEGW Caspase recruitment do - ACG/DOCK11 main-containing protein 10 (CARD - containing 55 742 LAWGTSPWLA Hypothetical protein Q6ZNX6 MAGUK protein 3) FLJ26930 (Carma 3) 743 LLAEEARKL Laminin gamma-1 chain P1104.7 precursor (Laminin B2 726 Caspase recruitment do - main-containing protein chain) 10 (CARD - containing 60 744 LIGICFCIA ATP-binding cassette Q96J66 MAGUK protein 3) transporter sub-family (Carma 3) C member 11 (Multidrug resistance-associated 727 ANWDAIWWSW Chromatin-specific protein 8) transcription elonga tion factor FACT 140 65 74. LMAEMGWHSW Uridine/cytidine O9NWZ5 kDa subunit kinase-like 1 US 7,919,467 B2 75 76 TABLE 2 - continued TABLE 2 - continued Description of Fragments, Parent Sequence Iden- Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification - Number for beptides 1791 and 1514-1533- 5 - NumberNBSE forESE ESBESSEpeotides 1-791 anda 1514-1533B. P Parent Parent Swiss- Swiss Prot Prot SEQ Identi- SE Identi ID Parent Sequence fication Q aen i NO: Fragment Identification No. 10 ID Parent Sequence fication NO: Fragment Identification No. 746 ISRLENITW Butyrophilin-like pro- Q6UX41 tein 8 precursor 765 YKTFSTSMMLL Hypothetical protein Q96I36 C12orf 62 747 MISLPGPLWTN Endothelial cell- O96AP7 Selective adhesion is 766 RLPPEGILHNV VPS13D-2A protein O7 O9C4 molecule precursor 767 LLGPRWLSP CDNA FLJ32O09 fis, Q96DN2 748 MLLDWMHTW Poly(A) - specific ribo- O95453 clone NT2RP7 OO9498, nuclease PARN (EC weakly similar to 3.1. 13. 4) (Polyadenyl- FIBULIN-1, ISOFORM A ate-specific ribonucle- 2O age) (Deadenylating nu- 768 FIILLWTYI Transient receptor po- Q9HBAO clease) (Deadenylation tential cation channel nuclease) subfamily V member 4 749 NWMNLIDFW Voltage-gated potassium Q96RP8 (TrpV4) (osm-9-like TRP channel KCNAA channel 4) (OTRPC4) 25 (Vanilloid receptor 750 NWTMKDNKI F-box protein 11 Q52ZP1 like channel 2) (Vanilloid receptor 751 PRSNIDWNI rythrocyte membrane O725S1 like protein 2) (VRL-2) protein band 4.1 like 5 769 FYDIEILK Wascular endothelial O43915 752 PSAOPLLSL CDNA FLJ45 O15 fis, Q6ZT3 O 30 growth factor D precur clone BRAWH3 O14639 sor (VEGF-D) (c-fos induced growth factor) 753 OLKESKLKI FAM13A1 v2 protein Q24.JPO (FIGF) 754 RDAPHLPDG Hypothetical protein Q62P7 O FLJ26432 35 77O WMAPESIFDKI Wascular endothelial P17948 growth factor receptor 755 RLPPEGILHNV VPS13D-1A protein O7 O9 C5 1 precursor (EC 2.7.10.1) (WEGFR-1) 756 SEGAEYDDOT Coagulation factor VIII POO451 (Vascular permeability precursor (Procoagulant factor receptor) (Tyro component) (Antihemo- 40 Sine-protein kinase re philic factor) (AHF) ceptor FLT) (Flt-1 (Tyrosine-protein 757 SLFERLWKW NFX1-type zinc finger- Q9P2E3 kinase FRT) (Fms-like containing protein 1 tyrosine kinase 1) 758 SLLDKIIGA Polymerase I and trans- Q6NZI2 45 cript release factor 771 LLDOONPDE Proto-oncogene C-crk. P46108 (PTRF protein) (P38) (Adapter molecule crk) 759 SMMDVDHOI T-complex protein 1 P4 8643 subunit epsilon (TCP-1- 772 VMFKKIKSFEV VDUP1 protein (Thiore- O9H3M7 epsilon) (CCT-epsilon) 50 doxin interacting protein) 76 O TLDEKIEKW Probable ATP-dependent Q96GO7 RNA helicase DDX27 (EC 773 KILLEGEESRISL Wimentin PO 867 O 3. 6.1. - ) (DEAD box protein 27) 774 KLLEGEESRISL HUMAN CTCL tumor anti- Q548L2 55 gen HD-CL- 06 (Vimentin 761 TLLEDGTFKW HSCARG Q9HBL8 variant)

762 TWLKTKFSS CDNA FLJ43956 fis, Q62U72 775 RILGAWAKW Winculin (Metavinculin) P182O6 clone TESTI4 O15681 763 WIFEDVGROVL, Mitochondrial-process - Q10713 776 SLSMVNHRL Integrin alpha-3 pre- P26 OO6 ing peptidase alpha cursor (Galactoprotein subunit, mitochondrial B3) (GAPB3) (WLA-3 precursor (EC alpha chain) (FRP-2) 3. 4. 24. 64) (Alpha-MPP) (CD49c antigen) Con tains: Integrin alpha-3 764 YILDINPLL CDNA FLJ45287 fis, Q6ZSRO 65 heavy chain; Integrin clone BRHIP3 OO2124 alpha-3 light chain US 7,919,467 B2 77 78 TABLE 2- Continued TABLE 2- Continued Description of Fragments, Parent Sequence Iden Description of Fragments, Parent Sequence Iden tification and Parent Swiss Prot Identification tification and Parent Swiss Prot Identification Number for peptides 1-791 and 1514-1533 Number for peptides 1-791 and 1514-1533 5 Parent Parent Swiss Swiss Prot Prot SEQ Identi SEQ Identi ID Parent Sequence fication ID Parent Sequence fication NO : Fragment Identification No. 10 NO: Fragment Identification No. 777 WGOADGGLSVLR Voltage-dependent T 791. ALWDKLFNL Nesprin. 2 (Nuclear en type calcium channel velope spectrin repeat alpha-1H subunit (Wol protein 2) tage-gated calcium channel alpha subunit 15 1514 KIMDQWOOA Adenomatous polypos is P25054 Cav3.2) (Low-voltage coli activated calcium channel alphal 3.2 1515 RLQEDPPAGV Ubiquitin conjugating P49459 subunit) enzyme E2

778 DWATILSRR Wiskott-Aldrich syn 1516 KLDWGNAEW B cell receptor Pssf2 drome protein family associated protein member 4 (WASP-family BAP31 (CDM protein) protein member 4) 6 c6-AG

779 PKFEVIEKPOA ATP synthase coupling P18859 17 F LYDDNORV Topoisomerase Il-alpha P.11388 factor 6 mitochondrial precursor (EC 3.. 6. 3. 14) 25 518 F LYDDNORV Topoisomerase II beta Q02880 (ATPase subunit F6) 519 A. Integrin beta8 subunit NCTTIDDSLAI Proto-oncogene protein precursor Wnt -3 precursor Replication Protein. A P27694 781 ILPIWILAN myloid beta A4 precur O99767 30 sor protein-binding 521. I LDDIGHGW Abl Binding protein 3 U3.1089 family A member 2 (Neuron-specific X11L, 5.22 L LDRFLATW Cyclin I protein) (Neuronal Munc 18-1-interacting 523 L IDDKGTIKL Cell Division Control PO 6493 protein 2) (Mint-2) Protein 2 (CDC2) (Adapter protein X11 35 beta) 524 R Septin 2 (NEDD5) Q15019

782 EFLELSAAQE Zinc finger CCHC do 52.5 K. LOELNYNL STAT1 alpha/beta P42224 main-containing protein 5 526 I IEHLYGL LDL Receptor-related OO 7954 40 protein (LRP) 783 SLTDKVOEA Myeloidl/ymphoid or mixed-lineage leukemia 27 Y IELIDRW TACE (ADAM17) (Trithorax homolog, Drosophila) variant 528 N LMEOPIKV Junction plakoglobin P14923 784 SKNSALEYQL Zinc finger protein 45 (gamma catenin) DZIP1 (DAZ-interacting protein 1/2) 529 F AEDALNTW EDDR1 QO8345

LODWEEVEI Hypothetical protein LNWIKSW IP3 receptor type II Q14571 DKFZp761O1618 50 531 M KDIIKEY Melanoma-associated 786 FLDEPTNHL ATP-binding cassette antigen D2 (MAGE-D2 sub-family F member 2 antigen) (MAGE-D) (Iron-inhibited ABC (Breast cancer-associ transporter 2) ated gene 1 protein) (BCG-1) (11B6) (Hepato 787 KMDDPDYWRTW Ribosome biogenesis Q14137 55 cellular carcinoma-as protein BOP1 (Block of sociated protein JCL-1 proliferation 1 protein) 1532 TSYWKWLEH Melanoma-associated P433.58 antigen 4 (MAGE-4 anti 788 LANVQQVOI CDNA FLJ13765 fis, gen) (MAGE-X2) clone PLACE4 OOO128 (MAGE-41) 60 789 KLDPTKTTL NDRG1 protein (N-myc O92597 1533 HEYLKAFKW HUMAN Retinoblastoma OO 89.99 downstream regulated like protein 2 (13 O gene 1 protein) kDa retinoblastoma associated protein) 79 O HLTYLNWYL Pre-mRNA splicing (PRB2) (P13O) (RR-2) factor ATP-dependent 65 RNA helicase PRP16 US 7,919,467 B2 79 80 TABLE 3 SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 792 BCL-6 corepressor long isoform Q6W2J9 793 E1B 19K/Bcl-2-interacting protein Nip3 Q12983 794 Similar to Heat shock protein HSP 90-beta (HSP 84) (HSP PO8.238 90) 795 Cytochrome P450 11B2, mitochondrial precursor P19099 796 2'-5"oligoadenylate synthetase 3 Q2HJ14 797 26S protease regulatory subunit 4 (P26s4) P62191 798 26S proteasome non-ATPase regulatory subunit 