AU2008200628A1 - Nucleic acid and corresponding protein entitled 24P4C12 useful in treatment and detection of cancer - Google Patents

Description

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AUSTRALIA

Patents Act 1990 V ~ii -i i FB RICE CO Patent and Trade Mark Attorneys AGENSYS, INC.

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Nucleic acid and corresponding protein entitled 24P4C12 useful in treatment and detection of cancer The following statement is a full description of this invention including the best method of performing it known to us:- 00 NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 24P4C12 USEFUL IN TREATMENT AND DETECTION OF CANCER CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of Australian Patent Application No. 2002352976 filed on November 27, 00 2002, the contents of which are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION 00 The Invention describedl herein relates to a gene and its encoded prot 'ein. terned 24P4C12, expressed in certain N cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 24P4C12.

BACKGROUND OF THE INVENTION 00Cancer is the second leading cause of human death next to coronary disease. Woritid, milions of people die from cancer every year. In the United States alone, aS reported by the American Cancer Society. cancer causes the death of well o ver a half-mnillion people arinually, with over 1.2 millon new cases diagnosed per year. While death from headt disease have been decining significantly; those resulting from can~cer generally are on the rise. In the early part of the next century, cowce is predicted tb become the Wodig cause of death.

Worldwide, several canr= stand out as the leaing killers. In paricular, carcinomas of the Iung prostate. breast colon, pancreas, and ovary represent the prdmay causes of cancer death. These and vitually all other carnomna share a common lethal feature- With very few exceptions, metastict diseas fromn a carcinoma is fatal. Moreover, even for those cancer patients who Witally survive their primary cances, common expellence has show that their fives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cance patients experience physical debllitations following treatment. Furthermtore, many cancer patients experience a recurrence.

Worldwide, prostate cancer Is the fut most prevalent canr in men. In North America and Northern Europe, it Is by far the most common cancer in males and is the second leading cause of cancer death In men. In the United States alone, well over 30,000 men die annually of Oti disease second only to lung cancer. Despite the magnitude of these figures, there Is still no effective treatment for metastatic iprostate cance. Surgical prostatectomny, radiation "hsay, hormone ablation therapy, surgical castration and ctrotherapy continue to be the main treahment, modalites.

Unfortunately. these treatments are Ineffective for many and are often associated with undesirable consequences.

On the dlagostic front the takof a prostate tumnor mader that can accurately detect eaty-stage, locallized tumors remains a significant Imitation in the diagnosis and management of this disease. Althoughi the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utiity Is widely regarded as lacldng In several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cance xenografts that can recapitulate different stages of the disease In mice. The LAPG &wp fpgeles Boistate Cancoer) xenografts are prostate canc= xenografts that have survive passage In sevee combined inine deficient (SClD) ic and have e4rIlted tie capacity to mln*c the kanitin from androgen dependence to androgen Independence (K0lin of at., 1997, Nat Mied. 3:402). More recently Identified pro state cancer markers Include PCTA-1 (Suetf 1996, Proc. Nat.

AcAd Sd. USA 93.7252), protate-secfic membrane (PSM) antigent (Pinto of aL. ami Cancer Res 1996 Sep 2 1445- 51), STEAP (Huber of at, Proc Nall Acad Sc U SA I W Doc 7.96(25): 14523-8)and prostate stem cellantigen(PSCA) (Reter etat, 1998, Prot. Nag. Acad. Sci USA 95:1t735).

00 While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and Streat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocardnoma and transitional cell carcinoma of the renal pelvis or ureter. The inddence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma Is less frequent, with an inddence of approximately 500 cases per year in the United States.

OO

C Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell cardnoma may be approached aggressively in appropriate patients with a possibility 0 of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

00 Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth Smost common neoplasm) and 3 percent in women (eighth most common neoplasm). The inddence Is Increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, induding 39,500 in men and 15,000 In women. The age-adjusted incidence in the United States Is 32 per 100.000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking pattems in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an Increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and Intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most musde-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women.

Incidence rates declined significantly during 1992-1996 per year). Research suggests that these declines have been due to ncreased screening and polyp removal, preventing progression of polyps to Invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S.

cancer deaths.

At present, surgery Is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S.

cancer diagnoses. The Inddence rate of lung and bronchial cancer is declining significantly In men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of Increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

00 Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths.

During 1992-1996, mortality from lung cancer declined significantly among men per year) while rates for women were still significantly increasing per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking 00 patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include 00 N surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery Is usually the treatment of choice.

IO Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung N cancer on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is 00 Showever, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

SAn estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000.

After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) In 2000 due to breast cancer.

Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may Involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy.

Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstrction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast andlor tamoxifen may reduce the chance ol DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop Into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary wil be removed, especially In young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

00 There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about per year) while rates have increased slightly among women.

0 Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

00 SUMMARY OF THE INVENTION The present invention relates to a gene, designated 24P4C12, that has now been found to be over-expressed in C the cancer(s) listed in Table I. Northern blot expression analysis of 24P4C12 gene expression in normal tissues shows a 0 restricted expression pattern in adult tissues. The nudeotide (Figure 2) and amino acid (Figure 2, and Figure 3) sequences Sof 24P4C12 are provided. The tissue-related profile of 24P4C12 In normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 24P4C12 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, andlor therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynudeotides corresponding or complementary to al or part of the 24P4C12 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynudeotides encoding 24P4C12-related proteins and fragments of 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino adds: at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino adds of a 24P4C12-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynudeotides or oligonucleotides complementary or having at least a 90% homology to the 24P4C12 genes or mRNA sequences or parts thereof, and polynudeotides or oligonudeotides that hybridize to the 24P4C12 genes, mRNAs, or to 24P4C12-encoding polynuclectides. Also provided are means for Isoating cONAs and the genes encoding 24P4C12.

Recombinant DNA molecules containing 24P4C12 polynudeotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 24P4C12 gene products are also provided. The invention further provides antibodies that bind to 24P4C12 proteins and polypeptide fragments thereof, including polydonal and monodonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent In certain embodiments, there is a proviso that the entire nucleic acid sequence of Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 is not prepared. In certain embodiments, the entire nuceic acid sequence of Figure 2 is encoded and/or the entire amino add sequence of Figure 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 24P4C12 polynudeotides and proteins in various biological samples, as web as methods for identifying cells that express 24P4C12. A typical embodiment of this invention provides methods for monitoring 24P4C12 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 24P4C12 such as cancers of tissues listed in Table I, induding therapies aimed at Inhibiting the transcription, translation, processing or function of 24P4C12 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for beating a cancer that expresses 24P4C12 In a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits 00 0 the production or function of 24P4C12. Preferably, the carrier is a uniquely human carrier. In another aspect of the Invention, the agent is a moiety that is immunoreactive with 24P4C12 protein. Non-limiling examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polydonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The Santibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as 00 defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 24P4C12 and/or one or more than 00 one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to eliidt an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In Sa further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or -N more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, 0 the one or more than one nuceic acid molecule may express a moiety that is immunologically reactive with 24P4C12 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits C1 production of 24P4C12. Non-limiting examples of such molecules Indude, but are not limited to, those complementary to a nucleotide sequence essential for production of 24P4C12 anisense sequences or molecules that form a triple helix with a nudeotide double helix essential for 24P4C12 production) or a ribozyme effective to lyse 24P4C12 mRNA.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLUX (collectively HLA Peptide Tables) respective to its parental protein, variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position WX", one must add the value *X 1I to each position In Tables VIII-XXI and XXII to XUX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 1, 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once In Tables VIII-XXI and at least once in tables XXII to XLX, or an oligonudeolide that encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonudeotide encoding the peptide region, that has one two, three, four, or five of the following characteristics: i) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that Includes an amino acid position having a value equal to or greater than 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophllldty profile of Figure i) a peptide region of at least 5 amino adds of a particular peptide of Figure 3, In any whole number Increment up to the ful length of that protein in Figure 3, that includes an amino add position having a value equal to or less than 0.5, 0.4, 0.3,0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; ill) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein In Figure 3, that includes an amino acd position having a value equal to or greater than 0.6,0.7,0.8,0.9, or having a value equal to 1.0, In the Percent Accessible Residues profile of Figure 7; 00 iv) a peptide region of at. least 5 amino acfd of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.6,0.7,0.8, 0.9, or having a value equal to 1.0. in the Average Flexibility profile of Figure 8; or v) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the tull length of that protein in Figure 3. that includes an amino acid position having a value equal to or greater than 00 c) 0.6,0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9.

00 NK BRIEF DESCRIPTION OF THE FIGURES Figure 1. The 2412412 SSH sequence of 160 nucleolides.

Figure 2. A) The cONA and amino acid sequence of 24P4C12 variant I (also called'24P4C 12 v.V or'24P4C12 ~~KI variant 1 I Is shown In Figure 2A. The start methionine is underlined. The open reading frme extends from nuclelc acid 6- 00 2138 indluding the stop codon.

B) The cDNA and amino acid sequence of 24P4C12 variant 2 (also called 14M412 Is shown In Figure 2B.

The codon for the start methionine Is underlined. The open reang frame extends from nucleic acid 6-2138 including the Stop codOn.

C) The cl)NA and amino acid sequence of 24P4IC12 variant 3 (also called "24P4CI2 v.31 is shown in Figure 2C.

The codon for the start methionine is underlined. The open reading frame extends from nucleic; acid 6-2138 InidclIng the stop codon.

D) The cD)MA and amino acid sequence of 24P4C1 2 variant 4 (also called '244C1 2 v.41) Is shown in Figure 2D.

The odon for the start methionine Is underlined. The open reading frame extends from nudleic acid 6-2138 Including the stop Woon.

E) The cONA and amino acid sequence of 24P4C12 variant 5 (also called '24134C1 2 is shown in Figure 2E.

The codon for the stat methionine is underlined. The open reading firam extends from nucleic; acid 6-2138 including the stop rodon.

F) The cOMA and amino acid sequence of 24134C12 variant 6 (also called '24154C12 Is shown in Figure 2F.

The codon for the start methionine is underlined. The open reading frame extends from nuclc acid 6-2138 Including the stop codon.

G) The cDNA end amino adid sequence of 24P4C1 2 variant 7 (also celled '24P4C1 2 v.7) Is shown In Figure 2G.

The codon for the start methionine Is underlined. The open readng frame extends from nudlc acid 6-1802 including the Stop cDon11.

H1) The cOMA and amino acid sequence of 24P4C12 variant 8 (also called 24P4C12 v.81) is shown in Figure 21-.

The codon for the start methionine is underlined. The open reading frame extends from nuclelc add 6-2174 including the Stop Codon.

IThe cDNA and amino acid sequence of 24P4C12 vaiant 9 (also called '24P4012 Is shown in Figure 21.

The codon for the stat inethionine is underlined. The open reading frame extends from nuclc acid 6-2l44 including the stop codon.

Figure 3.

A) Aino acid sequence of 24P4C 12 v.1I is shown In Figure 3A; It has 710 amino acids.

B) The arnino acid sequence of 24P4C1 2 v.3 is shown in Figure 36; it has 710 amino acids.

C) The amino acid sequence of 24P4C12 V.5 is shown in Figure 3C; It has 710 ami~no acids.

D) The amino acid sequence of 24P4C12 v.6 Is shown in Figure 313; iK has 710 amino acids.

00 E) The amino adid sequence of 24P4C12 v.7 is shown in Figure 3E; it has 598 amino acids.

F) The amino acid sequence of 24P4C12 v.8 is shown in Figure 3F; it has 722 amino acids.

G) The amino adid sequence of 24P4C12 v.9 is shown in Figure 3G; it has 712 amyino adds. As used herein, a reference to 24P4C1I2 includes all variants thereof, including those shown in Figures 2, 3, 10, and 11, unless the context 00cdearly Indicates otherwise.

Figure 4. Alignment or 24P4C12 with human cholne transporter-like protein 4 (CT"4) (gil14249468).

Hydrophificity amino acid profile of 24P4C1 2 determined by computer algorithmn sequence analysis 00 using the method of Hopp and Woods (Hopp Woods KRL, 1981. Proc. NatI. Azad. Sdi. U.S.A. 78:3824a32B) accessed CN on the Protscale website located on the World Wide Web at (.expasy.chlcgi-binlprotscae.p) through the ExPasy molecular ilg server, FigurelB. Hydropathicity amrino acid profile of 24P4C12 determined by computer algorithm sequence analysis 00 using the mnethod of Kyle and Doolittle (Kyte Doolittle 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-binlprotscaie.pl) through the ExPasy molecular biology server.

Figure 7. Percent accessible residues amino acid profile of 24P4C12 determined by computer algorithm sequence analysis using the method of Janin (Janmn 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.drlcgi-bnlprotscAle.p) through the ExPasy molecular biology serve.

Figure 8. Average flexibility, amino acid profile of 24P4C12 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran and Ponnuswamy 1988. Int. J. Pept Protein Res.

32:242-255) accessed on the ProtScale websita located on the World Wide Web at (.expasy.chlcgi-binlProtscale.pl) through the ExPasy molecular biology server.

Figurell. Bela-turn amino acid profile of 24P4C1 2 determined by computer algorithm sequence analyss using the method of Deleage and Roux (Deleage, Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.exasy.ch/gi-bin/pmtscale.p) through the ExPasy molecular biology server.

Figure 10. Schematic alignment of SNP variants of 24P4C12. Variants 24P4C12 v.2 through v.6 are variants with single nudeotide differences. Though these SN.P variants are shown separately, thy could also occuir In any combinations and in any transcript variants that contains the base pairs. Numbers correspond to those of 24 P4CI 2 v.1. Black box shows the same sequence as 24P4C 12 v.1. SNPs are indicated above the box.

Figure i1. Schematic allgnment of protein variants of 24P4C12. Protein variants correspond to nucleoide variants. Nucleofide variants 24P4C12 v.2, v.4 In Figure 10 code for the same protein as 24P4C1 2 v.1. Nudleotidle variants 24P4C1 2 v.7, v.8 and v.9 are spice variants of v. 1, as shown in Figure 12 Z Sngle amino acid differences were Indicated above the boxes. Black boxes represent the same sequence as 24P4C1 2 v. 1. Numbers underneath the box correspond to 24P4C12 FIgure 12. Exon compositions of transcript variants of 24P4C12. Variant 24P4C12 v.7, v.8 and v.9 are transcript variants of 24P4Ci2 v.1. Variant 24P4C12 v.7 does not have exons 10 and 11 of variant 24P4C1 2 v.1. Variant 24P4C12 v.8 extended 36 bp at the Y end of exon 20 of variant 24P4C12 v.1. Variant 24P4C12 v.9 had a longer axon 12 and shorter exon 13 as compared to variant 24P4C12 v.1. Numbers underneath the boxes correspond to those of 24P4C12 v.1.

Lengt of Introns and exons are not proportional.

Figure 13. Secondary structure and transmembrane domains predicin for 24P4C12 protein variant I (SEQ ID NO: 112). k The secondary structure of 24P4C12 protein variant 1 was predicted using the HNN Hierarchical Neural Network method (Guermeur, 1997, hroJibilJbcp.frkgib Jnpsa...automat~pl?pagerrps&.Lnn.hinl), accessed from the ExPasy molecular biology server (httpJ~vww.exqasy.chftoobd). This method priedicts the presence and locaion of alpha helices. extended strands, and random coils from the primary protein sequence. The percent of the protein In a given 00 secondary structure is also listed. B: Schematic representation of the probability of existence of transmembrane regions and Cl orientation of 24P4C12 variant 1 based on the TMpred algorithm of Hofmann and Stoffel which utlizes TMBASE (K.

f) Hofmann, W. Stoffel. TMBASE A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). C: Schematic representation of the probability of the existence of transmembrane regions and the extracellular and 00 intracellular orientation of 24P4C12 variant 1 based on the TMHMM algorithm of Sonnhammer, von Heine, and Krogh (Erik LL. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T.

00 Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998). The TMpred and TMHMM C algorithms are accessed from the ExPasy molecular biology server (httpJ/ww.expasy.chftools/).

Figure 14. 24P4C12 Expression by RT-PCR. First strand cDNA was generated from vital pool 1 (kidney, liver and 0 lung), vital pool 2 (colon, pancreas and stomach), a pool of prostate cancer xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD 00 and LAPC-9AI), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pod, ovary cancer pool, breast Scancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and 30 cycles of amplification. Results show strong expression of 24P4C12 in prostate cancer pool and ovary cancer pool. Expression was also detected in prostate cancer xenografts, bladder cancer pool kidney cancer pool, cdon cancer pool, breast cancer pool, cancer metastasis pool, vital pool 1, and vital pool 2.

Figure 15. Expression of 24P4C12 in normal tissues. Two multiple tissue northem blots (Clontech) both with 2 ug of mRNAlane were probed with the 24P4C12 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of 24P4C12 in prostate, kidney and colon. Lower expression is detected in pancreas, lung and placenta amongst all 16 normal tissues tested.