7 P51665 799 26S proteasome non-ATPase regulatory subunit 14 OOO487 800 40S ribosomal protein S16 P62249 8O1 40S ribosomal protein S6 (Phosphoprotein NP33) P62753 8O2 40S ribosomal protein S9 P46781 803 60S ribosomal protein L10a (CSA-19) P62906 804 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 Q16877 (6PF-2-K/Fru-2,6-P2ASE testis-type isozyme) 805 Cleavage and polyadenylation specificity factor, 73 kDa Q9UKF6 subunit (CPSF 73 kDa subunit) 806 Akinase anchor protein 10, mitochondrial precursor O43572 807 Actin, cytoplasmic 1 (Beta-actin) P60709 808 Activated T-cell marker CD109 Q6YHK3 809 Activin receptor type 2A precursor (EC 2.7.11.30) P27037 810 ADAM19 protein Q8TBU7 811 AP-1 complex subunit beta-1 (Adapter-related protein Q10567 complex 1 beta-1 subunit) (Beta-adaptin 1) 812 Adaptor-related protein NFO1019537 Q9BYI8 813 Lung alphabeta hydrolase protein 1 Q96SEO 814 Alpha-actinin-3 Q08043 815 Ankyrin-2 (Brain ankyrin) (Ankyrin-B) Q01484 816 Ankyrin repeat and SOCS box protein 17 Q8WXJ9 817 Anti-colorectal carcinoma heavy chain Q65ZQ1 818 APOBEC1 complementation factor (APOBEC1- Q9NQ94 stimulating protein) 819 Probable DNA dC->dU-editing enzyme APOBEC-3D (EC Q96AK3 3.5.4.—) 820 Apollipoprotein-L4 precursor (Apollipoprotein L-IV) Q9BPW4 821 Apoptosis stimulating of p53 protein 1 Q96KQ4 822 Nucleoporin 188 kDa (arachin) Q5SRE5 823 Protein ariadne-1 homolog (ARI-1) (Ubiquitin-conjugating Q9Y4X5 enzyme E2-binding protein 1) 824 Set1/Ash2 histone methyltransferase complex subunit Q9UBL3 ASH2 (ASH2-like protein) 825 ATP synthase FO subunit 8 Q85KZ3 826 Splice isoform 2 of Q9H7FOATPase family homolog up- Q9H7FO regulated in Senescence cells 827 Probable phospholipid-transporting ATPase IA (EC Q9Y2O0 3.6.3.1) (Chromaffin granule ATPase II) 828 ATP-binding cassette A10 Q8WWZ4 829 ATP-binding cassette Sub-family A member 2 (ATP- Q9BZC7 binding cassette transporter 2) (ATP-binding cassette 2) 830 Autoantigen RCD8 Q6P2E9 831 Xonemal dynein heavy chain 8 Q96JB1 832 Butyrophilin-like protein 8 precursor Q6UX41 833 Ubiquitin carboxyl-terminal hydrolase 20 (EC 3.1.2.15) Q9Y2K6 834 Bardet-Biedl syndrome 7 protein (BBS2-like protein 1) Q8IWZ6 835 Large proline-rich protein BAT2 (HLA-B-associated P48634 transcript 2) 836 Bcl-2 related ovarian killer Q9UL32 837 Lipopolysaccharide-responsive and beige-like anchor P50851 protein (CDC4-like protein) 838 Splice isoform 3 of P35612 P356.12-3 839 UDP-GlcNAc:beta Gal beta-1,3-N- Q9Y2A9 acetylglucosaminyltransferase 3 (EC 2.4.1.—) 840 Cell growth inhibiting protein 39 Q2TTR2 841 BH3-interacting domain death agonist (BID) P55957 842 CD48 antigen precursor (B-lymphocyte activation marker PO9326 BLAST-1) 843 Bone morphogenetic protein receptor type-2 precursor (EC Q13873 2.7.11.30) 844 Bullous pemphigoid antigen 1, isoform 7 Q8WXK8 845 BRCA1 associated RING domain 1 variant Q53F80 846 Breast cancer type 2 susceptibility protein (Fanconi anemia P51587 group D1 protein) US 7,919,467 B2 81 82 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 847 Protein BRE (Brain and reproductive organ-expressed Q9NXR7 protein) (BRCA1/BRCA2-containing complex subunit 45) 848 Breast cancer 1 early onset Q3LRJO 849 Breast and ovarian cancer Susceptibility protein Q7KYU6 850 BTG2 protein (NGF-inducible anti-proliferative protein P78543 PC3) 851 Nuclear protein 5qNCA Q7LBC6 852 CAD protein Includes: Glutamine-dependent carbamoyl- P27708 phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2); Dihydroorotase (EC 3.5.2.3) 853 Cadherin EGF LAG seven-pass G-type receptor 3 Q9NYQ7 precursor (Flamingo homolog1) (hFmi1) (Multiple epidermal growth factor-like domains 2) (Epidermal growth factor-like 1) 854 Cadherin FIB3 Q6UW70 855 integrin alpha-3 precursor (Galactoprotein B3) P26006 856 Calcineurin B homologous protein 2 (Hepatocellular O43745 carcinoma-associated antigen 520) 857 Calcium calmodulin-dependent protein kinase II inhibitor Q7Z7J9 alpha (CaMKIINalpha) 858 Calpain-11 (EC 3.4.22.—) Q9UMQ6 859 Alpha-1 catenin (Cadherin-associated protein) (Alpha E- P35221 catenin) 860 Neural cell adhesion molecule variant Q59FYO 861 Ribosomal L1 domain-containing protein 1 (Cellular O76021 Senescence-inhibited gene protein) 862 CENP-F kinetochore protein (Centromere protein F) P494.54 (Mitosin) 863 Centaurin-delta 1 (Cnt-d1) (Arf-GAP, Rho-GAP, ankyrin Q8WZ64 repeat and pleckstrin homology domain-containing protein 2) 864 Centrosomal protein 2 (Centrosomal Nek2-associated Q9BV73 protein 1) (C-NAP1) 865 Pericentriol material 1 Q15154 866 Cervical cancer Suppressor gene 5 Q8NFX8 867 T-complex protein 1 subunit Zeta-2 Q92526 868 Chemokine-like factor (C32) Q9UBR5 869 Vacuolar protein sorting 13A Q96RL7 870 Chromodomain-helicase-DNA-binding protein 2 (EC O14647 3.6.1. ) 871 FERM domain-containing protein 6 Q96NE9 872 Putative protein C21orf56 Q9HOA9 873 Adiponutrin (iPLA2-epsilon) Q9NST1 884 Coatomer Subunit gamma-2 Q9UBF2 875 Sodium/potassium calcium exchanger 2 precursor Q9UI40 876 Exportin-1 (Chromosome region maintenance 1 protein O1498O homolog) 877 CUB and Sushi domain-containing protein 3 precursor Q7Z407 878 Cullin-7 (CUL-7) Q14999 879 Cyclic AMP-dependent transcription factor ATF-4 P18848 880 S-phase kinase-associated protein 1A (Cyclin A/CDK2- P632O8 associated protein p19) 881 Cyclin-A1 P78396 882 Cyclin M3, isoform 1 Q8NEO1 883 CyStathionine beta-synthase human homolog of Q58H57 Cynomolgus monkey gene product 884 Cytochrome P450 2E1 (EC 1.14.14.1) P05181 885 Keratin, type II cytoskeletal 8 PO5787 886 CPEB2 protein Q3B8N6 887 Probable ATP-dependent RNA helicase DDX5 (EC 3.6.1. ) P17844 888 Dedicator of cytokinesis protein 1 Q1418.5 889 Development and differentiation-enhancing factor 2 O431SO 890 Probable ubiquitin carboxyl-terminal hydrolase FAF-Y (EC O00507 3.1.2.15) 891 G2 and S phase expressed protein 1 Q9NYZ3 892 HUMAN CDNA FLJ30829 fis, clone FEBRA2001790, Q96NI3 highly similar to Xenopus laevis RRM-containing protein SEB-4 mRNA 893 KIAA1799 protein Q96B95 US 7,919,467 B2 83 84 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 894 Peroxisomal proliferator-activated receptor A-interacting Q9BYK8 complex 285 kDa protein (EC 3.6.1.—) (ATP-dependent helicase PRIC285) 895 HUMANKLAA1922 Q96PW6 896 Transcription elongation factor SPT5 (DLC-1)(deleted in OOO267 iver cancer-1) 897 DNA damage-binding protein 1 (Damage-specific DNA- Q16531 binding protein 1) 898 DNA excision repair protein ERCC-6 Q03468 899 DNA polymerase alpha subunit B (DNA polymerase alpha Q14181 70 kDa subunit) 900 DNA replication licensing factor MCM2 P49736 (Minichromosome maintenance protein 2 homolog) 901 DNA2-like homolog (EC 3.6.1.—) (DNA replication ATP- P51530 dependent helicase-like homolog) 902 Estrogen response element binding protein (cotton-top O77798 Tarmarin), DNA2-like homolog (human) 903 DNA damage-inducible transcript 3 (DDIT-3) (Growth P35638 arrest and DNA-damage-inducible protein GADD153) 904 DNA-directed RNA polymerase I largest subunit (EC O956O2 2.7.7.6) 905 DnaJ homolog subfamily C member 1 Q96KC8 906 Splice isoform 2 of P35462 P3S462-2 907 RuvB-like 2 (EC 3.6.1.