Figure 16. Expression of 24P4C12 in Prostate Cancer Xenografts and Cel Lines. RNA was extracted from a panel of cell fines and prostate cancer xenografts (PrEC, LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, PC-3, DU145, TsuPr, and LAPC-4CL). Northern blot with 10 ug of total RNAlane was probed with 24P4C12 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The 24P4C12 transcript was detected in LAPC-4AD, LAPC-4AI, LAPC- 9AD, LAPC-9AI, LNCaP, and LAPC-4 CL Figure 17. Expression of 24P4C12 in Patient Cancer Specimens and Normal Tissues. RNA was extracted from a pool of prostate cancer specimens, bladder cancer specimens, colon cancer specimens, ovary cancer specimens, breast cancer specimens and cancer metastasis specimens, as well as from normal prostate normal bladder normal kidney and normal colon Northem blot with 10 pg of total RNA/lane was probed with 24P4C12 SSH sequence.

Size standards in kilobases (kb) are indicated on the side. Strong expression of 24P4C12 transcript was detected in the patient cancer pool specimens, and in normal prostate but not in the other normal tissues tested.

Figure 18. Expression of 24P4C12 in Prostate Cancer Patient Specimens. RNA was extracted from normal prostate prostate cancer patient tumors and their matched normal adjacent tissues (Nat). Northem blots with 10 ug of total RNA were probed with the 24P4C12 SSH fragment Size standards in kilobases are on the side. Results show expression of 24P4C12 in normal prostate and all prostate patient tumors tested.

Figure 19. Expression of 24P4C12 In Colon Cancer Patient Specimens. RNA was extracted from colon cancer cell lines (CL: Colo 205, LoVo, and SK-CO-), normal colon colon'cancer patient tumors and their matched normal adjacent tissues (Nat). Northern blots with 10 ug of total RNA were probed with the 24P4C12 SSH fragment Size standards In kilobases are on the side. Results show expression of 24P4C12 In normal colon and all colon patient tumors tested. Expression was detected in the cell lines Colo 205 and SK-CO-, but not in LoVo.

00 O Figure 20. Expressior of 24P4C12 in Lung Cancer Patient Specimens. RNA was extracted from lung cancer cell CN lines (CL: CALU-1, A427, NCI-H82, NCI-H146), normal lung lung cancer patient tumors and their matched normal adjacent tissues (Nat). Northern blots with 10 ug of total RNA were probed with the 24P4C12 SSH fragment Size r. standards in klobases are on the side. Results show expression of 24P4C12 in lung patient tumors tested, but not in normal 00 lung. Expression was also detected in CALU-1, but not in the other cell lines A427, NCI-H82, and NCI-H146.

0 Figure 21. Expression of 24P4C12 in breast and stomach human cancer specimens. Expression of 24P4C12 was assayed in a panel of human stomach and breast cancers and their respective matched normal tissues on RNA dot 00 blots. 24P4C12 expression was seen in both stomach and breast cancers. The expression detected in normal adjacent N tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may indicate that these IN tissues are not fully normal and that 24P4C12 may be expressed in early stage tumors.

SFigure 22. 24P4C12 Expression in a large panel of Patient Cancer Specimens. First strand cDNA was prepared 00 from a panel of ovary patient cancer specimens uterus patient cancer specimens prostate cancer specimens bladder cancer patient specimens lung cancer patient specimens pancreas cancer patient specimens colon Scancer specimens and kidney cancer specimens Normalization was performed by PCR using primers to actin.

Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and 30 cydes of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Expression was recorded as absent, low, medium or strong. Results show expression of 24P4C12 in the majority of patient cancer specimens tested, 73.3% of ovary patient cancer specimens, 83.3% of uterus patient cancer specimens, 95.0% of prostate cancer specimens, 61.1% of bladder cancer patient specimens, 80.6% of lung cancer patient specimens, 87.5% of pancreas cancer patient specimens, 87.5% of colon cancer specimens, 68.% of of dear cell renal carcinoma. 100% of papillary renal cell carcinoma.

Figure 23. 24P4C12 expression in transduced cells. PC3 prostate cancer cells, NIH-3T3 mouse cells and 300.19 mouse cells were transduced with 24P4C12 .pSRa retroviral vector. Cells were selected in neomydn for the generation of stable cell lines. RNA was extracted following selection in neomycin. Northern blots with 10 ug of total RNA were probed with the 24P4C12 SSH fragment. Results show strong expression of 24P4C12 in 24P4C12pSRa transduced PC3, 3T3 and 300.19 cells, but not in the control cells transduced with the parental pSRa construct Figure 24. Expression of 24P4C12 in 293T cells. 293T cell were transiently transfacted with either pCDNA3.1 Myc-His tagged expression vector, the pSRO expression vector each encoding the 24P4C12 variant 1 cONA or a control neo vector. Cells were harvested 2 days later and analyzed by Westem blot with antl-24P4C12 pAb or by Flow cytometry (B) on fixed and permeabilized 293T cells with either the ant-24P4C12 pAb or anti-His pAb followed by a PE-conjugated antirabbit IgG secondary Ab. Shown Is expression of the monomeric and aggregated forms of 24P4C12 by Western blot and a fluorescent shift of 24P4C12-293T cells compared to control neo cells when stained with the anti-24P4C12 and anti-His pAbs which are directed to the intracellular NH3 and COOH termini, respectively.

Figure 25. Expression and detection of 24P4C12 in stably transduced PC3 cells. PC3 cells were Infected with retrovirus encoding the 24P4C12 variant 1 cDNA and stably transduced cells were derived by G418 selection. Cells were then analyzed by Western blot or immunohistochemistry with anti-24P4C12 pAb. Shown with an arrow on the Western blot is expression of a -94 kD band representing 24P4C12 expressed In PC3-24P4C12 cells but not in control neo cells. Immunohistochemical analysis shows specific staining of 24P4C12-PC3 cells and not PC3-neo cells which Is competed away competitor peptide to which the pAb was derived.

Figure 26. Expression of recombinant 24P4C12 antigens in 293T cells. 293T cells were transiently transfected with Tag5 His-tagged expression vectors encoding either amino adds 59-227 or 319453 of 24P4C12 variant 1 or a control vector. 2 days later supematants were collected and cells harvested and lysed. Supematants and lysates were then subjected to Western blot analysis using an anti-His pAb. Shown is expression of the recombinant Tag5 59-227 protein in 0 antigens for generation of 24P4C1 2-specific antibodies.

Figure 27. Monoclonal antibodies detect 24P4C12 protein expression in 293T cells by flow cytometry. 293T cells were transfected with either pCDNA 3.1 His-tagged expression vector for 24P4C1 2 or a control neo vector and harvested 2 days later. Cells were fixed, permeabilized, and stained with a 1:2 dilution of supernatants of the indicated hyblomas 00 generated from mice immunized With 300.19-24P4C12 cells or with anti-His pAb. Cells were then stained with a PE- -conjugated secondary Ab and analyzed by Hlow cytomnetry. Shown is a fluorescent shift of 293T-24P4C12 cells but not control neo cells demonstrating specific recogniton of 24P4C12 protein by the hybrldoma supemnatants.

00 Figure 2B. Showis expression of 24P4C12 Enhances Proliferation. PC3 and 3T3 were grown overnight in low PBS. Cells were then incubated in low or 10% PBS as indicated. Proliferation was measured by Alamar Blue.

Figure 29. Detecion of 24P4C12 protein by immunohistochemistry In prostate cancer patent specimens.

Prostate adenocmcinoina lissue and Its matched normal adjacent tissue were obtained from prostate cancer patients. The 00 results showed strong expression of 24P4CI 2 in fie tumor cells and normal epithelium of the prostate cancer patients'lissue (panels low grade prostate adenocarcinoma, high grade prostate adenocarcinoma, normal tissue adjacent to tumor). The expression was detected mostly around the cell membrane indicating that 24P4C1 2 is membrane associated In prostate tissues.

Figure 30. Detection of 24P4C1 2 protein by imnmunohistochenmistry in vaious cancer patient specimens. Tissue was obtained from patents with colon adenocaranonia, breast ductl carcinoma, lung adenocarcinoma, bladder transitional cell carcinoma, renal clear cell carcinoma and pancresatic adenocaranoma. The results showed expression of 24P4C12 in the tumor cells of the cancer patients' tissue (panel colon adenocarioma, lung adenocarcinomra, breast ductal carcinoma, bladder transitional carcinoma, renal dear cell carcinomia, pancreatic adenocarcinonia).

Figure 31. Shows 24P4C1 2 Enhances Tumor Growth In SCID Mice. 1 x 106 PC3-24P4C1 2 cells were mixed with Matrigell and injected on the night and left subcutaneous fl anks of 4 male SCID mice per group. Each data point represents mean tumor volume Figure 32. Shows 24P4C12 Enhances Tumor Growth in SCID W~e. 1 x 106 3T3-24P4C12 cells were mixed with Matrigel and Injected on the right subouaneous flanks of 7 male SCID mice per group. Each data point represents mean tumor volume DETAILED DESCRIPTION OF THE INVENTION Outline of Sections Definitions 110) 24P4C12 Potlyniuceotides IA.) Uses of 24P4C12 Polyrucleoties I.AI.) Monitoring of Genetic Abnormalite ILA2.) Antlsense Embodiments IiA3) Primers and Primer Pairs I.A4.) lsolation of 24P34C2-Enooding Nuclec Adid Molecules Recombinant Nuclec Acid Molecules and Host-Vector Systems IlI.) 24P4Cl2-elsted Protein Motif -bearing Protein Embodiments lfl.B.) Expression of 24P4C1 2-related Proteins IlI.C.) Modifications of 24P4C12-relafed Proteins lll.D.) Uses of 24PAIC12-rellated Proteins 00 IV.) 24P4C12 Antibodies OV.) 24P4C12 Cellular Immune Responses VI.) 24P4C12 Transgenic Animals C) VII.) Methods for the Detection of 24P4C12 SVIII.) Methods for Monitoring the Status of 24P4C12-related Genes and Their Products 0 0 IX.) Identification of Molecules That Interact With 24P4C12 Therapeutic Methods and Compositions Anti-Cancer Vaccines 00 XB.) 24P4C12 as a Target for Antibody-Based Therapy 24P4C12 as a Target for Cellular Immune Responses X.C.1. Minigene Vaccines SX.C. Combinations of CTL Peptides with Helper Peptides 00 X.C.3. Combinations of CTL Peptides with T Cell Priming Agents X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor HTL Peptides Adoptive Immunotherapy Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 24P4C12.

XU.) Inhibition of 24P4C12 Protein Function XII.A) Inhibition of 24P4C12 With Intracelular Antibodies XII.B.) Inhibition of 24P4C12 with Recombinant Proteins XI.C.) Inhibition of 24P4C12 Transcription or Translation XII.D.) General Considerations for Therapeutic Strategies XIll.) Identification, Characterization and Use of Modulators of 24P4C12 XIV.) KITS/Artides of Manufacture Definitions: Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill In the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for carity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled In the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out In accordance with manufacturer defined protocols andlor parameters unless otherwise noted.

The terms "advanced prostate cancer', 'locally advanced prostate cancer', advanced disease" and locally advanced disease' mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Assocation (AUA) system, stage C1 C2 disease under the WhitmoreJewett system, and stage 13 T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having dinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically 00 identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the N prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the 3 tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

'Altering, the native glycosylation pattern" is intended for purposes herein to mean deleting one or more 00 carbohydrate moieties found in native sequence 24P4C12 (either by removing the underlying glycosylation site or by deleting Sthe glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 24P4C12. In addition, the phrase includes qualitative changes in the glycosylation of the native 00 proteins, involving a change in the nature and proportions of the various carbohydrate moleties present C The term "analog' refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule a 24P4C12-related protein). For example, an analog of a 24P4C12 protein can be spedfically bound by an antibody or T cell that specifically binds to 24P4C12.

00 The term 'antibody' Is used In the broadest sense. Therefore, an "antibody" can be naturally occurning or man-made such as monodonal antibodies produced by conventional hybridoma technology. Anti-24P4C12 antibodies comprise monodonal and polydonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An "antibody fragment is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, the antigen-binding region. In one embodiment it specifically covers single anti-24P4C12 antibodies and dones thereof (including agonist, antagonist and neutralizing antibodies) and ant-24P4C12 antibody compositions with polyepitopic specificity.

The term "codon optimized sequences' refers to nudeotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exonintron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences.' A 'combinatorial library' is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical 'building blocks' such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide mutein) library, is formed by combining a set of chemical building blocks called amino adds in every possible way for a given compound length the number of amino adds in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known to those of skill in the art Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, U.S. Patent No. 5,010,175, Furka, Pept Prot Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random blo- oligomers (PCT Publication WO 92100091), benzodiazepines (U.S.

Pat No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat Acad. Sd.

-USA 90.6909-6913 (1993)), vinylogous polypeptides (Haghara et al., J. Amer. Chem. Soc. 114.6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et aL, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem.

59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nudeic add libraries (see, Strategene, Corp.), peptide nudeic add libraries (see, U.S. Patent 5,539,083), antibody libraries (see, Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, Liang et al., Science 00 0 274:1520-1522 (1996), and U.S.-Patent No. 5,593,853), and small organic molecule libraries (see, benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S.

Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No.

S5,506, 337; benzodiazepines, U.S. Patent No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, 357 NIPS, 390 NIPS, 00 Advanced Chem Tech, Louisville KY; Symphony, Ranin, Wobum, MA; 433A, Applied Biosystems, Foster City, CA; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by 00 Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark SCorporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist Any of the above devices are suitable for use with the present invention. The nature and 0 implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art In addition, numerous combinatorial libraries are themselves commercialy available (see, Se.g., ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosdences, Columbia, MD; etc.).

The term 'cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells andlor causes destruction of cells. The term is intended to indude radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymaticaly active toxins of bacterial, fungal, plant or animal origin, Including fragments andlor variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinolds, yttrium, bismuth, ricin, ridn A-chain, combrestatin, duocarmydns, dolostatins, doxorubidn, daunorubicin, taxol, dsplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchidne, dihydroxy anthracin dlone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeocin A chain, alpha-sardn, gelonin, mitogellin, retstrictocin, phenomycn, enomycin, curicin, crotin, calicheamicn, Sapaonaria officinalis Inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At 2 131, 1125, YN, Re 1 8 SmS 3 BP12o 21, P32 and radioactive isotopes of Lu induding Lu'u. Antibodies may also be conjugated to an anticancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The "gene product" is sometimes referred to herein as a protein or mRNA. For example, a "gene product of the Invention' is sometimes referred to herein as a "cancer amino add sequence', 'cancer protein', "protein of a cancer listed in Table a "cancer mRNA", 'mRNA of a cancer listed in Table etc. In one embodiment, the cancer protein Is encoded by a nucleic add of Figure 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of Figure 2. In one embodiment, a cancer amino add sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic add of Figure 2. In another embodiment, the sequences are sequence variants as further described herein.

"High throughput screening' assays for the presence, absence, quantification, or other properties of particular nucleic adds or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, U.S. Patent No. 5,559,410 disdoses high throughput screening methods for proteins; U.S Patent No. 5,585,639 discloses high throughput screening methods for nudeic add binding in arrays); while U.S. Patent Nos. 5,576,220 and 5,541,061 disdose high throughput methods of screening for ligandlantibody binding.

In addition, high throughput screening systems are commercially available (see, Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fulerton, CA; Precision Systems, Inc., Natick, MA; etc.). These systems typically automate entire procedures, induding all sample 00 Sand reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, Se.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene 00 transcription, ligand binding, and the like.

The term thomolog" refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

00 "Human Leukocyte Antigen' or 'HLA' is a human class I or dass II Major Histocompatibility Complex (MHC) C protein (see, Stites, et al., IMMUNOLOGY, 8" M ED., Lange Publishing, Los Altos, CA (1994).

I The terms 'hybridize', 'hybridizing', "hybridizes" and the like, used in the context of polynudeotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization In 50% formamide/6XSSCI.1'% SDS1100 00 pg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1% SDS are above 55 degrees C.

C The phrases "isolated' or 'biologically pure' refer to material which is substantially or essentially free from components which normally accompany the material as It is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment For example, a polynucleotide is said to be isolated" when it is substantially separated from contaminant polynudeotides that correspond or are complementary to genes other than the 24P4C12 genes or that encode polypeptides other than 24P4C12 gene product or fragments thereof. A skilled artisan can readiy employ nucleic acid isolation procedures to obtain an isolated 24P4C12 polynudeotide. A protein is said to be Isolated,' for example, when physical, mechanical or chemical methods are employed to remove the 24P4C12 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 24P4C12 protein. Alternatively, an Isolated protein can be prepared by chemical means.

The tennmmammal" refers to any organism dassifled as a mammal, incding mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal Is a mouse. In another embodiment of the invention, the mammal is a human.

The terms "metastatic prostate cancer and "metastatic disease' mean prostate cancers that have spread to .regional lymph nodes or to distant sites, and are meant to Include stage D disease under the AUA system and stage TxNxM+ under the TNM system. As is the case with'locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment Initiation.

Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis Include lymph nodes, lung, liver and brain. Metastatic 'prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionudide scans, skeletal radiography, and/or bone lesion biopsy.

The term "modulator' or 'test compound' or 'drug candidate' or grammatical equivalents as used herein descibe any molecule, protein, oligopeptide, small organic molecule, polysaccharide, polynudeotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, a nucleic add or protein sequences, or effects of cancer sequences signaing, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By neutralize' is meant that an activity of a protein 00 Is Inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect Sof a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nudelc acds or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another Sembodiment, a modulator induced a cancer phenotype. Generally, a pluraity of assay mixtures is run in paralel with 0 different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, at zero concentration or below the level of detection.

Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are 00 organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.

Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen C bonding, and typically indude at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional 00 chemical groups. The candidate agents often comprise cyclical carbon or heterocydic strucures and/or aromatic or 0polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecutes C such as peptides, saccharides, fatty adds, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One dass of modulators are peptides, for example of from about five to about amino acids, with from about five to about 20 amino adds being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, Includes a non-transmembrane region, andlor, has an Nterminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free add and the N-terminus is a free amine to aid in coupling, to cystelne. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA The peptides of the Invention, of preferred lengths, can be linked to each other or to other amino adds to create a longer peptide/protein.

The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or 'based' random peptides. In a preferred embodiment, peptidelprotein-based modulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic adds. Nudeic acd modulating agents can be naturally occurring nucleic acids, random nucleic adds, or 'biased" random nudeic adds. For example, digests of prokaryotic or eukaryotic genomes .can be used in an approach analogous to that outlined above for proteins.

The term "monodonal anttiody refers to an antibody obtained from a population of substantially homogeneous antibodies, the antibodies comprising the population are identical except for possible naturally occurring mutations that are present In minor amounts.

A 'moti', as in biological motif of a 24P4C12-related protein, refers to any pattern of amino acds forming part of the primary sequence of a protein, that is assodated with a particular function protein-protein Interaction, protein-DNA interaction, etc) or modification that is phosphorylated, glycosylated or amidated), or localization secretory sequence, nudear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humoraly or cellulary. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, 'motif refers to the pattem of residues In a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a dass I HLA motif and from about 6 to about 25 amino adds for a class II HLA motif, which Is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA alele and differ in the pattern of the primary and secondary anchor residues.

00 SA "pharmaceutical excipienr comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering 1 agents, tonicity adjusting agents, wetting agents, preservative, and the like.

"Pharmaceutically acceptable' refers to a non-toxic, inert, and/or composition that is physiologically compatible with Shumans or other mammals.

00 The term 'polynudeotide' means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either 0ribonuceotides or deoxynudeotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA andlor RNA In the art, this term if often used interchangeably with oligonudeotide'. A 00 polynudeotide can comprise a nucleotide sequence disclosed herein wherein thymidine as shown for example In Figure N 2, can also be uracil this definition pertains to the differences between the chemical structures of DNA and RNA, in O particular the observation that one of the four major bases in RNA is uracil instead of thymidine SThe term'polypeptide' means a polymer of at least about 4, 5, 6, 7, or 8 amino adds. Throughout the 0 specification, standard three letter or single letter designations for amino adds are used. In the art, this term is often used 0 interchangeably with 'peptide" or "protein'.

SAn HLA "primary anchor residue' is an amino add at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an Immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA dass I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA dass II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

*Radioisotopes" include, but are not limited to the following (non-limiting exemplary uses are also set forth): Examples of Medical Isotopes: Isotope Description of use Actinium-225 (AC-225) See Thorium-229 (Th.229) Actinium-227 (AC-227) Parent of Radium-223 (Ra-223) which Is an alpha emitter used to treat metastases in the skeleton resulting from cancer breast and prostate cancers), and cancer radlolmmunotherapy Bismuth-212 (Bi-212) See Thorium-228 (Th-228) Bismuth-213 (Bi-213) See Thorum-229 (Th-229) Cadmium-109 (Cd-109) Cancer detection 00 (CoO) Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical supplies Copper-64 (Cu-64) 00 A positron emitter used for cancer therapy and SPECT imaging Copper-67 (Cu-67) 00 Betalgamma emitter used in cancer radicimmunotherapy and diagnostic studies breast and colon 00 cancers, and lyphoma) Dysprosium-166 (Dy-I66) Cancer radioimmunotherapy 00 Erbium-169 (Er-169) Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and toes Europlum--152 (Eu-I 52) Radiation source fo food Irradiation and for sterilization of medical supplies Europium-154 (Eu-154) Radiation source for food irradiation and for sterilization of medical supplies Gadolinfrr-153 (Gd-153) Osteoporosis detection and nuclear medical quality assurance devices Gold-iga (Au-198) Implant and intracavity therapy of ovarian, prostate, and brain cancers HomIrnum-166 (Ho-166) Multiple myelomra treatmnt in targeted skeletal therapy, cancer radioimmunotiierapy, bone marrow ablation, and rheumatoid arthritis treatmnent lodine-125 (1-125) Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatmyent, radiolabeling, tumor imaging, mapping of receptors In the brain, intersital radiation therapy, brechytherpy for treatment of prostate cancer, determination of glonierular filtration. rate (GFR), determinati of plasma volume, detecion of deep vein thrombosis of the legs todine-131 (1-131) Thyroid function evaluation, thyroid disease detection, treatmnent of thyroid cancer as well as other nonmalignant thyroid diseases Graves disease, goiters, and hyperthyroldism), treatment of leukemia, lymphoma. and other forms of cancer breast cancer) using rediolmmunotherapy Iridium-192 (lr-192) Brachytherapy, brain and spinal cord tumor treatmrent, treatment of blocked arteries arteriosolerosis and restenoisis), and implants for breast and prostate tumors Lutelium-177 (Lu-177) 00 Cancer radilmmunotherapy and- treatment of bloced arteries arteriosclerosis and restenosis) CI Motybenum-99 (Mo-99) Parent of Technetium-99m (Tc.99m) which is used for imaging the brain, liver, lungs, heart, and other organs.

Currently, To-99mn Is the most widely used radioisotope used for diagnostic imaging of various cancers and 00 diseases Involving the brain, heart, liver, lungs; also used In detection oftdeep vetn thrombosis of the Lgs Osmium-I 94 (Os-l94) Cancer radiommunotherapy 00 N- Palladlum-103 (Pd-103) Prostate cancer treatment Platinum-195m 00 (Pt-i Studies on blodistrIbution and metabolism of cisptatin, a chtemotherapeutic drug Phosp*iorus-32 (P-32) Polycyliiemia rubra vera (blood cell disease) and leukemia treatment bone cancer diagnosis/treatment colon, pancreatic, and liver cancer treatment; radiolabeling nuclekc adds for In vitro research, diagnosis of superficial tumors, treatment of blocked arteries arteriosclerosis and restenosis), and intrecavity therapy Phosphorus-33 (P.33) Leukemia treatment bone disease diagnosisltreatment, radiolabeling, and treatment of bloced arteries arteriosclerosis and restenosis) Radium-223 (Ra-223) See Actiniumn-227 (Ac-227) Rlienlumn-186 (Re-186) Bone cancer pain relief, rheumatoid arthits treatment and diagnosils and treatment of lymphoma and bone, breast, colon, and liyer cancer using radiolrmmunotherapy Rhtenium-188 -(Re-188) Cancer diagnosis and treatment using radoimmunotherapy. bone cancer pain relief, treatment of rheumatoid arthritis, and treatment of prostate cancer (Rh-105) Cancer radioimmunotherapy Samarium-145 (Sm-145) Ocular cane treatment Samalum-153 (Sm-153) Cancer radioklmwnotherepy and bone cancer pain relief Scandium-47 (Sc-47) Cancer radiolnunolterapy and bone cancer pain relief 00 Radiotraoer used in brain studies. imaging of adrenal cortex by gamma-sainfigraphy, lateral locations ot steroid secreting tumors, pancreatic scanning, detection of hyperactive paratiyrold glands, measure rate of (71 bile adid loss from the endogenous pool Bone cancer detection and brain scans 00 StrontiumM8 (Sr-89) Bone cancer pain relief, multiple myeloma treatment, and osteobilastic therapy 00 N7K Ted'rietium-99m IND (Tc-99m) See Molytbdenum-99 (Mo-99) (KI Thorium-228 00 (Th-228) Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimnmunolherapy (7KI Thodum-229 (Th-229) Parent of Actlnium-225 (Ac-225) and grandparent of Bsmuth-213 (Bi.213) which are alpha emnitters used in cancer radioimmunotherapy Thullum-i (Tm-170) Gamma source for blood irradiators, energy source for implanted medical devices Tin-hi1m (Sn-i 17m) Cancer immunotherapy and bone cancer pain relief Tungsten-188 (W-188) Parent for Rhenium- 188 (Re-I 88) which is used for cancer diagnoslics/trealment, bone cancer pain relief, rheumatoid arthritis treatment and treatment of blockced artenies arteriosclerosis and restenosis) Xenon-127 (Xe-127) Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, and cerebral blood flow studies Ytterbium-175 (Yb-175) Cancer radlobrimunodweapy Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment Yttriurn-911 (V.91) A gamma-emitting label for Yttflumn-90 (Y-90) which Is used for cance radiolmmunoflerpy lynmphoma, breast colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable fiver cancers) 00 O By 'randomized' or grammatical equivalents as herein applied to nudelc acids and proteins is meant that each nucleic acid and peptide consists of essentially random nudeotides and amino acids, respectively. These random peptides S(or nucleic acids, discussed herein) can incorporate any nuceotide or amino add at any position. The synthetic process can be designed to generate randomized proteins or nucleic adds, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bloactive proteinaceous 00 agents.

In one embodiment, a library is "fully randomized," with no sequence preferences or constants at any position. In another embodiment, the library is a "biased random' library. That is, some positions within the sequence either are held 00 N constant, or are selected from a limited number of possibilities. For example, the nudeotides or amino acid residues are O randomized within a defined dass, of hydrophobic amino adds, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic add binding domains, the creation of cysteines, for cross4inking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

00 A 'reambinanr DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manpulation in vitD.

Non-limiting examples of small molecules Include compounds that bind or interact with 24P4C12, ligands induding hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 24P4C12 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 24P4C12 protein; are not found in naturally occurring metabolic pathways; andlor are more soluble in aqueous than non-aqueous solutions "Stringency of hybridization reactions is readily deterninable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel e al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions', as defined herein, are identified by, but not limited to, those that employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citratel.1 sodium dodecyl sulfate at 50'C; employ during hybridization a denaturing agent, such as formamide, for example, 50% fonnamide with 0.1% bovine serum albumin/0.1% Flcoll/.1% polyvinylpyrrolidonel50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 or employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium dtrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardts solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42 CC, with washes at 42oC in 0.2 x SSC (sodium chloridelsodium. citrate) and 50% formamide at 55 oC, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 OC. "Moderately stringent conditions" are described by, but not limited to, those in Sambrook e al., Molecular Cloning: A Laboratory Manual, New Yorc Cdd Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions temperature, Ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight Incubation at 376C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium dtrate), 50 mM sodium phosphate (pH 5 x 00 Denhardrs solution, 10% dextran sulfate, and 20 mng/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37.50C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA'supermotif' is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

overall phenotypic frequencies of HLAsupertypes in different ethnic populations are set forth In Table IV The non- 0 A2 A'02DI, A-0202, A-0203, A-0204, A- 0205, A10206, A-6802, A-6901. A-0207 00 A3, A3, All. A31,A-3301, A-6801, A-0301, A-1101. A-3101 N=K BY. B7, 6*3501-03, 8*51, B*5301, B'5401, 8*5501, B*5502. B'560i, B-670i, B-1801, B-0702, B-5101, B-5602 INO B44; B-3701, B-4402, B*403, B60 (B-4001), B81 (8-4006) Al. A-01 02, A-2604, A-3601. A-4301. A-8001 00 A4 A-24, A-30, A-2403, A-2404. A'3002, A-3003 827: B81401-O2Z B-1503, B81509,8B1510, B-1518, W'3801-02, 0-3901,B-3902, B-3903-04, W~4801-02, B-7301, B*2701-08 B58. 8-1516. B-1517, B-5701, B-570, 858 §12-_B-4601, 852,13-15011 (1362), B-1502 (B75), B-1513 (8377) Calculated population coverage afforded by different HLA-supertype combinations are set forth in Tab~le IV As used herein 'to trear or 'therapeuic* end grammatically related terms, refer to any Improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality-, full eradication of disease Is not required.

A Iffrsgenic animal' a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal. an embryonic stage. A 'ransgene' is a DNA that is integrated into t genome of a cell from which a transgenic animal develops.

As used herein, an lILA or cellular immune response 'vaccine' Is a composition that contains or encodes one or more peptides of the invention. There are numerous embodinerits of such vaccines, such as a cocklail of one or more individual peptides; one or more peptides of the invention comprised by a potyepitoplc peptide; or nucleic acids that encode such Individual peptides or polypeptides, a minigene fht encodes a polyepltopic peplide. The 'one or more peptides' can Include any whole unit integer from 1-150 or more, at least 2, 3,4, 5.6,7, 8,9,10,11, 12,13, 14. 15, 16,17. 18, 19, 20, 21,22, 23, 24, 25, 28,27,28,29, 30,31,.32,33, 34,35,36, 37,38,39,40,41,42,43,44,45,46,47,48,49, 50, 65, 70,75. 80, 85, 90, 95,100, 105, 110,115,120, 125, 130,135,140,145, or 150 or more peptides of the Invention.

The peptides or polypeptides can optionally be modified, such as by lipidalion, adton of targeting or other sequences. HILA class I peptldes of the Invention can be adrrexed with, or inked to, HLA class 11 peptides, to facilitate acivation of both cytotDxic T lymphocytes and helper T lymphocytes. HIA vaccines can also comprie peptide-pulsed antigen presenting cells, dendritic; calls.

The trmn Nariant' refers to a molecule that exihlbts a variation from a descibed type or norm, such as a protein that has one or more different amino acdd residues In the corresponding position(s) cfia spedifically descibed protein the 24134C1 2 protein shown in Figure 2 or Fqgre 3. An analog Is an example of a variant protein. Splice isoforms and single nudeotides polymnorphisms (SNPs) are further examples of variants.

The 124P4C112-elated proteins' of the invention Incude those speolfically idengWie herein, as well as altetc variants, conservative substitution variants, analogs and homnologs that can be isolated/generaWe and charaicterized withot undue ieqpermentdri following lhe methods outlined herein or readily avallable In the art Fusion proteins that corrnbie parts of different 24134012 proteins or fragments thereof, as well as fusion proteins of a 24P4C1 2 protein and a heterologous polypeplide 00 are also incuded. Such 24P4C12 proteins are collectvely referred to as the 24P4C12-related proteins, the proteins of the (N2~ invention,or 24PC12. The termt '244C127related protein* refers bo a potypeptide fragment or a 24P4Cl2 protein sequence of 4, 5.6,7,8B.9,10,11, 12,13,14,15,16.17,18,19.20. 21, 2Z 23,24, 25, or more than 25 aino acids; or, at least 30, 35,40. 55, 60, 65, 70,80.,85,90, 95,100, 105, 110,115, 120,125,130,135,140,145, 150, 155, 160,165,170, 175, 180,185, 00 190, 195, 200, 225, 250, 275, 300, 325, 350. 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, or 664 or more 0 ~amino acids.

11.) 24P4C12 Polynucleoltides 00 One aspect of the invention provides palynucleotides corresponding or complementary to all or part of a 24P4C12 N ~gene, mRNA, andlor codng sequence, preferably in islated form, including polynudleotides encoding a 24P34C1 2-related protein and fragments thereof, DNA, RNA, DNAIRNA hybrid, and related molecules, polyrucleoddes or oigonudleolides complementary to a 24P4C1 2 gene or mRNA sequence or a part thereof, and polynudeotides or oligonucleotides that 00 hybridize to a 24PC12 gene, mRNA, orto a 24P4C12 encoding polynudleotide (collectively, '24P4C12 polynucleotides"). In all instances when referred to In this section, T can also be U in Figure 2.