—) (48-kDa TATA box-binding Q9Y230 protein-interacting protein) 908 DRE1 protein Q9NXT9 909 Dynactin-1 (150 kDa dynein-associated polypeptide) Q14203 910 Dynein heavy chain, cytosolic (DYHC) Q14204 911 Echinoderm microtubule associated protein-like 5 Q6UYC9 912 ECT2 protein (Epithelial cell-transforming sequence 2 Q9H8V3 Oncogene) 913 Endothelial differentiation-related factor 1 (EDF-1) O60869 914 Developmentally-regulated endothelial cell locus 1 protein) O43854 915 Elongation factor 2 (EF-2) P13639 916 domain protein C21orf55 Q9NX36 917 EMILIN-3 precursor (EMILIN-5) (Elastin microfibril Q9NT22 interface-located protein 5) 918 Synaptotagmin-like protein 4 (Exophilin-2) Q96C24 919 Enhancer of filamentation 1 (HEF1) Q14511 920 Band 4.1-like protein 3 (4.1B) (Differentially expressed in Q9Y2J2 adenocarcinoma of the lung protein 1) (DAL-1) 921 Epidermal growth factor receptor substrate 15 P42S66 922 Epithelial membrane protein 3 (EMP-3) (YMP protein) P54852 923 Zyxin (Zyxin-2) Q15942 924 Eukaryotic translation initiation factor 4 gamma 1 Q04637 925 F-actin capping protein beta Subunit P47756 926 Protocadherin Fat 2 precursor (hPat2) (Multiple epidermal Q9NYQ8 growth factor-like domains 1) 927 KIAA1752 protein Q9COB1 928 Fc alphamu receptor Q8WWV6 929 Low affinity immunoglobulin gamma Fc region receptor PO8637 II-A precursor (IgGFc receptor III-2) 930 FYVE, RhoGEF and PH domain-containing protein 2 (Zinc Q7Z6J4 finger FYVE domain-containing protein 4) 931 Fibroblast growth factor receptor-like 1 precursor (FGF Q8N441 receptor-like protein 1) 932 Fibroblast growth factor receptor 4 precursor (EC 2.7.10.1) P224.55 933 Fibroblast growth factor receptor 2 precursor (EC 2.7.10.1) P218O2 (FGFR-2) 934 FK506-binding protein 7 precursor (EC 5.2.1.8) Q9Y680 935 Glomulin (FKBP-associated protein) (FK506-binding Q92990 protein-associated protein) 937 Flavin containing monooxygenase 3 isoform 2 variant Q53FW 5 938 Protein flightless-1 homolog Q13045 939 Guanylate-binding protein 4 Q96PP9 940 Filamin-A (Alpha-filamin) (Filamin-1) (Endothelial actin- P21333 binding protein) 941 FLJ10101 protein Q8WU94 942 CDNA FLJ14503 fis, clone NT2RM1000252, weakly Q96T17 similar to H. sapiens E-MAP-115 mRNA 943 E2F8 protein QSBKY4 US 7,919,467 B2 85 86 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 944 Human Hypothetical protein Q2VPJ3 945 HUMAN CDNA FLJ34154 fis, clone FCBBF3013 058 Q8NB70 946 Hypothetical protein FLJ43654 (Hypothetical protein Q6ZUJ4 C3orf62) 947 HUMAN CDNA FLJ46180 fis, clone TESTI4004.031 Q6ZRQ5 948 Flotillin-2 (Epidermal Surface antigen) (ESA) Q14254 949 Serine/threonine-protein kinase ATR (EC 2.7.11.1) (Ataxia Q13535 elangiectasia and Rad3-related protein) (FRAP-related protein 1) 950 Frizzled 5 precursor (Frizzled-5) Q13467 951 Fructose-bisphosphate aldolase C (EC 4.1.2.13) PO9972 952 G protein pathway Suppressor 1 isoform 1 variant Q53HS2 953 KiSS-1 receptor (KiSS-1R) (Kisspeptins receptor) Q969F8 (Metastin receptor) (G-protein coupled receptor 54) 954 Probable G-protein coupled receptor 55 Q9Y2T6 955 Probable G-protein coupled receptor 35 Q9HC97 956 G-protein coupled receptor family C group 5 member C Q9NQ84 precursor (Retinoic acid-induced gene 3 protein) 957 Leucine-rich repeat-containing G-protein coupled receptor O75473 5 precursor (Orphan G-protein coupled receptor HG38) (G- protein coupled receptor 49) (G-protein coupled receptor 67) 958 Probable G-protein coupled receptor 133 precursor (G- Q6QNK2 protein coupled receptor PGR25) 959 G1 to S phase transition protein 1 homolog (GTP-binding P1517O protein GST1-HS) 960 Gamma-aminobutyric-acid receptor alpha-6 Subunit Q16445 precursor (GABA(A) receptor) 961 Ganglioside-induced differentiation-associated protein 1- Q96MZO ike 1 (GDAP1-L1) 962 Gap junction alpha-5 protein (Connexin-40) (CX40) P36382 963 GEM-interacting protein (GMIP) Q9P107 964 Golgin subfamily B member 1 (Giantin) Q14789 96S UDP-N-acetylhexosamine pyrophosphorylase (Antigen X) Q16222 966 Neutral alpha-glucosidase AB precursor (EC 3.2.1.84) Q14697 967 Probable dolichyl pyrophosphate Glc1Man9GlcNAc2 Q9BVK2 alpha-1,3-glucosyltransferase (EC 2.4.1.—) 968 Bifunctional aminoacyl-tRNA synthetase Includes: PO7814 Glutamyl-tRNA synthetase (EC 6.1.1.17) (Glutamate-- RNA ligase); Prolyl-tRNA synthetase (EC 6.1.1.15) (Proline--tRNA ligase) 969 Glycoproteinnmb-like protein Q8IXJ5 970 Prolactin-releasing peptide receptor (PrRP receptor) P49683 (PrRPR) (G-protein coupled receptor 10) 971 G protein-coupled receptor 112 Q5EGP2 972 Growth factor receptor-bound protein 14 (GRB14 adapter Q14449 protein) 973 GRIP and coiled-coil domain-containing protein 2 (Golgi Q8IWJ2 coiled coil protein GCC185) (CTCL tumor antigen se1-1) 974 GROS1-L protein Q9HC86 975 Growth hormone secretagogue receptor type 1 (GHS-R) Q92847 976 Glutathione S-transferase A4-4 (EC 2.5.1.18) O15217 977 GTP-binding protein Rhes (Rashomolog enriched in Q96D21 striatum) (Tumor endothelial marker 2) 978 GTP-binding protein Rit1 (Ras-like protein expressed in Q92963 many tissues) 98O Hypothetical protein HDLBP Q53QU2 981 Heat shock protein 75 kDa, mitochondrial precursor (HSP Q12931 75) (Tumor necrosis factor type 1 receptor-associated protein) 982 Heat shock protein apg-1 Q53ZP9 983 Tumor rejection antigen (Gp96) 1 Q5CAQ5 984 Heat-shock protein beta-3 (HspB3) (Heat shock 17 kDa Q12988 protein) 985 Low-density lipoprotein receptor-related protein 5 O75197 precursor 986 Regulator of telomere elongation helicase 1 (EC 3.6.1. ) Q9NZ71 (Helicase-like protein NHL) 987 Hematopoietic protein 1 Q52LWO 988 Heme oxygenase 1 (EC 1.14.99.3) (HO-1) PO96O1 US 7,919,467 B2 87 88 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 989 Heparan sulfate glucosamine 3-O-sulfotransferase 5 (EC Q8IZT8 2.8.2.23) 990 Hepatocellular carcinoma-associated antigen 66 Q9NYH9 991 Melanoma-associated antigen E2 (MAGE-E2 antigen) Q8TD90 (Hepatocellular carcinoma-associated protein 3) 992 26S proteasome non-ATPase regulatory subunit 10 (26S O75832 proteasome regulatory subunit p28) Hepatocellular arcinoma-associated protein p28-II 993 Hephaestin Q5JUU1 994 HECT domain and RCC1-like domain-containing protein 2 O9571.4 (HERC2) 995 Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP P22626 A2/hnRNP B1) 996 Heterogeneous nuclear ribonucleoprotein R (hnRNPR) O43390 997 HEXIM1 protein (HMBA-inducible) O94992 998 Histatin-1 precursor (Histidine-rich protein 1) P15515 999 Histone deacetylase 1 (HD1) Q13547 OOO Histone deacetylase 9 (HD9) (HD7B) (HD7) Q9UKVO OO1 Homeodomain-interacting protein kinase 2 (EC 2.7.11.1) Q9H2X6 OO2 Cullin-2 (CUL-2) Q13617 OO3 SWI/SNF-related matrix-associated actin-dependent O6O264 regulator of chromatin subfamily A member 5 (EC 3.6.1.—) OO4 HUMAN HSPCO27 26S proteasome non-ATPase Q9Y6E3 regulatory subunit 13 Synonyms 26S proteasome regulatory subunit S11 26S proteasome regulatory subunit p40.5 005 Hypothetical protein (Novel protein HSPC117) Q9Y3IO (DJ149A16.6 protein) (Hypothetical protein HSPC117) OO6 Claudin domain-containing protein 1 (Membrane protein Q9NY35 GENX-3745) Q9NY35 OO7 Large neutral amino acids transporter Small Subunit 2 (L- Q9UHIS type amino acid transporter 2) (hLAT2) OO8 Heterogeneous nuclear ribonucleoprotein C-like 1 (hnRNP O60812 core protein C-like 1) O09 Ornithine decarboxylase (EC 4.1.1.17) (ODC) P11926 O10 Regulator of nonsense transcripts 2 (Nonsense mRNA Q9HAU5 reducing factor 2) (Up-frameshift Suppressor 2 homolog) (hUpf2) O11 ATX10 HUMAN Ataxin-10 Q9UBB4 O KIAA1833 protein Q569G6 O13 HUMAN UDP-GalNAc:betaGlcNAc beta1,3- Q8NCRO galactosaminyltransferase, polypeptide 2 (Beta 1,3-N- acetylgalactosaminyltransferase-II) (MGC39558) O14 Hypothetical protein KIAA1033 Q2M389 O15 Activating signal cointegrator 1 complex subunit 3 (EC Q8N3CO 3.6.1. ) O16 Delta-interacting protein A (Hepatitis delta antigen- Q15834 interacting protein A) (Coiled-coil domain-containing protein 85B) O17 Hypothetical protein FLJ14466 Q96BP7 O18 interferon-inducible double stranded RNA-dependent O75569 protein kinase activator A O19 Hypothetical protein C9orf142 Q9BUH6 O2O Tetratricopeptide repeat protein 17 Q96AE7 O21 CDNA FLJ14058 fis, clone HEMBB1000554 Q9H7ZO O22 Anaphase promoting complex subunit 13 Q9BS18 O23 Hypothetical protein CCDC60 Q8IWA6 O24 Sphingosine kinase 2 (EC 2.7.1.—) Q9NRAO O25 Probable ATP-dependent RNA helicase DDX11 (EC Q96FC9 3.6.1.—) (DEAD H box protein 11) (CHL1 homolog) (Keratinocyte growth factor-regulated gene 2 protein) (KRG-2) O26 Protein KIAAO182 Q14687 O27 Ras GTPase-activating protein SynGAP (Synaptic Ras- Q96PVO GTPase-activating protein 1) (Synaptic Ras-GAP 1) (Neuronal RasGAP) O28 Fibrinogen C domain containing 1 Q8N539 O29 MGC39581 protein Q86XMO O3O Bcl-2-like 13 protein (Mill protein) (Bcl-rambo) Q9BXK5 O31 Myosin head domain containing 1 Q96H55 O32 WD-repeat protein 51A Q8NBTO O33 KIF27A (OTTHUMP00000021559) Q86VH2

US 7,919,467 B2 91 92 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. O87 Nischarin Q6PIB4 O88 FERM domain-containing protein 4A Q9P2O2 O89 Leucine-rich repeats neuronal protein 1 precursor Q6UXK5 (Neuronal leucine-rich repeat protein 1) (NLRR-1) O90 KIAA1512 protein Q9P216 O91 KIAA1598 protein Q9HCH4 O92 Phosphatidylinositol-3 phosphate 3-phosphatase adaptor Q96QU2 Subunit 093 KIAA1730 protein Q9COD3 O94 KIAA1786 protein Q96 JN9 O95 Hypothetical protein MGC20470 Q96EK3 O96 OACT1 protein Q86XC2 O97 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 Q16877 (6PF-2-K/Fru-2,6-P2ASE testis-type isozyme) O98 (kappaB kinase complex-associated protein (IKK complex- O951.63 associated protein) (p150) O99 mmune receptor expressed on myeloid cells 2 Q7Z7I3 OO High-affinity cAMP-specific and IBMX-insensitive 3',5'- O60658 cyclic phosphodiesterase 8A (EC 3.14.17) O1 Bone specific CMF608 Q6WRIO O2 importin alpha-7 Subunit (Karyopherin alpha-6) O60684 O3 naD-like protein (Inadl protein) (hINADL) (Pals1- Q8NI35 associated tightjunction protein) (Protein associated to ightjunctions) O4 Nitric oxide synthase, inducible (EC 1.14.13.39) P35228 05 Transcription elongation factor SPT5 (hSPT5) OOO267 O6 nositol-trisphosphate 3-kinase B (EC 2.7.1.127) (Inositol P27987 4,5-trisphosphate 3-kinase B) O7 Type I inositol-3,4-bisphosphate 4-phosphatase (EC Q96PE3 3.1.3.66) (Inositol polyphosphate 4-phosphatase type I) O8 insulin receptor beta Subunit Q9UCB7 09 insulin-like growth factor binding protein, acid labile Q8TAYO Subunit 10 integrin beta-4 precursor (GP150) (CD104 antigen) P16144 11 Splice isoform 2 of P35462 P3S462-2 12 interferon alpha 2 protein Q16055 13 interferon-induced protein with tetratricopeptide repeats 1 PO9914 (IFIT-1)) (Interferon-induced 56 kDa protein) (IFI-56K) 14 interleukin-20 precursor (IL-20) (Four alpha helix cytokine Q9NYY1 Zcyto10) 15 Steroid receptor RNA activator isoform 1 Q9HD15 16 intersectin-2 (SH3 domain-containing protein 1B) Q9NZM3 (SH3P18) (SH3P18-like WASP-associated protein) 117 TI-like protein (Inter-alpha (Globulin) inhibitor H5-like) Q6UXX5 18 Gap junction alpha-5 protein (Connexin-40) P36382 19 Kelch-like protein 8 Q9P2G9 2O Keratin, type II cytoskeletal 1 (Cytokeratin-1) PO4264 21 ADAM9 precursor (EC 3.4.24.—) (A disintegrin and Q13443 metalloproteinase domain 9) (Metalloprotease? disintegrin cysteine-rich protein 9) (Myeloma cell metalloproteinase) 22 Next to BRCA1 gene 1 protein (Neighbor of BRCA1 gene Q14596 protein) (Membrane component, chromosome 17, Surface marker 2) (1A1-3B) 23 Hypothetical protein DKFZp686K2075 Q6MZZ8 24 KIAAO100 protein Q14667 25 Pre-mRNA-splicing factor ATP-dependent RNA helicase Q92620 PRP16 (EC 3.6.1. ) (ATP-dependent RNA helicase DHX38) (DEAH box protein 38) 26 KIAA0251 protein Q8TBS5 27 HUMAN KIAA0342 protein O15050 28 KIAA0357 protein O15064 29 Hypothetical protein KIAA0372 Q6PGP7 30 KIAA0377 splice variant 2 Q86TE7 31 KIAA0386 protein Q9Y4F9 32 HUMAN CTCL tumor antigen HD-CL-04 Q548S1 33 importin-13 (Imp13) (Ran-binding protein 13) O94829 34 KIAAO769 protein O94.868 35 Hypothetical protein KIAAO863 Q6IQ32 US 7,919,467 B2 93 94 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 36 Zinc finger protein KIAA1196 - Q96KM6 37 CRSP complex subunit 3 (Cofactor required for Sp1 Q9ULK4 transcriptional activation Subunit 3) (Transcriptional coactivator CRSP130) (Vitamin D3 receptor-interacting protein complex 130 kDa component 38 Pyruvate dehydrogenase lipoamide-phosphatase 2, Q9P2J9 mitochondrial precursor (EC 3.1.3.43) 39 Protocadherin-10 precursor Q9P2E7 40 Leucine-rich repeats and calponin homology (CH) domain QSVUJ6 containing 2 41 Ankyrin repeat domain 18B Q5WOG2 42 Kin17 protein (HsKin17 protein) (KIN, antigenic O6087O determinant of recA protein homolog) 43 Kinesin-like protein KIF13A (Kinesin-like protein RBKIN) Q9H1H9 44 Putative RNA binding protein KOC OOO425 45 Keratin, type I cytoskeletal 18 (Cytokeratin-18) P05783 46 Kv3.2d voltage-gated potassium channel Q86WO9 47 Lethal (3)malignant brain tumor-like protein (L(3)mbt-like) Q9Y468 (L(3)mbt protein homolog) 48 Lactadherin precursor (Milkfat globule-EGF factor 8) Q08431 (MFG-E8) (HMFG) (Breast epithelial antigen BA46) (MFGM) 49 Lamin-A/C (70 kDa lamin) PO2545 50 Laminingamma-1 chain precursor (Laminin B2 chain) P11047 51 Low-density lipoprotein receptor-related protein 5 O75197 (CSO 52 Leptin receptor precursor (LEP-R) (OB receptor) P48357 53 Mitogen-activated protein kinase kinase kinase 13 (EC O43283 2.7.11.25) S4 Leukemia virus receptor 2 Q08357 55 Leukemia-associated protein with a CXXC domain Q8NFU7 56 RNA-binding protein 6 (RNA-binding motif protein 6) P78332 (RNA-binding protein DEF-3) (Lung cancer antigen NY LU-12) 57 Lung cancer oncogene 5 Q7Z5Q7 58 Heterogeneous nuclear ribonucleoprotein M (hnRNPM) P52272 59 Macrophage migration inhibitory factor (MIF) P14174 (Phenylpyruvate tautomerase) (EC5.3.2.1) 60 Mitotic spindle assembly checkpoint protein MAD2B Q9UI95 (MAD2-like 2) (hREV7) 61 Mitogen-activated protein kinase kinase kinase 4 (EC Q9Y6R4 2.7.11.25) (MAPK/ERK kinase kinase 4) 62 Serine/threonine?tyrosine-interacting-like protein 1 (Dual- Q9Y6.J8 specificity protein phosphatase 24) (Map kinase phosphatase-like protein MK-STYX) 63 Microtubule-associated serine/threonine-protein kinase 2 Q6POQ8 (EC 2.7.11.1) 64 Matrix metalloprotease MMP-27 Q9H306 65 MCM10 protein Q7L590 66 Interferon-induced helicase C domain-containing protein 1 Q9BYX4 (EC 3.6.1.