CN2IEmbodiments of a 24P4C12 polynucleotide Include: a 24P4C12 polynucleotide having the sequence shown in Figure 2, the nudleotide sequence of 24P4C1 2 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a potynucleotide having the sequence as shown In Figure 2; or, at least 10 contiguous nucleotides of a polynudeotide having the sequence as shown in Figure 2 where Tis U. For example, embodimeants of 24P4C12 nudleotides comprise, without limitation: a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in Figure 2, wherein T can also be U; (11) a polynudeotide comprising, consisting essentially of. or consisting of the sequence as shown in Figure 2A, from nucleotide residue number 6 through nucleotide residue number 2138, indluding the stop codon, wherein T can also be U; (Ill) a polynudleotide comprising, consisting essentially of, or consisting of the sequence as shown In Figure 2B, from nucleotide residue number 6 through nudeotide residue number 2138, indluding the stop codon, wherein T can also be U; (IV) a polynudeofide comprising, consisting essentially, of, or consisting of the sequenice as shown In Figure 2C, from nucleolide residue number 6 through nucleotide residue number 2138, Indluding the a stop codon, wherein T can also be U; M a polyniudeotde comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D), from nudeotide residue number 6 through nucleotide residue number 2138, including the stop codon, wherein T can also be U; (VI) a polynudeotie cornprising, consisting essentially of, or consisting of the sequence as shown in Figure 2E, from nudeotide reidue number 6 through nudeotide residue number 2138, Including the stop codon, wherein T can also be U; 00 (VII) a polynucleofide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2F, from nucleotide residue number 6 through nucleolide residue number 2138, indluding the stop codon, wherein T can also be U); (VIII) a polynucleolide compising, consisting essentially of, or consisting of the sequence as shown in Figure 00 2G. from nucleotide residue number 6 through nudleotide residue number 1802, indluding the stop codon, wherein T can also be U; (IX) a polynudleotide compuising, onsisting essentially of, or consisting of the sequence as shown in Figure 00 2H1, from nucleotide residue number 6 through nudleotide residue number 2174, Including the stop codon, wherein IND T can also be U; a polynucleotide comprising, consisting essentialy of, or consisting of the sequence as shown in Figure 00 21, from nudleolide residue number 6 through nucleolide residue number 2144, including the stop codon, wherein T can also be U; (AI) a polynudleotide that encodes a 24P4CI 2-related protein that is at least 90, 91. 92, 93, 94, 95, 96. 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in Figure 2A-1; (XII) a polynucleotide that encodes a 24P4C12-related protein that is at least 90, 91, 92,93, 94, 95,96, 97, 98, 99 or 100% Identical to an entire amino acid sequence shown in Figure 2A-1; (XIlI) a polynudleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLI (AIV) a polynudleotide that encodes a peptide region of at least 5, 6, 7, 8,9,10,11. 12,13.14,15, 16.17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27.,28, 29,30,31.,32, 33, 34, 35 amnino acids of apepide of Figure 3A-D in any whole number increment up to 710 thatindludes at least 1, 2,3,4, 5, 6,7, 8,9,10, 11, 12,13,14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35 amino acid position(s) having a value greater than in the Hydrophilicity profile of Figure (XV) a polynudeooide that encodes a peptide reion of at least 5, 6,7, 8, 9,10,11, 12,13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A-O in any whole number Increment up to 710 that includes 1,2Z,3, 4, 5, 6, 7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity, profile of Figure 6; (XM) a polynudleotide that encodes a peptide region of at least 6, 7,8.9, 10, 11, 12,13, 141, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 aminto acids of a peptide of Figure WD-f in any whole number Increment up to 710 that Includes 1, 2, 3.4,5,6,7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31,32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 In the Percent Accessible Residues proffle of Figure 7; (XVII) a polynudeotide that encodes a peptide region of at least 5, 6,7, 8,9, 10,11, 12,13,14, 15 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of apeptide of Figure 3A-D in any whole number Increment up to 710 that includes 1,2Z,3,4,5,.6,7, 8.,9,10, 11,.12, 13, 14, 15,16, 17, 18.,19, 00 21, 22, 23, 24, 25. 26, 27, 28. 29, 30, 31, 32, 33.,34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XVIII1) a polyn uceotie that encodes a peptide region of at le ast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27.,28, 29, 30, 31, 32, 33.34, 35 amino acids of a pepfide ofFigure 3A-DIn any 00 ~~~~whole number increment up t70tat ncudes, 2, 3,4, 5, 6,7, 8, 9,10,11, 12, 13,14, 15,16,17,18,19, 21, 2Z 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33,34, 35 amino acid position(s) having a value greater than 0.5 In the Beta-turn profile of Figure 9; 00 N a polynudleolide that encodes a peptide region of at least 5, 10, 11, 12,13,14,15,16, 17, 18, 0 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 amino acids cf a peptide ofFigure 3ElIn anywhole 0 ~~~~number incremient up to 598 thatlIncludes 1, 2, 3,4, 5,6,7, 8, 9,10,11, 12, 13, 14,15,16.17, 18,19, 20,21, 22, 00 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35 amino acid position(s) having a value greater than 0.5 In the 0 HNydrophilicity, profile of Figure 0N (XX) a potyntudeotide that encodes a peptida region of at least 5, 6, 7, 8,9, 10, 11, 12, 13.,14, 15, %a 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35 amino acids of apeptide of Figure 3E in any whole number Increment up to 598 that Includes 1, 2,3,4, 5, 6.7, 89, 1,11, 1 Z13, 14, 15 16, 17, 18, 19. 20.21.,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino add position(s) having a value less than 0.5 in the Hydropathicity, profile of Figure 6; (XXI) a polynucleotide that encodes a peptide region of at east5,6 67,8. 9, 10, 11, 12,13.14, 15, 16$,17.18.

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33,34, 35 amino adids of a peptide of Figure 3E in any whole number Increment up to 598 that includes 1, 2, 3,4, 5, 6, 7, 6,9,10, 11, 12,13, 14, 15,16,17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32Z 33,34, 35 amino acid position(s) having a value greater than 0.5 In the Percent Accessible Residues profile of Figure 7; (XXII) a polynudotde that encodes a peptide region of at least 5, 6, 10,11, 12,13,14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino adds of a peptide of Figure 3E In any whole number Incement up to 598 that includes 1,2Z,3,4, 5,6,7,8. 9, 10, 11, 12, 13, 14, 15,16, 17, 18,19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; QO(I) a polynudeofide that encodes a peptide region of at least 5,6, 7, 8,9,10,11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.,25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 amino acids of a peptide of Figure 3E In any whole number incremrnent up to 598 tatindludes 1, 2, 3,4,5,6, 7, 8, 910, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acdd position(s) having a value greater than 0.5 In the Beta turn profile of Figure 9 (XXIV) a polynudleotide that encodes a peptide region of at least 5, 6, 7,8,9,10, 11, 12,13,14,15,1617, 18, 19,20,21, 22, 23,24,25.26, 27.28,29.30,31.32, 33,34.35 amnino adids of apeptide of Figure 3F In any whole -number Increment up to 722 that includes 1, 2,3.4.5,6.7,8,9,10,1112,13.14,15.16, 17.18,19.2, 21, 22.

23,24,25, 26, 27, 28, 29, 30, 31, 32. 33,34, 35 amino add position(s) having a value greater Jhan 0.5 In the Hydropilicity profile of Figure 00 (XXV) a polynucleotide that encodesa Peptde region ofat least5, 6, 7,8, 9, 10,11, 12,13, 14,15,16.17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35 amino adds of a peptide of Figure 3F in any whole numberInrement up to722 that inludes1, 2, 3,4, 5,6, 7,8, 9,10.11, 12.13, 14, 15.16, 17. 18, 19, 20, 21, 22, 23, 24, 25, 26. 27, 28, 29, 30, 31, 32, 33, 34. 35 amino acid position(s) having a value less than 0.5 in the 00 Hydropathlcity profile of Figure 6; (XXVI) a polynucleofide that encodes a peptide region of at least 5, 6,7, 8, 9,10,11, 12,13,14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino adids of a peptide of Figure 3F in any whole 00 number increment up to 722 thatIndludes 1,2Z,3,4, 5,6,7, 8,9,1,11, 12,13,14,15,16,17,18,19, 20, 21,22, IND ~23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; 00 (XVII) a polynuceoide that encodes apeptide regin ofat least 5,6,7,8, 9, 10,11,12,13,14,15,16,17,18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34t,35 amino acids of a peptide of Figure 3F in any whole C]numberIncrement up to 722 that Includes 1,2Z 3, 4, 5, 6,7,8, 9,10,11, 12,13,14, 15,16, 17,18, 19,20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibdity profil of Figure 8; (XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7,8, 9,10,11, 12, 13,14,15,16,17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3Z, 33, 34. 35 amino acids of a peptide of Figure 3F In any whole number increment up to 722 thatlndcudes 1, 2, 3,4,5, 6,7, 8, 9,10,11, 12,13, 14,15 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Betaturn profio of Figure 9 (XXIX) a polynudeofide that encodes a peptide region of at least 5, 6,7,8,9, 10 11,12,13,14,15,16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 amino acids of a peptide of Figure 3G in any whole number increment up to 712 that includes 1, 2, 3,4,5,6,7,8, 9,10,11, 12,13, 14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amidno acid position(s) having a value greater than 0.5 in the HIydrophificity profile of Figure (XXX) a polynudleolide that encodes a peptide region of at least 5, 6, 7, 8, 910, 11, 1Z,13,14,15,16,17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 3Z 33, 34,35 amino acids of a peptide of Figure 3G in any whole number increment up to 712thatlnncudes 1, 2, 3,4,5,6,7, 8, 9,10,1.1, 12,13,14,15,16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6; (MI) a polynudleotide that encodes a peptide region of at least 5,6, 7,8, 9, 10,11,12,13,14,15,16,17,18, 19.,20, 21, 22, 23,24, 25, 26, 27,28,29,30, 31, 32,33, 34,35 amino adids of a peptide of Figure 3G in any whole numiber Increment up to 712 thatlIndudes 1, 2, 3,4,5,6, 7,8,9,1011 1213, 14,15,16,17,18,19, 20, 21, 22 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33.,34, 35 amnino acid position(s) having a value greater than 0.5 In the Percent Accessible Residues promol of Figure 7; (XXXII) a polynudeotide that encodes a peptido region of at least 5,6, 7, 8,9, 10,11,12,13,.14.15.16,17, 18, 19, 20, 21, 22,23, 24, 25, 26,27, 28, 29, 30, 31, 32,33, 34, 35 amino acids of a peptide of Figure 313 in any whole 00 ~number increment up to 712 that includes 1, 2,3.4,56,6, 7,89, 10, 11, 12,13,14.15, 16, 17.18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Aveage Flexibility profile of Figure 8; (XXXII a polynucleotide that encodes a peptie region of at least 5, 6,7, 8,9, 10, 11, 12,13, 14,15.,16, 17, 18, 00 19,.20, 21, 22,23, 24, 25, 26,.27,28, 29, 30,31,32Z 33 34, 35 amino acids of a peptide of Figure 3G in any whole number increment up to 712 that indudes 1,2, 3,4, 5,6.789, 10, 11 12.13,14,1516,17,1819,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta- 00 turn profile of Figure 9 ID(XXXIV) a polynudleoticie that is fully complementary to a polynucleotide of any one of CK1 (XXXV) a peptide that is encoded by any of to (XXXIII); and 00 (XXXVI) a Composition CDp1Jsing a polyniuceotide of any of (IHXXIV) or peptide of ()WV together with a pharmaceutical excipient andlor in a human unit dose form.

(XX(XVII) a method of using a polyniudeolide of any (IH XXXIV) or peptide of (X"XV or a composition of (XXXVI) in a method to modulate a cell expressing 24P4C12, ()X=III) a method of using a polynudeotide of any (IH XXXIV) or peplide of (XXXV) or a composition of (XXXVI) in a method to diagnose, prophytax, prognose, or treat an Individual who bears a CCll expressing NNWC1 (XX)g a method of using a polyniuceotide of any (1)4 XxIV) or peptide of (XXX(V) or a composition of (XXXVI) in a method to diagnose, prophylax, prognose, or treat an Individual who bears a cell expressing 24P4C12, said eel from a cancer of a tissue listed in Table I; (XL) a method of using a polynudleolide of any (I)JQOOV) or peptide of (XXXV) or a composition of (XXXVI) I a method to diagnose, prophylax, prognose, or treat a a cancer; (XLI) a method of using a polynudeotde of any (IX)OV) or peptide of (XXV or a composition of (XXXVI) in a method to diagnose, prophylax, prognose, or trat a a cancer of a tsue listed in Table 1; and, (XLII) a method of using a polynucleotie of any or peptide of (XXi(V) or a composition of (XXXVI) in a method to identify or characterize a modulator of a cell expressing 24P4C1 2.

As used herein, a range Is understood to disdose specifically all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 24P4C12 polynucteotides that encode specific portions of 24P4C1 2 mRNA sequences (aid those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: 4,5,6,7,8,910, 11, 12, 13,14,15, 16,17,18,19, 20,21, 22,23,24,25, 30, 35,40,45,50, 55,60,65,70, 80, 85.,90,95, 100, 105,110, 115, 120,125,130, 135,.140,145,150,155,160, 165,170. 175, '180, 185, 190,195,200, 225, 250, 275, 300,325, 350,375,400,425,450, 475,500,.525, 550, 575,600,825, 850,675, 700, 710 or more contiguous amino acids of 24P4C12 variant1; the maximal lengths relevant for other variants are: variant 3,710 amino acids; variant 710 amino adds, variant 8, 710 amino adids, variant 7, 598 amino adids, variant 8, 722 amino acids, and variant 9, 712 amino ads.

00 O For example, representative embodiments of the invention disclosed herein Include: polynudeotides and their encoded peptides themselves encoding about amino acid 1 to about amino add 10 of the 24P4C12 protein shown in Figure 0 2 or Figure 3, polynudeotides encoding about amino acid 10 to about amino add 20 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 20 to about amino acid 30 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotldes encoding about amino add 30 to about amino acid 40 of the 24P4C1 2 protein shown in Figure 02 or Figure 3, polynueotides encoding about amino acd 40 to about amino acid 50 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynuceotides encoding about amino add 50 to about amino add 60 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 60 to about amino acid 70 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino add 60 to about amino add 70 of the 24P4C12 protein shown in Figure 00 N 2 or Figure 3, polynudeotides encoding about amino acid 70 to about amino acid 80 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 80 to about amino acid 90 of the 24P4C12 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 90 to about amino add 100 of the 24P4C12 protein shown in 00 Figure 2 or Figure 3, in increments of about 10 amino adds, ending at the carboxyl terminal amino acid set forth In Figure 2 00 or Figure 3. Accordingly, polynuceotides encoding portions of the amino acid sequence (of about 10 amino adds), of amino aciadds, 100 through the carboxyl terminal amino acid of the 24P4C12 protein are embodiments of the invention. Wherein it is understood that each particular amino add position discloses that position plus or minus five amino acid residues.

Polynudeotides encoding relatively long portions of a 24P4C12 protein are also within the scope of the Invention.

For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or or 50 etc.) of the 24P4C12 protein "or variant' shown In Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynudeotide fragments can include any portion of the 24P4C12 sequence as shown in Figure 2.

Additional illustrative embodiments of the invention disclosed herein include 24P4C12 polynudeotide fragments encoding one or more of the biological motifs contained within a 24P4C12 protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 24P4C12 protein "or variant set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment typical polynudeotide fragments of the invention encode one or more of the regions of 24P4C12 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynudeotide fragments can encode one or more of the 24P4C12 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth in Tables VIIl-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide In an HLA Peptide Table, and the Search Peptides listed in Table LVII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant The position of each Search Peptide relative to its respective parent molecule is listed In Table VII. Accordingly, if a Search Peptide begins at position one must add the value "X minus 1" to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides In their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 1, 149 to each HLA peptide amino add position to calculate the position of that amino add in the parent molecule.

Uses of 24P4C12 Polvnucleotides IIA.1.) Monitoring of Genetic Abnormalities The polynuceotides of the preceding paragraphs have a number of different specific uses. The human 24P4C12 gene maps to the chromosomal location set forth In the Example entitled "Chromosomal Mapping of 24P4C12." For example, because the 24P4C12 gene maps to this chromosome, polynudeotides that encode different regions of the 24P4C12 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being assocated with various cancers. In certain genes, a variety of chromosomal abnormalities including 00 0 rearrangements have been identified as frequent cytogenetic abnormalities In a number of different cancers (see e.g.

SKrajnovic et el., Mutat Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger eta/., P.NAS. 85(23): 9158-9162 (1988)). Thus, polynudeotides encoding specific regions of the 24P4C12 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 24P4C12 that may contribute to the malignant phenotype. In this context, these

O

polynudeotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans etal., Am. J. ObsteL Gynecol 171(4): 1055-1057 00 (1994)).

N Furthermore, as 24P4C12 was shown to be highly expressed in bladder and other cancers, 24P4C12 Spolynudeotides are used in methods assessing the status of 24P4C12 gene products in normal versus cancerous tissues.

Typically, polynudeotides that encode specific regions of the 24P4C12 proteins are used to assess the presence of Sperturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific 00 regions of the 24P4C12 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR Sassays as well as single-strand conformation polymorphism (SSCP) analysis (see, Marrogi et al., J. Cutan. Pathol.