—) (Melanoma differentiation-associated protein 5) 67 Melanoma ubiquitous mutated protein Q2TAK8 68 Melanoma antigen family D, 2 QSBJF3 69 Melanocyte protein Pmel 17 precursor (Melanocyte P40967 lineage-specific antigen GP100) 70 GPI-anchored protein p137 (p137GPI) (Membrane Q14444 component chromosome 11 Surface marker 1) Cytoplasmic activation proliferation-associated protein 1 71 Hepatocyte growth factor receptor precursor (EC 2.7.10.1) PO8581 (HGF receptor) (Scatter factor receptor) (SF receptor) (HGF/SF receptor) (Met proto-oncogene tyrosine kinase) 72 Mitogen-activated protein kinase 14 (EC 2.7.11.24) Q16539 73 Mitogen-activated protein kinase kinase kinase kinase 2 Q12851 (EC 2.7.11.1) (MAPK/ERK kinase kinase kinase 2) 74 Mitotic kinesin-related protein Q96Q89 75 Sperm-associated antigen 5 (Astrin) (Mitotic spindle- Q96RO6 associated protein p126) 76 Myeloid lymphoid or mixed-lineage leukemia protein 4 Q9UMN6 (Trithorax homolog2) 77 Putative helicase MOV-10 (EC 3.6.1.—) (Moloney leukemia Q9HCE1 virus 10 protein) US 7,919,467 B2 95 96 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 78 MOZ/CBP protein Q712H6 79 Calgranulin B (Migration inhibitory factor-related protein PO6702 4) (MRP-14) (P14) 8O MUC2 HUMAN Mucin-2 precursor (Intestinal mucin 2) Q02817 81 Mucin-5B precursor (Mucin 5 subtype B, tracheobronchial) Q9HC84 82 Multiple PDZ domain protein (Multi PDZ domain protein O7597O ) (Multi-PDZ-domain protein 1) 83 RUFY2 (Run and FYVE domain-containing protein Rabipa. Q8IW33 84 Multidrug resistance-associated protein 7 Q8NHX7 85 Multiple copies in a T-cell malignancies (Malignant T cell Q9ULC4 amplified sequence 1) (MCT1) 86 DNA mismatch repair protein Msh3 P20585 87 Protein CBFA2T2 (MTG8-like protein) (MTG8-related O43439 protein 1) (Myeloid translocation-related protein 1) 88 Myomesin-1 (190 kDa tiltin-associated protein) (190 kDa P521.79 connectin-associated protein 89 Myosin heavy chain, cardiac muscle beta isoform (MyHC- P12883 beta) 90 Myosin-13 (Myosin heavy chain, skeletal muscle, Q9UKX3 extraocular) (MyHC-eo) 91 Tumor suppressor candidate 3 (N33 protein) Q13454 92 Nebulin-related anchoring protein Q8TCHO 93 Neural cell adhesion molecule 1, 1 P13592 94 Neurotrimin precursor Q9P121 95 Ninein Q8N4C6 96 Notch homolog 2 Q5VTDO 97 Neurogenic locus notch homolog protein 1 precursor P46531 (Notch 1) (hN1) (Translocation-associated notch protein TAN-1) Contains: Notch 1 extracellular truncation: Notch 1 intracellular domain 98 Neurogenic locus notch homolog protein 3 precursor Q9UM47 (Notch 3) Contains: Notch 3 extracellular truncation; Notch 3 intracellular domain 99 Neurogenic locus notch homolog protein 4 precursor Q994.66 (Notch 4) (hNotch4) Contains: Notch 4 extracellular truncation; Notch 4 intracellular domain 2OO Plexin-A1 precursor (Semaphorin receptor NOV) Q9UIW2 2O1 HUMANNPDO11 Q9H2R7 2O2 Nuclear autoantigen Sp-100 (Speckled 100 kDa) (Nuclear P23497 dot-associated Sp100 protein) 2O3 Nuclear factor erythroid 2-related factor 1 (NF-E2-related Q14494 actor 1) (NFE2-related factor 1) (Nuclear factor, erythroid derived 2, like 1) (Transcription factor 11) (Transcription actor HBZ17) (Transcription factor LCR-F1) (Locus control region-factor 1) 204 Nuclear factor of activated T-cells, cytoplasmic 1 (NFAT O95644 transcription complex cytosolic component) (NF-ATc1) 205 Nuclear receptor coactivator 2 (NCoA-2) (Transcriptional Q15596 intermediary factor 2) 2O6 Ubiquitin-like PHD and RING finger domain-containing Q96T88 protein 1 (EC 6.3.2.—) 2O7 Nucleic acid helicase DDXx Q8IWW2 208 Nucleoporin 62 kDa (NUP62 protein) Q6GTM2 209 Nuclear pore complex protein Nup98-Nup96 precursor PS2948 Contains: Nuclear pore complex protein Nup98 (Nucleoporin Nup98) (98 kDa nucleoporin); 210 Nucleoprotein TPR P12270 211 Nuclear pore complex protein Nup107 P57740 212 Nuclear pore complex protein Nup205 Q92621 213 ODF2 protein Q6PJQ8 214 Trophoblast glycoprotein precursor (5T4 oncofetal Q13641 trophoblast glycoprotein) 215 Dynamin-like 120 kDa protein, mitochondrial precursor O60313 (Optic atrophy 1 gene protein) 216 Orexin receptor type 2 (OX2r) (Hypocretin receptor type 2) O43614 217 Transmembrane emp24 domain-containing protein 10 P49755 precursor (Transmembrane protein Tmp21) 218 Orphan nuclear receptor TR2 (Testicular receptor 2) P13056 219 MKL/myocardin-like protein 1 (Myocardin-related Q969V6 transcription factor A) (MRTF-A) (Megakaryoblastic leukemia 1 protein) (Megacaryocytic acute leukemia protein) US 7,919,467 B2 97 98 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 220 Ovarian cancer related tumor marker CA125 - Q8WXI7 221 Oxysterol-binding protein-related protein 8 (OSBP-related Q9BZF1 protein 8) 222 Centrosomal protein of 70 kDa (Cep70 protein) (p10- Q8NHQ1 binding protein) 223 Leucine carboxyl methyltransferase 2 (EC 2.1.1.—) O6O294 (p21WAF1/CIP1 promoter-interacting protein) 224 F-box/LRR-repeat protein 5 (F-box and leucine-rich repeat Q9UKA1 protein 5) (F-box protein FBLA/FBL5) 225 inhibitor of growth protein 3 Q9NXR8 226 P53 inducible protein Q9UN29 227 DNA polymerase alpha catalytic subunit (EC 2.7.7.7) PO9884 228 Chloride intracellular channel protein 4 (Intracellular Q9Y696 chloride ion channel protein pé4H1 229 Paired mesoderm homeobox protein 2B (Paired-like Q99453 homeobox 2B) (PHOX2B homeodomain protein) (Neuroblastoma Phox) 230 PRB3 protein P81489 231 Protein patched homolog 1 (PTC1) (PTC) Q13635 232 Rap guanine nucleotide exchange factor 2 (Neural RAP Q9Y4G8 guanine nucleotide exchange protein) (nRap GEP) (PDZ domain-containing guanine nucleotide exchange factor 1) (PDZ-GEF1) 233 Pecanex-like protein 1 (Pecanex homolog) - Q96RV3 234 GC-1-related estrogen receptor alpha coactivator short Q8TDE4 isoform 235 PHD finger Q86U89 236 Hypothetical protein DKFZp686C07187 Q6NO38 237 Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic P42338 subunit beta isoform (EC 2.7.1.153) (PI3-kinase p110 subunit beta) (PtdIns-3-kinase p110) 238 Phosphodiesterase 8A, isoform 1 Q6P9H3 239 Serine/threonine-protein kinase SMG1 (EC 2.7.11.1) Q96Q15 (SMG-1) (hSMG-1) (Lambda iota protein kinase C interacting protein) (Lambda-interacting protein) ( 240 PiggyBac transposable element derived 3 Q8N328 241 PIWIL3 protein Q7Z3Z3 242 Homeobox protein PKNOX1 (PBX/knotted homeobox 1) P55347 243 Transmembrane protein 115 (Protein PL6) Q12893 244 Plakophilin-2 Q99959 245 Plectin 6 Q6S380 246 Plectin 1 (PLTN) (PCN) (Hemidesmosomal protein 1) Q15149 (HD1) 247 Plexin B1; plexin 5; semaphorin receptor O43157 248 Pleiotropic regulator 1 O43660 249 Blood vessel epicardial substance (hBVES) (Popeye Q8NE79 domain-containing protein 1) (Popeye protein 1) 250 Carboxypeptidase-like protein X2 precursor Q8N436 251 YIF1B protein QSBJH7 252 Melanoma antigen preferentially expressed in tumors P78395 (Pr4eferentially expressed antigen of melanoma) (OPA interacting protein 4) 253 Splice isoform 2 of Q9H7FO Q9H7FO-2 2S4 P2Y purinoceptor 13 (P2Y13) (G-protein coupled receptor Q9BPV8 86) (G-protein coupled receptor 94) 255 Putative pre-mRNA-splicing factor ATP-dependent RNA O43143 helicase DHX15 (EC 3.6.1. ) (DEAH box protein 15) (ATP-dependent RNA helicase #46) 2S6 HUMAN RNA-binding protein 34 (RNA-binding motif P42696 protein 34) 257 Prolyl 4-hydroxylase alpha-1 subunit precursor (EC P13674 .14.11.2) (4-PH alpha-1) (Procollagen-proline, 2 oxoglutarate-4-dioxygenase alpha-1 subunit) 258 Profilin-1 PO7737 259 Programmed cell death protein 5 (TFAR19 protein) (TF-1 O14737 cell apoptosis-related gene 19 protein) 260 Propionyl-CoA carboxylase beta chain, mitochondrial P05166 precursor (EC 6.4.1.3) 261 26S proteasome non-ATPase regulatory subunit 1 (26S Q994.60 proteasome regulatory subunit RPN2) (26S proteasome regulatory subunit S1) (26S proteasome subunit p112) US 7,919,467 B2 99 100 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 1262 26S proteasome non-ATPase regulatory subunit 3 (26S O43242 proteasome regulatory subunit S3) (Proteasome subunit p58) 1263 Proteasome activator complex subunit 3 (Proteasome P61289 activator 28-gamma Subunit) (PA28gamma) (PA28g) (Activator of multicatalytic protease subunit 3) (11S regulator complex gamma Subunit) (REG-gamma) (Ki nuclear autoantigen) 264 Protein C14orf166 Q9Y224 26S Protein KIAA1219 Q86X10 266 Protein KIAA1688 Q9COH5 267 Protein Plunc precursor (Palate lung and nasal epithelium Q9NP55 clone protein) (Lung-specific protein X) (Nasopharyngeal carcinoma-related protein) (Tracheal epithelium-enriched protein) (Secretory protein in upper respiratory tracts) 268 Protein transport protein Sec23B Q15437 269 Liprin-alpha-2 (Protein tyrosine phosphatase receptor typef O75334 polypeptide-interacting protein alpha-2) (PTPRF interacting protein alpha-2) 270 Protocadherin gamma A12 precursor (PCDH-gamma-A12) O6033O (Cadherin-21) (Fibroblast cadherin 3) 271 Protocadherin gamma A10 precursor (PCDH-gamma-A10) Q9Y5H3 272 Leucine carboxyl methyltransferase 2 (EC 2.1.1.—) O6O294 (p21WAF1/CIP1 promoter-interacting protein) 273 KIAA1636 protein Q9HCD6 274 Probable G-protein coupled receptor 160 Q9UJ42 275 Protein C21orf245 Q9NYP9 276 Periodic tryptophan protein 2 homolog Q15269 277 Rab-like protein 2B Q9UNT1 278 Cell cycle checkpoint protein RAD17 (hRad17) (RF- O75943 Cactivator 1 homolog) 279 DNA repair protein RAD50 (EC 3.6.--) (hRAD50) Q92878 28O Ras GTPase-activating protein 1 (GTPase-activating P20936 protein) (GAP) (Ras p21 protein activator) (p120GAP) (RasGAP) 281 Rasguanine nucleotide exchange factor 2 Q9UK56 282 Ras-GTPase-activating protein-binding protein 1 (EC Q13283 3.6.1. ) (ATP-dependent DNA helicase VIII) (GAPSH3 domain-binding protein 1) (G3BP-1) (HDH-VIII) 283 Ras-related protein Rab-27A (Rab-27) (GTP-binding P51159 protein Ram) 284 Ras-related protein Rab-3D O95716 285 Nuclear pore complex protein Nup107 (Nucleoporin P57740 Nup107) (107 kDa nucleoporin) 286 Receptor-interacting factor 1 Q86XS4 287 Regulating synaptic membrane exocytosis protein 3 (Nim3) Q9UJDO (Rab-3 interacting molecule 3) (RIM3) (RIM3 gamma) 288 Regulator of G protein signaling protein (Regulator of G- Q86UVO protein signalling like 1) 289 MHC class II regulatory factor RFX1 (RFX) (Enhancer P22670 actor C) (EF-C) 290 Retinoblastoma-associated protein (PP110) (P105-RB) PO64OO 291 Roundabout homolog 3 precursor (Roundabout-like protein Q96MSO 3) 292 Retinoblastoma-associated protein HEC (Kinetochore O14777 associated 2) 293 Retinoblastoma-associated protein RAP140 Q9UK61 294 AT-rich interactive domain-containing protein 4A (ARID P29374 domain-containing protein 4A) (Retinoblastoma-binding protein 1) 295 umonji/ARID domain-containing protein 1A P29375 (Retinoblastoma-binding protein 2) (RBBP-2) 296 RhoGTPase regulating protein variantARHGAP20-1ad Q6RJUS 297 40S ribosomal protein S4, Y isoform 2 Q8TD47 298 RNA binding motif Q13380 299 RNA binding protein (Autoantigenic, hnRNP-associated Q2M365 with lethal yellow), long isoform - 3OO RNA-binding protein Q8NI52 301 Roribonucleoprotein-binding protein 1 (SIAHBP1 protein) Q9UHX1 3O2 HUMAN OTTHUMPOOOOOO3O902 Q5JYR6 US 7,919,467 B2 101 102 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 303 Ryanodine receptor 2 (Cardiac muscle-type ryanodine Q92736 receptor) (RyR2) (RYR-2) (Cardiac muscle ryanodine receptor-calcium release channel) (hRYR-2) 3O4 SEC14-like protein 1 Q92503 305 Secreted and transmembrane protein 1 precursor (Protein Q8WVN6 K12) 306 Neudesin precursor (Neuron-derived neurotrophic factor) Q9UMX5 307 P-selectinglycoprotein ligand 1 precursor (PSGL-1) Q14242 (Selectin P ligand) (CD162 antigen) 3O8 Semaphorin-6D precursor Q8NFY4 309 Serine/threonine?tyrosine-interacting protein (Protein Q8WUJO tyrosine phosphatase-like protein) 310 Olfactory receptor 8G5 (Olfactory receptor OR11-298) Q8NG78 311 Shb-like adapter protein, Shf Q7M4L6 312 Signal transducer and activator of transcription 1-alphabeta P42224 (Transcription factor ISGF-3 components p91p84) STAT1 313 Signal transducer and activator of transcription 3 (Acute- P4O763 phase response factor) 314 40S ribosomal protein S7 P62O81 315 60S ribosomal protein L35 P42766 316 60S ribosomal protein L7 P18124 317 Thrombospondin-2 precursor P3S442 318 C3 and PZP-like alpha-2-macroglobulin domain containing 8 Q8IZJ3 319 ATP-binding cassette Sub-family F member 2 (Iron- Q9UG63 inhibited ABC transporter 2) 32O Ribosome biogenesis protein BOP1 (Block of proliferation Q14137 1 protein) 321 CDNA FLJ13765 fis, clone PLACE4000128, weakly Q9H8C5 similar to Mus musculus putative transcription factor mRNA 322 CD200 cell Surface glycoprotein receptor isoform 2 variant 2 Q6O8B3 323 LRRC58 protein Q96CX6 324 Claudin-6 (Skullin 2) P56747 325 T-box transcription factor TBX18 (T-box protein 18) O95935 326 (NTS7 protein Q8WUH5 327 FRAS1-related extracellular matrix protein 2 precursor Q5SZK8 (ECM3 homolog) 328 Zinc finger protein 318 (Endocrine regulatory protein) Q5VUA4 329 Eukaryotic translation initiation factor 3 subunit 8 (eIF3 Q996.13 p110) (eIF3c) 330 HUMAN LOC196394 protein Q8IY45 331 Hypothetical protein FLJ44216 Q8NDZ2 332 Heat shock protein HSP 90-beta (HSP 84) (HSP 90) PO8.238 333 Sarcoma antigen NY-SAR-41 (NY-SAR-41) Q5T9S5 334 Protein FAM86A Q96GO4 335 Ras-like family 11 member A (OTTHUMP00000018162) Q6T310 336 Keratin, type I cytoskeletal 18 (Cytokeratin-18) (CK-18) P05783 337 U3 Small nucleolar RNA-associated protein 14 homolog A Q9BVJ6 (Antigen NY-CO-16) 338 Hypothetical protein DKFZp781D1722 Q68DMO 339 Chromosome-associated kinesin KIF4A (Chromokinesin) O95239 340 Kinesin-like protein KIF6 Q6ZMV9 341 Myosin-10 (Myosin heavy chain, nonmuscle IIb) P3558O (Nonmuscle myosin heavy chain IIb) 342 Hypothetical protein C17orf57 Q8IY85 343 Similar to peptide N-glycanase homolog (S. cerevisiae) Q9BVR8 344 Peptidyl-prolyl cis-trans isomerase A (EC5.2.1.8) P62937 345 Serpin A13 precursor Q6UXR4 346 40S ribosomal protein SA (p40) (34f67 kDa laminin PO8865 receptor) (Colon carcinoma laminin-binding protein) (NEM/1CHD4) (Multidrug resistance-associated protein MGr1-Ag) 347 N-acetylglucosamine-1-phosphotransferase subunit gamma Q9UJJ9 precursor 348 Liprin-beta-1 (Protein tyrosine phosphatase receptor type f Q86W92 polypeptide-interacting protein-binding protein 1) 349 40S ribosomal protein S3a P61247 350 40S ribosomal protein S3a P61247 351 LOC124512 protein (Fragment) Q86XAO 532 Hypothetical protein MGC26744 Q96KX1 535 Hypothetical protein LOC122258 Q96KW.9 3S4 Sulfiredoxin-1 (EC 1.8.98.2) Q9BYNO US 7,919,467 B2 103 104 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 355 Basalin Q5QJ38 356 Protein FAM86A Q96GO4 357 Transmembrane protein 16F Q4KMO2 358 TEB4 protein O1467O 359 SLC1 OAS Q5PT55 360 Serine/threonine-protein kinase SNF1-like kinase 1 (EC P57059 2.7.11.1) 361 LOC391257 protein Q6PO94 362 Zinc finger protein 161 (Putative transcription factor DB1) Q14119 363 Slithomolog 2 protein precursor (Slit-2) O94813 364 FYN-binding protein (FYN-T-binding protein) O15117 365 Jumonji/ARID domain-containing protein 1C (SmcX P41229 protein) (Xeló9 protein) 366 Jumonji/ARID domain-containing protein 1D (SmcY Q9BY66 protein) (Histocompatibility Y antigen) 367 