26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

IIA.2.) Antisense Embodiments Other specifically contemplated nudeic add related embodiments of the Invention disclosed herein are genomic DNA, cONAs, ribozymes, and antisense molecules, as wel as nudeic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and indude molecules capable of Ihibiting the RNA or protein expression of 24P4C12. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nudeic add molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic add molecules using the 24P4C12 polynudeotides and polynudeotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonudeotides that bind to a target polynudeotide located within the cells. The term "antisense' refers to the fact that such oligonudeotides are complementary to their intracellular targets, 24P4C12. See for example, Jack Cohen, Oligodeoxynudeolides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 24P4C12 antisense oligonucleotides of the present invention indude derivatives such as S-oligonudeolides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. Soligos (nudeoside phosphorothioates) are isoelectronic analogs of an oligonudeotide (Ooligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding -oligos with 3H-1,2benzodlthlol-3-one- ,1-dioxide, which is a sulfur transfer reagent. See, lyer, R. P. el al., J. Org. Chem. 55:4693-4698 (1990); and lyer, R. P. et aL, J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 24P4C12 antisense oligonudeotides of the present invention include morpholino antisense oligonucleotides known in the art (see, Partridge et al., 1996, Antisense Nudeic Acid Drug Development 6: 169-175).

The 24P4C12 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 24P4C12 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 24P4C12 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 24P4C12 antisense oltgonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence 00 that hybridizes to 24P4C1 2 mRNA. Optionally, 24P4C1 2 antisanse oligonucleolide is a 30-mer oligonudleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 24P4C12. Alternatively, the antisense molecules are modified to employ nibozymes in the inhibition of 24P4C1 2 expression, see, L. A. Couture D. T. Stinchcomb; Trends Genet 12: 5 10-515 (1996).

00 ll.A.3.) Primers and Primer Pairs Further specific embodiments of these nucleotides of t invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or 00 specifically hybridize to nucleic acid molecules of the invention or to ay part thereof. Probes can be labeled with a NK detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a INO chemrlluminescent compound, metal chelator or enzyme. Such probes and primers are used. lo detect the presence of a 24P4C1 2 polynudeotie in a sample and as a means for detecting a cell expressing a 24P4C12 protein.

00Examples of such probes indlude polypeptides comprising all or part of the human 24P4C1 2 cDNA sequence shown In Figure 2. Examples of primer pairs capable of specifically amplifying 24P4C1 2 mRNAs are also descibed In the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 24P4C12 mRNA.

The 24P4C12 polynucleotldes of the Invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 24P4C12 gene(s), mRNA(s), or fragments thereof, as reagents for the diagnosis andfor prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 24P4C12 polypeptfides; as tools for modulating or inhibiting the expression of the 24P4C12 gene(s) and/or translation of the 24P4C1 2 transcript(s); and as therapeutic agents.

The present invention includes the use of any prolbe as described herein bo identify and isoate a 24P4C12 or 24P4C1 2 related nucleic acid sequence from a naturally occuring source, such as humans or other mammals, as well as the Isolated nucleic acid sequence per se, which would comprise all or most of the sequences found In the probe used.

II.A.4.) Isolation of 24P34C1 2-Encoding Nucleic Acid Molecules The 24P34C1 2 cONA sequences described herein enable the Isolation of other pol) lucleotides enooding 24P4C12 gene product(s), as well as the isolation of polynucleolides encoding 24P4C1 2 gene product homologs, alternatively spliced isoforms allelic variants, and mutant forms of a 24P4C12 gene product as wel as polynucleotides that encode analogs of 24P4C1 2-i'elated proteins. Various molecular cloning methods that can be employed to isolate full length cONAs encodng a 24P4C12 gene are well known (see, for example, Samnbrook, J. et aL, Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press.

New York 1989; Current Protocols in Molecular Eliology. Ausubel et at, Eds.. Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning system Lambda ZAP Express, Stratagene). Phage clones containing 24P4C1 2 gene cONAs can be Identified by probing with a labeled 24P4CI12 cONA or a fragment thereof. For example, in one embodiment, a 24P4C12 cDNA (eag., Fgure 2) or a portion thereof can be syintheszed andused as apRobe torerieve overapplng and full-ength cDN~scorresponding to a24P4C2 gene. A 24P4C12 gene itself can be Isolated by screening genornic DNA libraries, bacterial aftficial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the Ike, with 24P4C1 2 DNA probes or printers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems The Invention also provides recombinant DNA or RNA molecules containng a 24P4C1 2 polyriucleotide, a fragment analog or homologue thereof, Including but not limited to plnages, plasrnkls, phagerrids, cosnrids, YACs. BACs, as well as various vira and non-viral vectors well known 'in thne art and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods forgenerating suich molecules are well known (see, for example. Sambrookc et al., 1989, supra).

00 SThe invention further provides a host-vector system comprising a recombinant DNA molecule containing a 24P4C12 C polynudeotide, fragment analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of Ssuitable eukaryoic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various 00 prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transdudble prostate cancer cell lines, primary 0 cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins COS, CHO, 293, 293T cells). More particularly, a polynudeotide comprising the coding sequence of 24P4C12 or a fragment, analog 00 or homoog thereof can be used to generate 24P4C12 proteins or fragments thereof using any number of host-vector systems N routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 24P4C12 proteins or fragments thereof are available, Osee for example, Sambrook et el., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for 00 mammalian expression Include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 24P4C12 can be expressed in several C prostate cancer and non-prostate cell ines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 24P4C12 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 24P4C12 end 24P4C12 mutations or analogs.

Recombinant human 24P4C12 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 24P4C12-related nudeotide. For example. 2931 cells can be transfected with an expression plasmid encoding 24P4C12 or fragment, analog or homolog thereof, a 24P4C12-related protein is expressed in the 293T cells, and the recombinant 24P4C12 protein is isolated using standard purification methods affinity purification using anti-24P4C12 antibodies). In another embodiment, a 24P4C12 coding sequence is subdoned into the retroviral vector pSRuMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 24P4C12 expressing cell lines. Various other expression systems wel known in the art can also be employed. Expression constructs encoding a leader peptide Joined in frame to a 24P4C12 coding sequence can be used for the generation of a secreted form of recombinant 24P4C12 protein.

As discussed herein, redundancy In the genetic code permits variation in 24P4C12 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host For example, preferred analog codon sequences typically have rare codons codons having a usage frequency of less than about 20% hi known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizng codon usage tables available on the INTERNET such as at URL dna.affrc.go.jpl-nakamura/codon.html.

Additional sequence modifications are known to enhance protein expression in a cellular host. These indude elimination of sequences encoding spurious polyadenylation signals, exon/lntron splice site signals, transposon-like repeats, andlor other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host as calculated by reference to known genes expressed in the host cel.

Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications Include the addition of a translational Initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. CeO Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes Initiate translation exdusively at the 5' proximal AUG codon is abrogated only under rare conditions (see, Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

Il) 24P4Ci2.related Proteins 00 Another aspect of the present invention provides 24P4C12-related proteins. Specific embodiments of 24P4C12 proteins comprise a polypeptide having all or part of the amino add sequence of human 24P4C12 as shown in Figure 2 or Figure 3. Altematively, embodiments of 24P4C12 proteins comprise varant, homolog or analog polypeptides that have alterations in the amino acid sequence of 24P4C12 shown in Figure 2 or Figure 3.

C Embodiments of a 24P4C12 polypeptide indude: a 24P4C12 polypeptide having a sequence shown in Figure 2, a 00 peptide sequence of a 24P4C12 as shown in Figure 2 wherein T is U; at least.10 contiguous nudeotides of a polypeptide having the sequence as shown in Figure 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 24P4C12 peptides comprise, withoutlimitation: 00 a protein comprising, consisting essentially of, or consisting of an amino add sequence as shown in Figure 2A-I or Figure 3A-G; S(II) a 24P4C12-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97,98, 99 or 100% homologous to San entire amino add sequence shown in Figure 2A-I; C (III) a 24P4C12-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-I or 3A-G; (IV) a protein that comprises at least one peptide set forth in Tables VIII to XUX, optionally with a proviso that it is not an entire protein of Rgure 2; a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX. collectively, optionally with a proviso that it is not an entire protein of Figure 2; (VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of Figure 2; (VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2; (VIII) a protein that comprises at least one peptide selected from the peptides set forth In Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino add sequence of Figure 2; (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino adds of a protein of Figure 3A. 3B, 30, 30, 3E, 3F, or 3G In any whole number Increment up to 710, 710, 710, 710, 598, 722, or 712 respectively that indudes at least 1, 2, 3, 4, 6,7,8, 9, 10, 11, 12, 13, 14, 15,16,17,18, 19, 20,21,22,23,24,25, 26, 27,28,29,30, 31,32,33,34,35 amino add position(s) having a value greater than 0.5 In the Hydrophilidty profile of Figure apolypeptide comprising at least 5,6,7, 8,9,10,11,12,13,14,15,16,17,18,19,20, 21, 22,23,24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino adds of a protein of Rgure 3A, 3B, 3C. 3D, 3E, 3F, or 3G in any whole number incremenl up to 710, 710, 710, 710, 598, 722, or 712 respectively, that Indudes at least 1, 2, 3, 4, 6,7,8, 9,10, 11,12,13,14,15,16,17,18,19,20,21, 22, 23, 24,25,26. 27,28,29,30,31,32,33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathidty profile of Figure 6; 00 0 (XI) a polypeptide comprising at least 5,6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21,22,23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acds of a protein of Figure 3A, 3 3, 3C, D, 3E, 3F, or 3G in any Swhole number increment up to 710, 710, 710, 710, 598, 722, or 712 respectively, that includes at least 1,2, 3, 4, 6,7, 8, 9,10,11, 12,13,14,15, 16,17, 18.19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33.34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; 00 (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21,22, 23, 24, 26, 27, 28, 29, 30, 31,32.33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, 3E, 3F, or 3G in any 00 whole number increment up to 710, 710, 710, 710, 598, 722, or 712 respectively, that includes at least 1, 2, 3, 4, O 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33. 34, 35 amino add position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; 00 (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,11, 12,13,14, 15,16, 17,18,19, 20, 21,22,23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino adds of a protein of Figure 3A, 3B, 3C, 3D, 3E, 3F, or 3G in any C whole number increment up to 710, 710, 710, 710, 598, 722, or 712 respectively, that includes at least 1,2, 3, 4, 6,7,8,9, 10,11, 1213,14,15,16,17, 1819, 20,21,22, 23,24,25,26,27,28,29, 30,31,32 33,34,35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9; (XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively; (XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX. collectively; (XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively; (XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX. collectively; (XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX (XIX) a peptide that occurs at least once In Tables VIII-XXI, and at least twice in tables XXII to XLIX; (XX) a peptide that occurs at least twice in Tables VIII-XXi, and at least once in tables XXII to XLIX; (XXI) a peptide that occurs at least twice In Tables VIII-XXI, and at least twice in tables XXII to XLIX (XXII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonudeotide encoding such peptide: i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number Increment up to the full length of that protein in Figure 3, that indudes an amino add position having a value equal to or greater than 0.5, 0.6,0.7, 0.8, 0.9, or having a value equal to 1.0, In the Hydrophilcity profile of Figure i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein In Figure 3, that Includes an amino add position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; ii) a region of at least 5 amino adds of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that indudes an amino add position having a value equal to or greater than 0.5,0.6,0.7,0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a region of at least 5 amino adds of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino add position having a value equal to or greater than 0.5,0.6, 0.7,0.8, 0.9, or having a value equal to 1.0, in the Average Fexibility profile of Figure 8; or.

00 v) a region of at least 5 amino acids of a particular peptIde of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9; (XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a 00 pharmaceutical excipient and/or in a human unit dose form.

(XXIV) a method of using a peptide of (I)XII, or an antibody or bindcing region thereof or a composition of 00 (XXIII) in a method to modulate a cell expressing 24P4C12, (XXV) a method of using a peptide of (I)-XXII) or an antibody or binding region thereof or a composition of (XXIII) In a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressinig 24P4C1 2 00(XXVI) a method of using a peptidle of (l)-{XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a call expressing 24P34C1 2, said cell from a CKI cancer of a tissue listed in Table 1; (XXVII) a method of using a peptide of (1NXXII) or an antibody or binding region thereof or a composition of (XXIII) in a method Io diagnose, prophylax, prognose, or treat a a cancer, (XXVIII) a method of using a peptide of (I1X40I) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophytax, prognose, or treat a a cancer of a tissue listed In Table 1; and, (XXX) a method of using a a peptide of (1)4{0(11) or an antibody or binding region thereof or a composition (XXIII) in a method to identify or characterize a modulator of a cell expressing ARMC1.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include M4PCI 2 polynucleofides that encode specific portioris of 24P4C12 mRNA sequences (and those which are complem~entary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: 4, 5, 10, 11, 12, 13, 14, 15, 16,17,18,19, 20, 21, 22, 23, 24, 25, 30. 35, 40, 45, 50.,55, 60, 65, 75.80, 85, 90.,95,100,105, 110, 115,120,125,130. '135,140,145,150,155,i60, 165 170,175, 180,185 190, 195, 200, 225,250, 275, 300, 325,350,375,.400, 425, 450, 475, 500, 525,550, 575, 600, 625,650,675,700,710crmore contiguous amino acids of 24P34C2 variant 1; the maximal lengths reevant for other variants are: variant 3,710 amino acids; varliant 710 amino acids, variant 6, 710, variant?, 598 a mino acids, vaiant 8, 722 aihino acids, and variant 9, 712 amino acids..

In general, naturally occrrning aflelic variants of human 24P4C12 share a high degree of structural identity and homology W0A or more homnology). Typically, afielic variants of a 24P4C1 2 protein contan conservaiv amino add substitutions within the 24P4C1 2 sequences described herein or contain a substitution of an amidno acid from a corresponding poslIlonin ahomologueof'24P4Cl2. One ass o124P4Cl2 alldicvariansare proteins that share ahigh degree of homogy withi atllast a small moocn ot a particiular 24P4C12 amtino acid sequence, but further contain a radical departure from tMe sequence, such as a noncansevative substiUtion, truncation, Insertion or frame shtft In comparisons of protein sequenims, the terms, similaty, Identity, ard homology eacht have a distinct meaning as appreciated In the field of genetics Moreover, orthology and paraloy can be hrpofltw ocepts descrtbing the relationship of menbers of a given protein family In one oranwsm to the members of the same family in other organism..

00 Amino add abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made cr in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2 ,C 3, 4,5,6,7, 8, 9,10, 11,12,13,14,15 conservative substitutions. Such changes include substituting any of isoleucine valine and leudne for any other of these hydrophobic amino acids; aspartic add for glutamic acid and vice 00 versa; glutamine for asparagine and vice versa; and serine for threonine and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the threedimensional structure of the protein. For example, glydne and alanine can frequently be interchangeable, as can 00 alanine and valine Methionine which is relatively hydrophobic, can frequently be interchanged with leudne and Sisoleucne, and sometimes with valine. Lysine and arginine are frequently interchangeable in locations in which the \0 significant feature of the amino add residue is its charge and the differing pCKs of these two amino add residues are not 0 significant. Still other changes can be considered 'conservative' in particular environments (see, e.g. Table III herein; pages 00 13-15'Biochemistry 2d ED. Lubert Stryer ed (Stanford University); Henikoff e al., PNAS 1992 Vol 89 10915-10919; Lei et Sal., J Biol Chem 1995 May 19 270(20):11882-6).

K Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 24P4C12 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 24P4C12 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Sitedirected mutagenesis (Carter et at., Nuct. Acids Res., 13:4331 (1986); Zoller etal., Nud. Acids Res., 10.6487 (1987)), cassette mutagenesis (Wells et Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R.

Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the doned DNA to produce the 24P4C12 variant DNA.

Scanning amino acid analysis can also be employed to Identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino adds. Such amino acids include alanine, glycine, serine, and cysteine.

Alanine is typically a preferred scanning amino acid among this group because it eiminates the sidechain beyond the belacarbon and is less likely to alter the main-chain conformation of the variant Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, Freeman Co., Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant an isosteric amino add can be used.

As defined herein, 24P4C12 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is "cross reactive' with a 24P4C12 protein having an amino acid sequence of Figure 3. As used In this sentence, 'cross reactive' means that an antibody or T cell that specifically binds to a 24P4C12 variant also specifically binds to a 24P4C12 protein having an amino acid sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 24P4C12 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino adds, contiguous or not is regarded as a typical number of amino adds in a minimal epitope. See, Nair et J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other dasses of 24P4C12-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino add sequence of Figure 3, or a fragment thereof. Another specific dass of 24P4C12 protein variants or analogs comprises one or more of the 24P4C12 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 24P4C12 fragments (nucleic or amino acid) that have altered functional (e.g.

00 O immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the Sart are to be applied to the nucleic or amino add sequences of Figure 2 or Figure 3.

SAs discussed herein, embodiments of the claimed invention indude polypeptides containing less than the full amino acid sequence of a 24P4C12 protein shown in Figure 2 or Figure 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 00 24P4C12 protein shown in Figure 2 or Figure 3.

Moreover, representative embodiments of the invention disclosed herein indude polypeptides consisting of about amino acid 1 to about amino add 10 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about S0 K amino add 10 to about amino add 20 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about Samino acid 20 to about amino add 30 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptldes consisting of about amino acid 30 to about amino acid 40 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about 0amino acid 50 to about amino acid 60 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about Oamino add 60 to about amino acd 70 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino ad 80 to about amino acid 90 of a 24P4C12 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino add 90 to about amino acid 100 of a 24P4C12 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 24P4C12 amino add sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 24P4C12 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

24P4C12-related proteins are generated using standard peptide synthesis technology or using chemical deavage methods well known in the art Alternatively, recombinant methods can be used to generate nucleic add molecules that encode a 24P4C12-related protein. In one embodiment nucleic add molecules provide a means to generate defned fragments of a 24P4C12 protein (or variants, homologs or analogs thereof).

III.A.) Motif*bearing Protein Embodiments Additional Illustrative embodiments of the invention disclosed herein Include 24P4C12 polypeptides comprising the amino add residues of one or more of the biological motifs contained within a 24P4C12 polypeptide sequence set forth In Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publidy available Intemet sites (see, URL addresses: pfam.wustl.edul; searchlauncher.bcm.tmc.edulseqsearchstruo-predicthtml; psortims.u-tokyo.ac.jpl; cbs.dtu.dkl; ebi.ac.uk/interprolscan.html; expasy.ch/tools/scnpsitl.html; Eplmatrix T M and Epimer, Brown University, brown.eduReseardhVB-HIV_Lablepima epimathtml; and BIMAS, birmas.datnih.gov/.).

Motif bearing subsequences of all 24P4C12 variant proteins are set forth and identified in Tables VIII-XXI and XXII-

XIX.

Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/).

The columns of Table V list motif name abbreviation, percent identity found amongst the different member of the motif family, motif name or description and most common function; location information is induded if the motif is relevant for location.

Polypeptides comprising one or more of the 24P4C12 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 24P4C12 motifs discussed above are associated with growth dysregulation and because 24P4C12 is overexpressed in certain cancers (See, Table Casein kinase II, 00 0 cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with 0 the development of the malignant phenotype (see e.g. Chen et al, Lab Invest, 78(2): 165-174 (1998); Gaiddon et at., Endocrinoogy 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzlel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem.

0 0 Blophys. Acta 1473(1):21-34 (1999); Raju etf a, Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et J. Nail. Cancer Inst Monogr. (13): 169-175 (1992)).

00 In another embodiment, proteins of the invention comprise one or more of the immunoreactive epltopes identified D in accordance with art-aocepted methods, such as the peplides set forth in Tables VIll-XXI and XXII-XIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 24P4C12 protein that are capable of optimally binding to 0 specified HLA alleles Table IV; Ephmabix T and Epimer

T

Brown University, URL brown.edu/ResearcTB- 00 0 HIVLablepimatix/epimatrix.hml; and BIMAS, URL bimas.dcrtnih.gov/.) Moreover, processes for identifying peptides that have 0sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known In the art, and are carried out without undue experimentation either in vtro or In vivo.

Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino add at one of the specified positions, and replacing It with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue In favor of any other residue, such as a preferred residue; substitute a lesspreferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, Table IV.

A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut etal.; Sette, Immunogenetics 1999 50(3-4): 201- 212; Sette at al., J. Immunol. 2001 166(2): 1389-1397; Sidney et Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunot. 1996 157(8): 3480-90; and Falk el al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol 149:3580-7 (1992); Parker et al., J. Immunol.

152:163-75 (1994)): Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander of al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.

Immunol. 1991 147(8): 2663-2669; Alexander at al., Immunity 1994 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXI-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which Indude a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, indude a greater portion of the potypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino add residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino add residues.

24P4C12-related proteins are embodied In many forms, preferably in isolated form. A purified 24P4C12 protein molecule will be substantially free of other proteis or molecules that impair the binding of 24P4C12 to antibody, T cell or 00 other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 24P4C1 2related proteins include purified 24P4C12-related proteins and functional, soluble 24P4C12-related proteins. in one r-K1 embodiment a functional, soluble 24P4C12 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The Invention also provides 24P4C12 proteins comprising biologically active fragments of a 24P4C1 2 amino acid 00 sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the stAtng 24P4C1 2 protein. such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 24P4C12 protein; to be bound by such antibodies; to elicit the activation of HfL or CTR; and/or, to be recognized by 1ITL or CTL that also specifically 00 bind to the starting protein.

N 24P74C 12-related polypeptides that contain particularly interesting structures can be predicted andlor identified using various analytical techiques well known in the art including, for example, the methods of Chou-Fasman, Gamier-Robson, Kyle- Doolittle, Elseeg, Karplus-Sdiultz or Jameson-Wolf analysis, or based on inimunogeicity. Fragments that contain such 00 structures are partctlauty useful in generating subunit-specific antl-24P4C12 antibodies orT cells or in ldentiyng cellular factors that bind to 24P4C12. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, TP. and Woods. 1981, Proc. Nall. Acad. Sci. U.SA 78:3824-3828. Hydropathlaty profiles can be generated, and lrmunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, 1982, J.

Mol. Biol. 157:105-132. Percent Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and Immunogenic peptide fragments identified, using the method of Bhaskaran Ponnuswamy P.K, 1988, Int. J. Pept Protein Res. 32:242-255. Beta-turn profiles can be generated, and Immunogenic peptide fragments identified, using the method of Deleage, Roux 1987, Protein Engineering 1:289-294.

CTIL epitopes can be determined using specific algortms to identitly peptides within a 24P4C1 2 protein that are capable of optimally bindig to specified HLA alleles by using the SYFPEITHI site at World Wide Web URL syfpelh.bmiheldelberg.comf;l- the listings in Table Eplmatrixm and Epimerml, Brown Universit, URL (brown.edu/ReserTB- HIV..LablepimatixopmatixhM; and BIMAS, URL bImas.dcrtnih.govO. Illustrating this, peptide epitopes from 24P4C12 that are presented in the context of human MHC Class I molecules, HLA-A1, A2, A3, All. A24. 87 and 635 were predicted (see, Tables VIII-XXI, XXII-XLX). Specifically, the complete amino acid sequence of the 24P4C12 protein and relevant portions of other variants, Le., for HLA Class I predictions 9 flanking residues on either side of a point mutation or axon judion, and for HLA Class 11 predictions 14 flanking residues on either side of a point mutation or axon junction corresponding to that variant were entered into the HLA Peptide Motif Search algorithm found In the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; In addition to the site SYFPEITHI, at URL syfpeithlbrnlheidelberg.coo/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular lILA-A2 (see, Falk of al., Nature 351: 2904(1991); Huntot aL, Science 255:1261-3 (1992); Parker of aL, J. Immunol. 149:.3580-7 (1992); Parker at at, J. Immunol. 152:163-75 (1994)). This algortm allows location and ranking of 8-mar, 9-mar, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I bining peptides are 10 or 11 1-mars. For example, for Class I HLA-A2, the epitopes preferably contain a leucine or methionine at position 2 and a valine MV or leucine at the C-terminus (see, Parker et J. Immunol. 149:35W07 (11992)) Selected results of 24P4C12 predicted binding peptides are shown In Tables VlI-XXI and XXII-XLIX herein. In Tables VIII- XXI and XWI-XLV~I, selected candidates, 9-mars end 10O-mers, for each famnily member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-LX. selected 00 candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific Speptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 370C at pH 6.5. PeptIdes with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic Stargets for T-cell recognition.

00 Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigenprocessing defective cell line T2 (see, Xue et al., Prostate 30:73-8 (1997) and Peshwa e al., Prostate 36:129-38 (1998)). Immunogenicty of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) 00 in the presence of antigen presenting cells such as dendritic cells.

\O It is to be appreciated that every epitope predicted by the BIMAS site, EpimerT and Epimarix T sites, or specified by the HLA class I or dass II motifs available in the art or which become part of the art such as set forth in Table IV (or Cl determined using World Wide Web site URL syfpelthl.bmi-heidelberg.com/, or BIMAS, bimas.dcrLnh.gov/) are to be 'applied' 00 Sto a 24P4C12 protein in accordance with the invention. As used in this context applied" means that a 24P4C12 protein is 0evaluated, visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art Every subsequence of a 24P4C12 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

Ill.B.) Expression of 24P4C12-related Proteins In an embodiment described in the examples that follow, 24P4C12 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 24P4C12 with a C-terminal 6XHis and MYC tag (pcDNA3.iknycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 24P4C12 protein in transfected cells. The secreted HIS-tagged 24P4C12 In the culture media can be purified, using a nickel column using standard techniques.

II.C.l Modifications of 24P4C12-related Proteins Modifications of 24P4C12-related proteins such as covalent modifications are included within the scope of this Invention. One type of covalent modification includes reacting targeted amino add residues of a 24P4C12 polypeptide with an organic derivatizing agent that Is capable of reacting with selected side chains or the N- or C- terminal residues of a 24P4C12 protein. Another type of covalent modification of a 24P4C12 polypeptide included within the scope of this invention comprises altering the native glycosylation pattem of a protein of the invention. Another type of covalent modification of 24P4C12 comprises linking a 24P4C12 polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 24P4C12-related proteins of the present invention can also be modified to form a chimeric molecule comprising 24P4C12 fused to another,, heterologous polypeptide or amino add sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumorassociated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 24P4C12 sequence (amino or nudeic add) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic add sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of 24P4C12. A chimeric molecule can comprise a fusion of a 24P4C12-related protein with a polyhstidlne epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with 00 cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a 24P4C12 protein. In an alternative embodiment the chimeric molecule can comprise a fusion of a 24P4C1l 2-related protein with an Ic Immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeic molecule (also referred to as an 'immuneadhesin"), such a fusion could be to the Fc region of an lgG molecule. The 19 fusions preferably hidude Owe substitution of a soluble (transmembrane domain deleted or inactivated) form of a 24P4C 12 polypeptide In place of at least 00 one variable region within an Ig molecule. In a preferred embodiment the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 region of an IgGI molecule. For the production of immunoglobulin fusions see, 00 U.S. Patent No. 5.428,130 issued June 27, 1995.

IND10.0.1 Uses of 24P4C1 2-related Proteins The proteins of the invention have a number of different specific uses. As 24P4C1 2 Is highly expressed in prostate 00 and othe cancers, 24P4C1 2-related proteins are used in methods that assess the status of 24P4C1 2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 24P4C1 2 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 24P41 2-related proteins comprising the amino acid residues of one or more of the biological motifs; contained within a 24P4C12 polypeptide sequence in order to evaluate the characteristcs of this region in normal versus cancerous tissues or to elicit an Immune response loathe epitope. Alternatively, 24P14C12-related proteins that contain the amino acid residues of one or more of the biological motifs in a 24P4C1 2 protein are used to screen for factors that interact with that region of 24P4C1 2.

244C 1 2 protein fragments/subsequences are particularly useful in generating and characterizing domain-sefli antibodies antibodies recognizing an extaceiular or Intracellular epitope of a 24P4C1 2 protein), for Identifying agents or cellular factors that bind to 24P4C1 2 or a particular structua domain thereof, and in various therapeutic and diagnostic contexts, incuding but not limited to diagnostic essays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 24P4C12 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for Idenlifying Igans and other agents and celiuar constituents that bind to a 24P4C12 gene product. Antibodies raised against a 24P4C12 protein or fragmenitthereof are useful in diagnostic end prognostic assays, and imaging methodologies In t management of human cancers characterized by expession of 24P34C1 2 protein, such as thnose listed in Table 1. Such antibodies can be expressed intraceilutarty and used In methods of treating patients with such cancers. 24P4C1 2-related nucleic adds -or proteins are also used in generating HTIL or CTIL responses.

Various lImmunological assays useful for the detection of 24P4C 12 proteins are used, including but not limited to various types of radimhmunoassays, enzyme-linked imunosorbent assays (EUSA), ermime-finked Inimunofluomemet assays (ELIFA), immunocyjtochemnical methods, and the like. Antbodies can be laeled and used as immunological imaging reagents capable of detecting 24P4C12.expressing call in radioscintigraphic rnaging methods). 24P4C12 proteins ame also particularly useful In generating cancer vaccines, as futher descfieid herein.

IV.) 24P4C12 Anibodies Anotiler aspect of the invention pviAdes antibodies that bind to 24P4C1 2-etated proteins. Preferred antibodies specifically bind to a 24P4C1 2-reated protein end do not bind (or bind weaidy)i to peptides or proteinis that are not 24P4C1 2.

related proteins. For exarroe, antbod'es tht bind 24P4C1 2 can bind 24P4C1 2-related proteins such as the homologs or -ntg thereof.

00 24P4IC1 2 antibodies of the invention are particularly useful in cancer (see, Table 1) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 24P4C1 2 is also expressed or overexpressed In these other cancers. Moreover, intracellularly expressed antibodies single chain antibodies) are therapeutically useful in treating cancers In which the expression of 24P4C 12 is Involved, such as advanced or metastatic prostate cancers.

0 mutant 24P4C12-related pteins. Such assays cant comprise one or more 24P4C12 antibodies capable of recgnizing and binding a 24PAIC12-related protein, as appropriate. These assays are perfoirmed within various Immunological assay 1ormats well 00 known In the art including but not imited to various types of rallolrmunciassays, enzymneined inimunosorbent assays (ELISA), enzyme-fiked immunotflucrescent assays (ELIFA), and the Wue.

bImunological non-antibiody assays of the invention also corise T call iramunigenticty assays (inhibitory or 00 stimulatory) as well as major histocompalblity complex (MHC) binding assays.

00 In addition, Immunological imaging methods capable of detecting prostate cancer arnd other cancers expressing 24P4C12 are also provided by tile invention, including but not limited to radoscindgraphic Imaging methods using labeled 24P14C1 2antibodlies. Such assays are cinically useful In the detection, monitoring, and prognosis of 24P4C12 expressing cancers such as prostate cancer.

24P4C1 2 antibodies are also used In methods for purifying a 24P34CU -related protein and for Isolatig 24P4C12 homologues and related molecules. For example, a method or purifying a 24PC112-related protein comprises incubating a 24P4C1 2 antibody, which has been cxrsloled to a solid mabiry with a lysateor othersoution containing a 24P4C1 2-related protein under conditions that permitft a24P4C1 2 antibody to bind to the 24P4C12-related protein; washing the solid matrix to eliminate impurities; and eluting the 24P4C1 2-related protein from the coupled antibody. Other uses of 24P4C1 2 antibodies in accordance with the Irvention include generating anti.4dioitypic antibodies that mimic a 24P4C12 protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 24P34Cl 2-related protein, peplide, or fragment in Isolated or inimunoonjugated form (Antibodies: A Labortory Manual, CSH Press, Eds., Hartow, and Lane (1988); Hatlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteinsdof24P4C12 cai also be used, such as a 24P4C12 GST4fuslon protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 Is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment a 24P4C1 2-related protein Is synthesized and used as an Immunogen.

In addition, naked DNA Imnizaion techniqlues known in the arl are used (with or without purified 24P4C12-related protein or 24P34C1 2 expressing cells) to generate an Immune response to the encoded mimunogan (fbr review, see Donnelly et at.. 1997, Ann. Rev. Immnrr. 15. 617-648).

The amino acid sequence of a 24P4C1 2 protein as shown In Figure 2 or Figure 3 can be analyzed to select specific regions of te24P4C2 proten for geatinantibodies. For example, hydrophioucity anid hydropilidty analyses of a 24P4C12 amino acid seqluence are used to Identify hydrapili regions in the 24P4C12 structure. Regions of a 24P4C12 protein that show immogenic structure, as wet as other regions and domnains, can readily be Identified using various otter methods known in the art such as Chou-fasman, Gamnier-Robson, Kyte-Doollittle, Elsenberg, Karpftis-Schuttz: or Jameson-Wolf aalysis, Hydrophuiiaty profies can be generated using the method of Hopp, T.P. and Woods, KR., 1981, Proc. Nail. Mad. Sol. U.SA 78:3824- 3828. Hydropathicity profiles can be generated using the method of Kyle. J. and Doolittle, 1982, J. Mc iol. 10157:105- 132. Perent Accessible Residues profiles can be generated using the mnethod of Janin 1979, Nature 277:491-492 Average Flexibility profiles can be generated using the method of Ohaskaran Ponnuswamy P.K, 1988, It. J. Pept P rotein Res. 32-242-255. Beta-turn profiles can be generated using the method of Deleage, Roux 1987, Protein 0 invention. Methods for the generation of 24P4C12 antibodies are fu~rther illustrated by way of the examples provided herein.

Methodsforpepag aproteinopypepf e for use asan m unogen arewell known ithe aft Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA. KLH or oter carder protein, In some circumstances, direct conjugation using, for example, carbiodlirale reagets are used; in other instances finking reagets such as 00 those supplied by Pierce Chemical Co., Rockford, It, are effective. Amrniisfration of a 24P4C12 immunogen is often conducted by Injection over a suitable time period and with use of a suitable adjuvant as is undlerstood in the ait During the immrunization schedule, titers of antibodies can be taken to determine adequacy of antibodly bomaion.