Monocarboxylate transporter 3 (MCT 3) O95907 368 Solute carrier family 4 sodium bicarbonate cotransporter- Q6U841 like member 10 369 Sorting nexin 14, isoform a Q6NUI7 370 Sorting nexin-4 O95219 371 Spectrin beta chain, brain 4 (Spectrin, non-erythroid beta Q9NRC6 chain 4) 372 Spermatogenesis-associated protein 7 (Spermatogenesis- Q9POW8 associated protein HSD3) 373 Non-POU domain-containing octamer-binding protein Q15233 (NonC) protein) (54 kDa nuclear RNA- and DNA-binding protein) (p54(nrb)) (p54nrb) (55 kDa nuclear protein) 374 Cohesin subunit SA-1 (Stromal antigen 1) (SCC3 homolog Q8WVM7 1) 375 Steroid receptor RNA activator isoform 1 Q9HD15 376 Structure-specific recognition protein 1 (SSRP1) Q08945 (Recombination signal sequence recognition protein) (T160) (Chromatin-specific transcription elongation factor 80 kDa subunit) 377 Suppressor of hairy wing homolog 2 (5'OY11.1) (Zinc Q86YH2 finger protein 632) 378 Transcription elongation factor SPT5 (hSPT5) (DRB OOO267 sensitivity-inducing factor large subunit) (DSIF large subunit) (DSIF pl60) (Tat-cotransactivator 1 protein) (Tat CT1 protein)- 379 Synaptogyrin-3 O43761 380 Synaptoanin-2-binding protein (Mitochondrial outer P57105 membrane protein 25) 381 Synemin Q8TE61 382 Talin-1 Q9Y490 383 TAR RNA loop binding protein (TAR (HIV) RNA binding Q13395 protein 1) 384 Taste receptor type 2 member 3 (T2R3) Q9NYW6 385 Taste receptor type 2 member 40 (T2R40) (T2R58) (G- P59535 protein coupled receptor 60) 386 Oxidoreductase HTATIP2 (EC 1.1.1. ) (HIV-1 TAT- Q9BUP3 interactive protein 2) 387 Transcription initiation factor TFIID subunit 6 P49848 (Transcription initiation factor TFIID 70 kDa subunit) (TAF(II)70) (TAFII-70) (TAFII-80) (TAFII30) 388 TRAG) protein Q6PIP7 389 T-complex protein 1 subunit beta (TCP-1-beta) (CCT-beta) P78371 390 Telomerase-binding protein EST1A (Ever shorter Q86US8 telomeres 1A) (Telomerase subunit EST1A) (EST1-like protein A) (hSmg5/7a) 391 Tumor endothelial marker 6 (Hypothetical protein TEM6) Q96PEO 392 Ras GTPase-activating-like protein IQGAP2 Q13576 393 Tetratricopeptide repeat protein 15 (TPR repeat protein 15) Q8WVT3 394 Myosin-18A (Myosin XVIIIa) (Myosin containing PDZ Q92614 omain) (Molecule associated with JAK3 N-terminus) (MAJN) 395 Polycystic kidney and hepatic disease 1 precursor Q8TCZ9 (Fibrocystin) 396 TMC4 protein Q7ZSM3 397 MDC-3.13 isoform 1 (TNFAIP8 protein) Q9UER5 398 Toll-like receptor 8 precursor Q9NR97 399 Tolloid-like protein 1 precursor (EC 3.4.24-) O43897 US 7,919,467 B2 105 106 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt ID Identification NO: Parent Sequence Identification No. 14OO DNA topoisomerase I, mitochondrial precursor (EC Q969P6 5.99.1.2) (TOP1mt) 1401 PAP associated domain-containing protein 5 (EC 2.7.7.—) Q8NDF8 (Topoisomerase-related function protein 4-2) (TRF4-2) 1402 Plastin-3 (T-plastin) P13797 1403 Translocated promoter region (To activated MET Q5SWYO Oncogene) 1404 P2Y purinoceptor 13 (P2Y13) (G-protein coupled receptor Q9BPV8 86) (G-protein coupled receptor 94) 1405 Transcript Y 5 Q9BXH6 1406 Transcription factor Sp4 (SPR-1) Q02446 1407 Transcription initiation factor TFIID subunit 1 (EC P21675 2.7.11.1) (Transcription initiation factor TFIID 250 kDa subunit) (TAF(II)250) (TAFII-250) (TAFII250) (TBP associated factor 250 kDa) (p.250) (Cell cycle gene 1 protein) 1408 Transcriptional repressor CTCFL (CCCTC-binding factor) Q8NI51 (Brother of the regulator of imprinted sites) (Zinc finger protein CTCF-T) (CTCF paralog 1409 Transducer of regulated CREB protein 3 Q6UUV7 1410 Transmembrane channel-like protein 4 Q7Z404 1411 Transcription initiation factor TFIID subunit 6 P49848 (Transcription initiation factor TFIID 70 kDa subunit) (TAF(II)70) (TAFII-70) (TAFII-80) (TAFII30) 1412 Trophinin-associated protein (Tastin) (Trophinin-assisting Q12815 protein) 1413 Tryptophanyl-tRNA synthetase (EC 6.1.1.2) (Tryptophan-- P23381 tRNA ligase) (TrpRS) (IFP53) (hWRS) 1414 Tubulin, gamma complex associated protein 3 Q5T9Y2 1415 Tumor necrosis factor ligand Superfamily member 6 (Fas P48O23 antigen ligand) (Fasligand) (CD178 antigen) (CD95L protein) (Apoptosis antigen ligand) (APTL) Contains: Tumor necrosis factor ligand Superfamily member 6, membrane form 1416 Tumor necrosis factor, alpha-induced protein 1, endothelial Q13829 (B12 protein) 1417 Netrin receptor DCC precursor (Tumor suppressor protein P431.46 DCC) (Colorectal cancer suppressor) 1418 Adipocyte-derived leucine aminopeptidase precursor (EC Q9NZO8 3.4.11.—) (A-LAP) (ARTS-1) (Aminopeptidase PILS) (Puromycin-insensitive leucyl-specific aminopeptidase) (PILS-AP) (Type 1 tumor necrosis factor receptor shedding aminopeptidase 419 U1 Small nuclear ribonucleoprotein A (U1 SnRNP protein PO9012 A) (U1A protein) (U1-A 420 U6 snRNA-associated Sm-like protein LSm8 O95777 421 Ubiquitin-protein ligase E3A (EC 6.3.2.—) (E6AP ubiquitin- Q05086 protein ligase) (Oncogenic protein-associated protein E6 AP) (Human papillomavirus E6-associated protein) 422 Ubiquitin carboxyl-terminal hydrolase 3 (EC 3.1.2.15) Q9Y614 (Ubiquitin thioesterase 3) (Ubiquitin-specific-processing protease 3) (Deubiquitinating enzyme 3) 423 UBX domain-containing protein 2 Q92575 424 UNC93 homolog B1 (UNC-93B protein) (hUNC93B1) Q9H1C4 425 Splice isoform 5 of Q9H171 Q9H171-5 426 CDNA FLJ46282 fis, clone TESTI4031066 Q6ZRK6 427 CCDC73 protein Q6P5Q7 428 Caspase recruitment domain-containing protein 10 (CARD- Q9BWT7 containing MAGUK protein 3) (Carma 3) 429 Chromatin-specific transcription elongation factor FACT Q9YSB9 140 kDa subunit 430 Beta-defensin 120 precursor Q8N689 431 Alpha-catulin (Catenin alpha-like protein 1) (Alpha- Q9UBT7 catenin-related protein) 432 Ribonuclease III (EC 3.1.26.3) (RNase III) Q9NRR4 433 Seizure related 6 homolog Q53EL9 434 Granulocyte colony-stimulating factor precursor (G-CSF) PO9919 (Pluripoietin) (Filgrastim) (Lenograstim) 435 Lysyl-tRNA synthetase Q9HB23 436 Protein C6orf130 Q9Y530 437 Melanophilin (Exophilin-3) (Synaptotagmin-like protein Q9BV36 2a) (Slp homolog lacking C2 domains a) US 7,919,467 B2 107 108 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 438 Novel protein (Possible ortholog of mouse Q5VTR6 phosphoinositide-3-kinase adaptor protein 1 (Pik3ap1) 439 AER61 glycosyltransferase Q6P985 440 Heat shock protein HSP 90-alpha (HSP 86) PO7900 441 60S ribosomal protein L19 P84098 442 PDZ domain-containing protein 11 Q5EBL8 443 Dedicator of cytokinesis 11-; Cdc42-associated guanine Q5JSL3 nucleotide exchange factor ACG/DOCK11 444 Hypothetical protein FLJ26930 Q6ZNX6 445 Laminingamma-1 chain precursor (Laminin B2 chain) P11047 446 ATP-binding cassette transporter sub-family C member 11 Q96J66 (Multidrug resistance-associated protein 8) 447 Uridine? cytidine kinase-like 1 Q9NWZ5 448 Butyrophilin-like protein 8 precursor Q6UX41 449 Endothelial cell-selective adhesion molecule precursor Q96AP7 450 Poly(A)-specific ribonuclease PARN (EC 3.1.13.4) O95453 (Polyadenylate-specific ribonuclease) (Deadenylating nuclease) (Deadenylation nuclease) 451 Voltage-gated potassium channel KCNA7 Q96RP8 452 F-box protein 11 Q52ZP1 453 rythrocyte membrane protein band 4.1 like 5 Q7ZSS1 454 CDNA FLJ45O15 fis, clone BRAWH3O14639 Q6ZT30 455 FAM13A1 v2 protein Q24.JPO 456 Hypothetical protein FLJ26432 Q6ZP70 457 VPS13D-1A protein Q709C5 458 Coagulation factor VIII precursor (Procoagulant POO4S1 component) (Antihemophilic factor) (AHF) 459 NFX1-type zinc finger-containing protein 1 Q9P2E3 460 Polymerase I and transcript release factor (PTRF protein) Q6NZI2 461 T-complex protein 1 subunit epsilon (TCP-1-epsilon) P48643 (CCT-epsilon) 462 Probable ATP-dependent RNA helicase DDX27 (EC Q96GQ7 3.6.1.