00 24P4C 12 monoclonal antibodlies can be produced by varbou means well known in the art For example, immortalized N cell ines that Reaele a desired monoclonal antibody are 'prepared using the standard hytbddoma technology of Kohler and Milstein or modifications that imnmortafize andlbody-produing 8 calls, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by eirnunoassay in which the antigen is a 24P34CI 2-related protein. Whien the appropriate 00 Immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in Wiro cultures or from asites fluid.

The antibodies or fragments of the Invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 24P4C 12 protein can also be produced In the context of chieric or complementaiitydetermining region (CDR) grafted antibodies of multiple spaes origin. Humanized or human 24P4C12 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanzig murine and other non-human antibodies, by substituting one or more of the non-human antibody CORs for corvespoindirtg human antibody sequences, are well known (see for example, Jones et 1986, Nature 321: 522-525; Riechmain at 9t., 1988, Nature 332: 323-327; Vertioeyen et al., 19.88, Science 239. 1534-1536). See also, Carter et al, 1993, Proc. NOd. Aced. Sci. USA 89: 4285 and Sims etal, 1993, J. Imniunol. 151: 2296.

1Methods for producing fully human monodlonal antibodies Include phage display and transgenic methods (for review, see Vaughan et 1998, Nature Biotechnology 18: 535-539). Fully human 24134C 12 monoclonal antibodies can be generated using dloning technologies employing large human Ig gene combinatorial libraries, phage display) (Giffts and Hoogenboom, Building an i immrune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications 'in Man, Clark, M. Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial ibraries. jo., pp 65-82). Fully human 24P4C12 monodlonal antibodies can also be produced using transgenic mice engineered to contain human uimmunogloboulin gene loci as described In PCT Patent Application W098/24893, Kudierlapai and Jakobovits of al., pubfished December 3.1997 (see also, Jakoibovits, 1998, E.V. Oin. inest.

Drug 607-614; U.S. patents 6,162,963 Issued 19 December 2000; 6,150,5814 issued 12 November 2000; and, 6,114598 issued 5 September 2000). ibis method! avoids tie in vit manipulation required with phage dlisplay technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 24P4C1 2 antibodies with a 24P4C1 2-related protein can be established by a number of well known means, inducting Western blot Immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 24P4C 1 2-related proteins, 24P4C12-expressing cells or extracts thereof. A 24P4C12 antibody or fragment thereof can be labeled with a detectabe marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bloluminescent compound, ctremiuminescent compound, a metal dielator or an enzyme. Further, bil-specific antibodies specific for two or more 24P4C12 epltopes are generated using methods generally known in the art Homodimeric antibodies can also be generated by cross-lnking techiques known in the art Wolff at al., Cancer Res. 53:256-2565).

24P4012 Cellular Immune Responses 00 The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the wod.

wide population. For an unders tanding of the value and efficacy of compositions of the Invention that Induce cellular Immune responses, a brief review of immunology-related technology is provided.

00 A complex of an HLA molecule and a peplidic antigen acts as the ligand recognized by HLA-restilcted T cells 0 ~(Buus, S.eat Coll 47:1071, 1986; Babbitt, B. P. et Nature317:359, 1985; Townsend, A. and Bodmer, Annu. Rev.

Immnol. 7:601, 1989; Germain, R, Annu. Rev. Immunol. 11:403,1993). Through the study of single amino acdd 00 substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that CN1 correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV IND (see also. Southwood, et al.. J. lmmunol. 160:3363, 1998; Rammensee, etaa., Irnmunogenetics 41:178, 1995; Rammensee etaa., SYFPEITHI, access via World Wide Web at URL (I134.2.96.22llsaipts.haserver.dlibome.hm); Sette, A 00 and Sidney, J. Cuff. Opin. Immunot. 10:478, 1998; Engelhard, V. Gunf. Opin. Immunol. 6:13,1994; Sette, A and Grey, H.

Cuff. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Gun'. Bil. 6:52, 1994; Ruppert et aL, Ceog 74:929-937, 1993; Konkdo eta)., J. Inimunoi. 155:,4307-4312,1995; Sidney ot al., J. ImmunoL 157:34860-3490, 1996; Sidney of aL, Humnan Invnunol. 45:79-93, 1996; Sette, A and Sidney, J1. Immunogenetics 1999 Nov,; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes haoe revealed pockets within the peptide binding cleft/groove of HLA molecuiles which accommodate, in an allele-spedflc mode, residues borne by peptide figands; these residues in turn determine the HLA binding capacity of the peptides in which they are present (See, Madden, D.R. Annu. Re. Immunol. 13:587,1995; Smith, et al.. Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stem el Stnue 2:245, 1994; Jones, E.Y. Gur. Opin. Immunol. 9:75,1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C.

et al., Proc Natl. Aced. Sc. USA 90:803, 1993; Guo, H. C. ea) Nature 360:364,1992; Silver. M. Le at Nature 360:.367, 1992; Matsurnura, M. at al., Science 257:927,1992; Madden at al., Cell70.1035, 1992; Fremront, D. Hi. et Science 257:91 9, 1992; Saper, M. A. Bjorkman, P. J. and Wiley, D. J. Mot. Bil. 219:.277, 1991.) Accodingly, the definition of class I and class 11 allee-specific HIA binding motifs, or class I or class 11 supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA anfigen(s).

Thus, by a process of HIA motif identification, candidates for epitope-based vaccines have been Identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and Its corresponding HLA molecule. Additional confirmatr work can be performed to select amongst thesw vaccine candidates, epitopes; with preferred characteristics in term of populationi coverage, and/or iminunogenicity.

Various strategies can be utiized to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal Individuals (see, Wentworth, P. Kqftl, Mot. limurro.

32:603, 1995; Cells, E. eta!t, Pmc Nagl. Aced. Sci. UiSA 91:2105,1994; Tsai, V. at al., I. Immunof. 158-.1796,1997; Kawashima, 1. etaat, Human Immuraol 59:1, 1998). This procedure involves the stinulation of peripheral blood lymphocytes (PBL) from normal subject with a test peptide In the presence of antigen presenting cells In Wfto over a period of several weeks. T cells specific for the peptie become activated during this lime and are detected using, a lymphoidne- or 1 Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, Wentworth, P. A. of at, J. Immunol. 26:97, 1996; Wentworth, P.

A al at., Int. Immunol. 8:651, 1996;, Alexander, J. ef at., J. Immunol. 159:4753, 1997). For example, in such methods peptides In incomplete Freund~s adjuvant are administered subcutaneously to HIA transgenic mice. Several weeks following immurilzatiori, splenocytes are removed and cultured in iY*w in the presence of test peptide for approximately one week.

00 SPeptide-specific T cells are detected using, a 51 Cr-release assay involving peptide sensitized target cells and target Scells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated d) andor from chronically ill patients (see, Rehermann, B. et J. Exp. Med. 181:1047, 1995; Doolan, D. L. et a., Immuniy 7:97, 1997; Bertoni, R. et al., J. Cin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response naturaly", or from 00 patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of C test peptide plus antigen presenting cells (APC) to allow activation of 'memory" T cells, as compared to "naive" T cells. At Sthe end of the culture period, T cell activity is detected using assays induding 51Cr release Involving peptide-sensitized Stargets, T cell proliferation, or lymphokine release.

00 VI.) 24P4C12 Transsenic Animals SNucleic acds that encode a 24P4C12-related protein can also be used to generate either transgenic animals or 'knock out" animals that, in turn, are useful In the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 24P4C12 can be used to done genomic DNA that encodes 24P4C12. The doned genomic sequences can then be used to generate transgenlc animals containing cells that express DNA that encode 24P4C12. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in thb art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 24P4C12 transgene incorporation with tissue-specfic enhancers.

Transgenic animals that indude a copy of a transgene encoding 24P4C12 can be used to examine the effect of increased expression of DNA that encodes 24P4C12. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the Invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic Intervention for the pathological condition.

Alternatively, non-human homologues of 24P4C12 can be used to construct a 24P4C12 'knock out animal that has a defective or altered gene encoding 24P4C12 as a result of homologous recombination between the endogenous gene encoding 24P4C12 and altered genomic DNA encoding 24P4C12 Introduced Into an embryonic cell of the animal. For example, .cNA that encodes 24P4C12 can be used to done genomic DNA encoding 24P4C12 in accordance with established techniques. A portion of the genomic DNA encoding 24P4C12 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are Included in the vector (see, Thomas and Capecch, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is Introduced into an embryonic stem cell line by electroporation) and cells In which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, U et al., Cell. 69915 (1992)). The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras (see, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Pracical Approach, E. J. Robertson, ed. (IRL, Oxford. 1987), pp. 113-152). A cimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out animal. Progeny harboring the homologously recombined DNA in their germ cells can be Identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, 00 for example, for their ability to defend against certain pathological conditions or for their development of pathological r~l conditions due to absence of a 24P4C12 polypeptidle.

V11.1 Methods for the Detection of 24134C12 00 Another aspect of the present invention relates to methods for detecting 24P4012 polynudeotides and 24P4C1 2related proteins, as wel as methods for identifying a cell that expresses 24P4C12. The expression profile of 24P4C1 2 makes it a diagnostic nmarkcer for metastasized disease. Accordingly. the status of 24P4C12 gene products provides information useful 00 for predicting a variety of factors Including suscetiblity to advanced stage disease rate of progression, andlor tumor aggressiveness. As discussed in detail herein the status of 24P4C12 gene products in patient samples can be analyzed by a variety protocols that are well known in the art Including Imuiihstodiernical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dlissected samples), Western blot analysis 00 and fissue array analysis.

More particularly, the invention provides assays for the detection of 24P4C1 2 polynudeotides in a biological sample, CI such as serum, bone, protstate, and other tissues, urine, semen, coil preparations, and the like. Detectable 24P4C1 2 polynudeoties include, for example, a 24P4C1 2 gene or fragment thereof, 24P4C 12 mRNA, alternative splice variant 24P4C1 2 mRNAs, and recombinant DNA or RNA molecules tMat contain a 24P4C1 2 polyriucleotidle. A number of methods for amplifyn and/or detectinig the presence of 24P4C1 2 polynudeotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 24P4C12 mRNA in a bioloical sample comprises producing cONA from the sample by reverse transcription using at least one primer amplifying the cONA so produced using a 2412C02 polynucleotides as sense and antisense primers to amplify 24P4C12 cDNAs therein; and detecting the presence of the amplified 24P4C12 cDNA. Optionally, the sequence of the amplified 24P4C12 cONA can be determined.

In another embodiment, a method of detecting a 24P4C12 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 24P4C1 2 polynudleotides as sense and antisense primers; and detecting the presence of the amplified 2415402 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 2402 nucleotide sequence (see, Figure 2) and used for this purpose.

The invention also provides asspyfor detecing the presence of a 24P4C1 2 protein in a issue or other biological sample such as serum, semen, bone, prostate, urine, cell preparatis, and the like Methods for detecting a 24P4C1 2-related protein awe also wel know and include, for example, immunopreiptaion, irrviunohlstodierical analysis, Western blot analysis, molecular binding assays, EUISA, ELIFA and the like. For example, a method of detecting the presence of a 24P4C1 2-related protein In a biological sample comprises first contacting the sample with a 24P4C1 2 antibody, a 24P4C1 2-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 24P4C12 antibody; and then detecting the binding of 24P4.C12-related protein in the sample.

Methods for idenitifyng a cell that expresses 24P4C12 are also within the scope of the Invention. In one embodirwent, an assay for Identifying a cell that expresses a 24P4C1 2 gene comprises detectng the presence of 24P4C1 2 niRNA in the cap.

Methods for tie detection of particular mRNAs in cells are well known and include, for example, hyt 'izton says usig complementary DNA probes (such as In silu hybridization using latele 24P4C12 riboprobes, Northern blot and related techniques) and various nudeic acid amplification assays (such as RT-PCR using complementary primers specific for 24134C12, and other amplification typ detaon methods, such as, for example, branched DNA. SISBA, lilA and the lik). Alternatively, an assay for idenfftfyg a call that expresses a 24P4C1 2 gene comprises detecting the presence of 24P4C I 2related protein In the 00 cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the Sdetection of 24P4C12-related proteins and cells that express 24P4C12-related proteins.

24P4C12 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 24P4C12 gene expression. For example, 24P4C12 expression is significantly upregulated in prostate cancer, and Is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 24P4C12 expression or over- 00 0 expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 24P4C12 expression by RT-PCR, nucleic acid hybridization or antibody binding.

00 VIII. Methods for Montorinq the Status of 24P4C12-related Genes and Their Products O Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and Scells progress from a normal physiological state to precancerous and then cancerous states (see, Alers et al., Lab C Invest..77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23:19-32 (1995)). In this context examining a biological 00 Ssample for evidence of dysregulated cell growth (such as aberrant 24P4C12 expression in cancers) allows for early detection Sof such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 24P4C12 in a biological sample of interest can be compared, for example, to the status of 24P4C12 in a corresponding normal sample a sample from that individual or alternatively another individual that Is not affected by a pathology). An alteration in the status of 24P4C12 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, Grever et al., J. Comp. Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 24P4C12 status in a sample.

The term "status" in this context is used according to its art accepted meaning and refers to the codition or state of a gene and its products. Typically. skiled artisans use a number of parameters to evaluate the ondition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (includng the location of 24P4C12 expressing cells) as well as the level, and biological activity of expressed gene products (such as 24P4C12 mRNA, polynudeotidas and polypeptides). Typically, an alteration In the status of 24P4C12 comprises a change in the location of 24P4C12 and/or 24P4C12 expressing cells and/or an increase in 24P4C12 mRNA and/or protein expression.

24P4C12 status in a sample can be analyzed by a number of means well known in the art, incuding without limitation, immunohistochemical analysis, in stu hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 24P4C12 gene and gene products are found, for example In Ausubel at al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 24P4C12 in a biological sample is evaluated by various methods utlized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 24P4C12 gene), Northern analysis and/or PCR analysis of 24P4C12 mRNA (to examine, for example alterations in the polynudeotide sequences or expression levels of 24P4C12 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations In polypeptde sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 24P4C12 proteins and/or associations of 24P4C12 proteins with polypeptide binding partners). Detectable 24P4C12 polynudeotides ndcude, for example, a 24P4C12 gene or fragment thereof, 24P4C12 mRNA alternative spice variants, 24P4C12 mRNAs, and recombinant DNA or RNA molecules containing a 24P4C12 polynudeoide.

The expression profile of 24P4C12 makes it a diagnostic marker for local and/or metastasized disease, and provides Infomation on the growth or oncogenic potential ofa biological sample. In particular, the status of 24P4C12 provides 00 Sinformation useful for predicting susceptibility to particular disease stages, progression, andlor tumor aggressiveness. The CK invention provides methods and assays for determining 24P4C12 status and diagnosing cancers thatexpress 24P4C12, such as ^Q cancers of the tissues listed in Table I. For example, because 24P4C12 mRNA is so highly expressed in prostate and other T cancers relative to normal prostate tissue, assays that evaluate the levels of 24P4C12 mRNA transaipts or proteins in a biological 00 sample can be used to diagnose a disease associated with 24P4C12 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 24P4C12 provides information including the presence, stage and location of dysplastic, 00 precancerous and cancerous cells, predicting susceptblity to various stages of disease, and/or for gauging tumor C aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease.

I Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 24P4C12 in biological samples such as those from individuals suffering from, or suspected of suffering from a 00 pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 24P4C12 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 24P4C12 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 24P4C12 expressing cells those that express 24P4C12 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 24P4C12-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 24P4C12 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin.

(such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, Murphy et al., Prostate 42(4): 315-317 (2000);Su etal. Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 Aug 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 24P4C12 gene products by determining the status of 24P4C12 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 24P4C12 gene products in a corresponding normal sample. The presence of aberrant 24P4C12 gene products in the test sample relative to the normal sample provides an Indication of the presence of dysregulated cell growth within the cells of the Individual.

In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant Increase In 24P4C12 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 24P4C12 mRNA can, for example, be evaluated in tissues Including but not limited to those listed in Table I. The presence of significant 24P4C12 expression in any of these tissues is useful to indicate the emergence, presence andlor severity of a cancer, since the corresponding normal tissues do not express 24P4C12 mRNA or express it at lower levels.

In a related embodiment, 24P4C12 status Is determined at the protein level rather than at the nucleic add level. For example, such a method comprises determining the level of 24P4C12 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 24P4C12 expressed in a corresponding normal sample. In one embodiment, the presence of 24P4C12 protein is evaluated, for example, using immunohistochemical methods. 24P4C12 antibodies or binding partners capable of detecting 24P4C12 protein expression are used In a variety of assay formats well known in the art for this purpose.

00 In a further embodiment one can evaluate the status of 24P4C12 nuceotide and amino acd sequences in a biological C' sample in order to identify perturbations in the structure of these molecules. These perturbations can indude insertions, deletions, substitutions and the Ike. Such evaluations are useful because perturbations in the nudeotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, Marrogi et al. 1999, J.

00 Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 24P4C12 may be indicative of the presence or 0promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 24P4C12 indicates a potential loss of function or increase in tumor growth.