—) (DEAD box protein 27) 463 HSCARG Q9HBL8 464 CDNA FLJ43956 fis, clone TESTI4.015681 Q6ZU72 465 Mitochondrial-processing peptidase alpha Subunit, Q10713 mitochondrial precursor (EC 3.4.24.64) (Alpha-MPP) 466 CDNA FLJ45287 fis, clone BRHIP3002124 Q6ZSRO 467 Hypothetical protein C12Orf62 Q96I36 468 VPS13D-2A protein Q709C4 469 CDNA FLJ32009 fis, clone NT2RP7009498, weakly Q96DN2 similar to FIBULIN-1, ISOFORMA 470 Transient receptor potential cation channel subfamily V Q9HBAO member 4 (TrpV4) (osm-9-like TRP channel 4) (OTRPC4) (Vanilloid receptor-like channel 2) (Vanilloid receptor-like protein 2) (VRL-2) 471 Vascular endothelial growth factor D precursor (VEGF-D) O43915 (c-fos-induced growth factor) (FIGF) 472 Vascular endothelial growth factor receptor 1 precursor P17948 (EC 2.7.10.1) (VEGFR-1) (Vascular permeability factor receptor) (Tyrosine-protein kinase receptor FLT) (Flt-1) (Tyrosine-protein kinase FRT) (Fms-like tyrosine kinase 1) 473 Proto-oncogene C-crk (P38) (Adapter molecule crk) P46108 474 VDUP1 protein (Thioredoxin interacting protein) Q9H3M7 475 Vimentin P08670 476 HUMAN CTCL tumor antigen HD-CL-06 (Vimentin Q548L2 variant) 477 Vinculin (Metavinculin) P182O6 478 Integrin alpha-3 precursor (Galactoprotein B3) (GAPB3) P26006 (VLA-3alpha chain) (FRP-2) (CD49c antigen) Contains: Integrin alpha-3 heavy chain; Integrin alpha-3 light chain 479 Voltage-dependent T-type calcium channel alpha-1H O9518O Subunit (Voltage-gated calcium channel alpha subunit Cav3.2) (Low-voltage-activated calcium channel alpha1 3.2 subunit) 480 Wiskott-Aldrich syndrome protein family member 4 Q8IV90 (WASP-family protein member 4) 481 ATP synthase coupling factor 6, mitochondrial precursor P18859 (EC 3.6.3.14) (ATPase subunitF6) 482 Proto-oncogene protein Wnt-3 precursor P56703 US 7,919,467 B2 109 110 TABLE 3-continued SEQID NO, Parent Protein Identification and SwissProt Identification Number for full-length sequences 792-1513

Parent SEQ SwissProt D Identification NO: Parent Sequence Identification No. 483 Amyloid beta A4 precursor protein-binding family A Q99767 member 2 (Neuron-specific X11L protein) (Neuronal Munc18-1-interacting protein 2) (Mint-2) (Adapter protein X11beta) 484 Zinc finger CCHC domain-containing protein 5 Q8N8U3 485 Myeloid/lymphoid or mixed-lineage leukemia (Trithorax Q59FF2 homolog, Drosophila) variant 486 Zinc finger protein DZIP1 (DAZ-interacting protein 1/2) Q86YF9 487 Hypothetical protein DKFZp761O1618 Q69YS5 488 ATP-binding cassette Sub-family F member 2 (Iron- Q9UG63 inhibited ABC transporter 2) 489 Ribosome biogenesis protein BOP1 (Block of proliferation Q14137 protein) 490 CDNA FLJ13765 fis, clone PLACE4000128 Q9H8C5 491 NDRG1 protein (N-myc downstream regulated gene 1 Q92597 protein) 492 Pre-mRNA splicing factor ATP-dependent RNA helicase Q92620 PRP16 493 Nesprin 2 (Nuclear envelope spectrin repeat protein 2) Q9NU50 494 Adenomatous polyposis coli P25054 495 Ubiquitin conjugating enzyme E2 P49459 496 B cell receptor-associated protein BAP31 (CDM protein) P5572 6c6-AG 497 Topoisomerase II-alpha P11388 498 Topoisomerase II beta Q02880 499 integrin beta8 Subunit precursor P26O12 500 Replication Protein A P27694 5O1 Abl Binding protein 3 U3.1089 502 Cyclin I Q14094 503 Cell Division Control Protein 2 (CDC2) PO6493 SO4 Septin 2 (NEDD5) Q15019 505 STAT1 alpha/beta P42224 SO6 LDL Receptor-related protein (LRP) Q07954 507 TACE (ADAM17) NP-068604 SO8 Junction plakoglobin (gamma catenin) P14923 509 EDDR1 Q08345 510 IP3 receptor type II Q14571 511 Melanoma-associated antigen D2 (MAGE-D2 antigen) Q9UNF1 (MAGE-D) (Breast cancer-associated gene 1 protein) (BCG-1) (11B6) (Hepatocellular carcinoma-associated protein JCL-1) 512 Melanoma-associated antigen 4 (MAGE-4 antigen) P43358 (MAGE-X2) (MAGE-41) 513 HUMAN Retinoblastoma-like protein 2 (130 kDa Q08999 retinoblastoma-associated protein) (PRB2) (P130) (RBR-2)

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US07919467B2). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

We claim: go 1517, 1519, 1521, 1527, 1528, and 1529 in an amount suffi cient to induce a CTL response to said tumor cells. 1. A method for eliciting a CTL response against tumor 2. The method of claim 1, wherein said composition further cells presenting at least one of the following epitopic pep comprises an adjuvant. tides: SEQ ID NO: 1516, 1517, 1519, 1521, 1527, 1528, or 3. The method of claim 2, wherein said adjuvant is selected 1529 in a subject, comprising administering to said subject a from the group consisting of complete Freund's adjuvant, composition comprising at least one polypeptide comprising 65 incomplete Freund's adjuvant, Montanide ISA-51, LAG-3, an epitopic peptide comprising an amino acid sequence aluminum phosphate, aluminum hydroxide, alum, and Sapo selected from the group consisting of SEQ ID NO: 1516, 1. US 7,919,467 B2 111 112 4. The method of claim 1, wherein said composition further 11. The method of claim 1 wherein said tumor cells are part comprises a cytokine. of an ovarian carcinoma. 5. The method of claim 4, wherein said cytokine is GM 12. The method of claim 1 wherein said polypeptide com CSF. prises at least two epitopic peptides. 6. The method of claim 1, wherein said composition further 13. The method of claim 12 wherein said polypeptide com comprises a vehicle. prises at least three epitopic peptides. 7. The method of claim 6, where said vehicle is selected from the group consisting of a liposome, an immunostimu 14. The method of claim 12, wherein said polypeptide lating complex (ISCOM), and slow-releasing particles. comprises a first epitopic peptide and a second epitopic pep 8. The method of claim 7, wherein said liposome comprises tide, wherein said first epitopic peptide comprises an amino an emulsion, a foam, a micelle, an insoluble monolayer, a 10 acid sequence selected from the group consisting of SEQID liquid crystal, a phospholipid dispersion, or a lamellar layer. NO: 1516, 1517, 1519, 1521, 1527, 1528, and 1529 and said 9. The method of claim 1, wherein said polypeptide con second epitopic peptide comprises an amino acid sequence sists of an amino acid sequence selected from the group selected from the group consisting of SEQ ID NO: 1516, consisting of SEQ ID NO: 1516, 1517, 1519, 1521, 1527, 1517, 1519, 1521, 1527, 1528, and 1529. 1528, and 1529. 15 10. The method of claim 1 wherein said tumor cells are part of a carcinoma. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,919,467 B2 Page 1 of 1 APPLICATIONNO. : 1 1/426161 DATED : April 5, 2011 INVENTOR(S) : Ramakrishna et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page: Item (75) should read Inventors: Venky Ramakrishna, Riegelsville, PA (US); Mark M. Ross, Charlottesville, VA (US); Ramila Philip, Ivyland, PA (US)

“Lorraine H. Keller has been deleted as an inventor.

Signed and Sealed this Seventeenth Day of May, 2011

David J. Kappos Director of the United States Patent and Trademark Office