00 A wide variety of assays for observing perturbations in nudeotide and amino acid sequences are well known in the art CN For example, the size and stucture of nucleic acid or amino add sequences of 24P4C12gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing 0 perturbations in nudeotide and amino add sequences such as single strand confomation polymorphism analysis are well known 00 in the ar (see, U.S. Patent Nos. 5,382,510 issued 7 September 1999, and 5,952,170 issued 17 January 1995).

Additionally, one can examine the methylation status of a 24P4C12 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the p-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration In prostate carcinomas (De Marzo t at., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least of cases of high-grade prostatic intraepithellal neoplasla (PIN) (Brooks et al., Cancer Epidemlol. Blomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot deave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which wit convert all unmethylated cytosines to uracl) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving mefhytation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et l. eds., 1995.

Gene amplification is an additional method for assessing the status of 24P4C12. Gene amplification Is measured in a sample directly, for example, by conventional Southern blotting or Northem blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Nal. Acad. Sc. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize spedfic duplexes, induding DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 24P4C12 expression. The presence of RT-PCR amplifable 24P4C12 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known In the art RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these indude RT-PCR assays for the detection of cells expressing PSA and PSM (Verka et at.,1997. Urol.Res. 25:373-384; Ghosseineofal., 1995, J. Clin. Oncol. 13:1195-2000; Hestonat al., 1995, Olin. Chem. 41:1687- 1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developig cancer. tIn one embodiment a method for predicting susceptibility to cancer compnises detecing 24P4C1 2 mRNA or 24P4C1 2 protein in a 00 tsesapeItprsneidicating susceptibility to cancer, wherein the degree of 24P4C1 2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment the presence of 24P4C12 in prostate or other tisse is examined, with the presence of 24P4C12 In the sample providing an Indication of prostate cancer sus~epitllty (or the emergence or existence of a 00 prostate tumor). Similarly, one can evaluate the integrity 24134C12 nudeotide and amino add sequences ina biological sample, in NI order to identify perturbations In the structure of these molecles sudh as isertions, deletions, substitutions and the like. The IND presence of one or more perturbations in 24P4C1 2 gene products in the sample is en indication of cancer susceptibility (or the emoergence or existence of a tumor).

00 The invention also comprises methods for gauging tumor aggressiveness. in one ernbod'iment a method for gauging aggressiveness of a tumor comprises determning the level of 24P4C12 mRNA or 24P4C12 protein expressed by tumor cell, comparinglthe level. so determnined to the level of 24P4C1 2 mRNA or 24P4C 2 protein expressed in acorresponding normal tisue taken from the same individual or a normal Issue reference sample, wherein the degree of 24P4C1 2 mRNA or 24P4C1 2 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific emnbodliment aggressiveness of a tumor is evaluated by deternng the extent to which 24P4C1 2 is expressed in the tumnor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 24P4C1 2 nudeotide and amino ad sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertion, deletions, substitutions and the Ike. 'The presence of one or more perturbations indicates mole aggressive tumors.

Another embodiment of the Invention Is directed to methods for observing the progression of a malignancy in an individual ove time. In one embodiment methods for observing the progression of a malignancy in an individual over time comprise determining the level of 24P4C1 2 mRNA or 24P4CI 2 proteln expressed by cell In a sample of the tumor, comparing the level so determined to the level of 24P4C1 2 mRNA or 24P4C12 protein expressed in en equivalent tissue sample taken from the same inlviual alta different time, wherein the degree of 24P4C12 mRNA or 24P4C1 2 protein expression In the tumor sample over tIme provides information on the progression of the cance. In a specific embodiment the progression of a cancer Is evaluated by delemdrIlng 24P4C1 2 expression In the tumor cells over time, where Increased expression over time indicates a progression of the cancer. Also, one can evaluate the Integrity 24P4C1 2 nudleotide and amino acid sequences In a biological sample in order to identify perturbations in the structure of these molecules sudh as Inusertions, deleions, substitutions and the Ike, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic end diagnostic protocols; known.in the art For example, another embodilment of the Invention Is directed to methods for observing a coincidence between the expression of 24P4C129gen and 24P4C12 gene products (or perturbations in 2415402 gene and 2415402 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a Issue sWOpl. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy PSA. PSCA and PSM expression for prostate cancer etr.) as well as gross cytologicallobservations (see, eg., Bodding etal., 1984, Anal. Wuant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Palhol. 26(2):223-9; Thorson eta)., 1998, Mod.

Pathol. 11(6):543-51: Baisdeneta., 1999,Am.J. Surg. Path.23(8):918-24). Methods for obsarviga coincidence between t1he expression of 24134C12 gene and 24P4C12 gene products (or perturbations In 24P4C12 gene and 24P4C12 gene products) andl another flactor that is associated with malignancy are useful, for exmple, because the presence of a set of specific factors Ithat coincide with diseas providles Information crucial for diagnosing and prognosticating the status of a tue sample.

00 0 In one embodiment, methods for observing a coincidence between the expression of 24P4C12 gene and 24P4C12 C gene products (or perturbations in 24P4C12 gene and 24P4C12 gene products) and another factor associated with malignancy Sentails detecting the overexpression of 24P4C12 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA Sor protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 24P4C12 mRNA or protein and PSA 00 mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 24P4C12 and PSA 0mRNA in prostate tissue is examined, where the coincidence of 24P4C12 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

00 Methods for detecting and quantifying the expression of 24P4C12 mRNA or protein are described herein, and standard N nudeic acid and protein detection and quantification technologies are well known in the ait Standard methods for the detection and quantification of 24P4C12 mRNA include in sfu hybridization using labeled 24P4C12 riboprobes, Northern blot and related 0 techniques using 24P4C12 polynudeotide probes, RT-PCR analysis using primers specific for 24P4C12, and other amplification 00 type detection methods, such as, for example, branched DNA, SISBA, TMA and the Eke. In a specific embodiment, semiquantitative RT-PCR is used to detect and quantify 24P4C12 mRNA expression. Any number of primers capable of amplifying 24P4C12 can be used for this purpose, Including but not limited to te various primer sets specifically described herein. In a specific embodiment, polydonal or monodonal anibodies spedfically reactive with the wild-type 24P4C12 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 24P4C12 The 24P4C12 protein and nudeic add sequences disdosed herein allow a skilled artisan to Identify proteins, small molecules and other agents that interact with 24P4C12, as well as pathways activated by 24P4C12 via ay one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the 'two-hybrid assay"). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, U.S. Patent Nos. 5,955,280 issued 21 September 1999, 5,925,523 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, Marcotte, et a., Nature 402 4 November 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 24P4C12 protein sequences.

In such methods, peptides that bind to 24P4C12 are identified by screening libraries that encode a random or controlled collection of amino adds. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 24P4C12 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 24P4C12 protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 Issued 31 March 1998.

Alte'matively, cell lines that express 24P4C12 are used to identify protein-protein Interactions mediated by 24P4C12. Such interactions can be examined using immunoprecipitation techniques (see, Hamilton etal.

Biochem. Biophys. Res. Commun. 1999, 261:646-51). 24P4C12 protein can be Immunopredpitated from 24P4C12expressing cell ines using anti-24P4C12 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 24P4C12 and a His-tag (vectors mentioned above). The Immunoprecpitated complex can be examined for protein association by procedures such as Westem blotting, sS-methionine labeling of proteins, protein microsequencng, slver staining and two-dimensional ge electrophoresis.

00 Small molecules and ligands that interact with 24P4C12 can be identified through related embodiments of such Sscreening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 24P4C12's ability to mediate phosphorylation and de-phosphorylation, interaction with DNAor RNA Smolecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small Smolecules that modulate 24P4C12-related ion channel, protein pump, or cell communication functions are identified and 0 0 0 used to treat patients that have a cancer that expresses 24P4C12 (see, Hille, Ionic Channels of Excitable Membranes 2d Ed., Sinauer Assoc., Sunderland, MA. 1992). Moreover, ligands that regulate 24P4C12 function can be identified based on their ability to bind 24P4C12 and activate a reporter construct Typical methods are discussed for 00 example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligands in which at least D one figand is a small molecule. In an illustrative embodiment cels engineered to express a fusion protein of 24P4C12 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library Stranscriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the

(O

Sproximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target 0sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 24P4C12.

An embodiment of this invention comprises a method of screening for a molecule that Interacts with a 24P4C12 amino add sequence shown In Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 24P4C12 amino acid sequence, allowing the population of molecules and the 24P4C12 amino acid sequence to interact under conditions that facilitate an Interaction, determining the presence of a molecule that interacts with the 24P4C12 amino add sequence, and then separating molecules that do not interact with the 24P4C12 amino acid sequence from molecules that do. In a specfic embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 24P4C12 amino acid sequence. The identified molecule can be used to modulate a function performed by 24P4C12. In a preferred embodiment, the 24P4C12 amino add sequence is contacted with a library of peptides.

X.t Therapeutic Methods and Compositions The identification of 24P4C12 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 24P4C12 functions as a transcrption factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a 24P4C12 protein are useful for patients suffering from a cancer that expresses 24P4C12. These therapeutic approaches generally fall into two classes. One class comprises various methods for Inhibiting the binding or association of a 24P4C12 protein with its binding partner or with other proteins.

Another class comprises a variety of methods for inhibiting the transcription of a 24P4C12 gene or translation of 24P4C12 mRNA.

Anti-Cancer Vaccines The invention provides cancer vaccines comprising a 24P4C12-related protein or 24P4C12-related nucleic add. In view of the expression of 24P4C12, cancer vadines prevent and/or treat 24P4C12-expresslng cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cel-mediated immune responses as anticancer therapy is wel known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et at, 1995, Int J. Cancer 63231-237; Fong e at., 1997, J. Immunol. 159:3113.3117).

00 Such methiods can be rjadity practiced by employing a 24P4C1 2-related protein, or a 24P4C1 2-encoding nucleic CK1acid molecule and recombinant vectors capable of expressing and presenting the 24P4C12 immunogen (which typicalfly comprises a number of antib~ody or T cell epitopes). Skilled artisants understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, HeryIn et Ann Med 1999 Feb 31(1):66-78; Maruyama 00 el al., Cancer Immunol lmrnunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an Immune response (e.g.

humoral and/or cell-mediated) in a mammal, comprise the steps af exposing the marnmal's immune system to an irnmunoreactive epltope an epitope present In a 24P4C12 protein shown in Figure 3 or analog or homnolog thereat) so 00 that the mammal generates an immune response that is specific for that epitope generates antibodies that specifically N recognize that epitope). In a preferred method, a 24P4C12 Inimunogen contains a biological motif, sea Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 24P4C12 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.

The entire 24P4C1 2 protein, Immunogenic re~ons; or epitopes thereof can be combined and delivered by various 00 means. Suchd vaccine compositions can include, for example, lipopeptides (e.g.,Vitiello, A. et J. Guin. Invest. 95:341, 1995), peptide compositions encapsulated In poly(DIL-laclde-co-gtycollde) microspheres (see, Eldridge, et Malec. Irnmunol. 28:287-294, 1991: Alonso et Vaccine 12-299-306,1994; Jones otfa!., Vaccine 13:675-681, 1995), peptide compositions contained in immnune stimulating complexes (ISCOMS) (see, Takahashi et al., Nature 344:873- 875, 1990; Hu et aL, Ctin Exp Inmmunol. 113:235-243,1998), multiple antigen peptide systems (MAPs) (see Tam, J. P., Proc. Nail. Acad. Sd U.S.A. 85:54D9-5413, 1985; Tam, J. lmmuno. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Porkus, M. E et at, In: Concepts in vaccine development, Kaufmann, S. H. ad., p. 379,1996; Chakrabarti, S. eta., Nature 320:535. 1986: Hu, S. L eta., Nature 320:537, 1986; ieny, et at, AIDS Bio/Technology4:790,11986; Top, F.

H. t al, J. Infect. Dis. 124:148,1971; Chanda, P. K eta., Viologyl175:35, 1990), particles of viral or synthetic origin Kofler, N. et al., J. lmmuinol. Methods. 192:25, 1996; Eldridge, J. H. ofta., Sam. Henmtol. 30:16, 1993; Falo, L. Jr. et Nature Med..7:649, 1995), adjuvants (Warren, H. Vogel, F. and Chedid, L A. Annu. Rev. Immunol. 4:369,1986; Gupta, R. K ef al, Vaccine 11:293, 1993), Uposom (Reddy, R. etat., J. Immunol 148:1585,1992; Rock, K. Immunal.

Today 17:131, 1996), or, naked or particle absorbed cONA (Ulmer, J. B. eta)., Science 259:1745,1993; Robinson, H. L, Hunt L A, and Webster, R. Vaccine 11:957,1993; Shiver, J. W. at al., In: Concepts In vaccine development, Kaufrnann, H. ed., p. 423, 1996; Cease, K and Berzofsky, J. A, Annu. Rev. Immimuol. 12.-923, 1994 and Elrldge, J. H. ot al., Sam. Hemetol. 30:16,1993). ToxIn-targeted delivery technologies, also known as receptor mnediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

In patients with 24P4C1 2-assocaad cancer, the vaccne compositions of the invention can also be used in conjunction with other treatments used for cancer, surgery, chemotherapy, drug therapies, radiation therapies, etc.

including use in comnbination with Immune adjuvants such as IL-2, IL-i1 2, GM-CSF, and the like.

Cellular Vaccines: CTIL epitopes can be determined using specific algorithms to Identify peptides within 24P4C1 2 protein that bind corresponding FLA alleles (see Table IV, Epkner 7 aid Epimabi~l', Brown University (URL brown.edulResearch/T13- HIVjabtepimratixtepirratrihlffnt); and, BIMAS, (URL blmas.datnh.govl; SYFPEITHI at URL syfpeithi.bmi-heidelbergcoml).

In a preferred embodiment a 24P4C11 2 immunogen contains one or more amino acid sequences identified using techniques well known In the art such as the sequences shown In Tables VIII-XXI and X)UI-XILIX or a peptide of 8, 9, 10 or 11I amino adds specified by an HLA Class I motif/supermotif Table IV Table IV or Table IV and/or a peptide of at least 9 amino acids that comprises an HILA Class 11 motif/supermotif Table IV (B3) or Table IV As is appreciated In the art the HIA Class I binding groove is essentially dosed ended so that peptides; of only a particular size range can fit into the groove and be bound, generally HIA Class I epitopes are 8, 9, 10, or I1I amino acids long. In contrast the HIA Class 11 00 Sbinding groove is essentially operi ended; therefore a peptide of about 9 or more amino adds can be bound by an HLA Class CN. II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., o position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, additional amino adds can be 00 attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12,13, 14,15,16,17,18, 19, 20, 21, 22, 23, 24, or 25 amino adds long, or longer than 25 amino adds.

Antibody-based Vaccines 0 A wide variety of methods for generating an immune response in a mmmal are known in the art (for example as N the first step In the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing Sthe mammal's Immune system to an immunogenic epitope on a protein a 24P4C12 protein) so that an immune 0response is generated. A typical embodiment consists of a method for generating an immune response to 24P4C12 in a 0 host by contacting the host with a sufficient amount of at least one 24P4C12 B cell or cytotoxic T-cell epitope or analog 0 thereof; and at least one periodic interval thereafter re-contacting the host with the 24P4C12 B cell or cytotoxic T-ceO epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 24P4C12related protein or a man-made multiepitopic peptide comprising: administering 24P4C12 Immunogen a 24P4C12 protein or a peptide fragment thereof, a 24P4C12 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, U.S. Patent No.

6,146,635) or a universal helper epitope such as a PADRE peptide (Epimmune Inc., San Diego, CA; see, Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., immunity 1994 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 24P4C12 Immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 24P4C12 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, U.S. Patent No.

5,962,428). Optionally a genetic vaccine facilitator such as anionic lpids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea Is also administered. In addition, an antildiotyplc antibody can be administered that mimics 24P4C12, in order to generate a response to the target antigen.

Nudeic Add Vacnes: Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient Genetic Immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 24P4C12.

Constructs comprising DNA encoding a 24P4C12-related proteinlimmunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 24P4C12 proteinlimmunogen. Alternatively, a vacine comprises a 24P4C12-related protein.

Expression of the 24P4C12-related protein Immunogen results In the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 24P4C12 protein. Various prophylactic and therapeutic genetic Immunization techniques known In the art can be used (for review, see Information and references published at Intemet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff of. al., Science 247:1465 (1990) as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNAbased delivery technologies include "naked DNA', facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ('gene guni) or pressure-mediated delivery (see, U.S. Patent No. 5,922,687).

00 SFor therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the pracce of the invention include, but are not limited p to, vaccnia, fowIpox, canarypox, adenovirus, influenza, pollovirus, adeno-assodated virus, lentivirus, and slndbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et a. J. Natl. Cancer Inst 87982-990 (1995)). Non-viral delivery systems 00 can also be employed by Introducing naked DNA encoding a 24P4C12-related protein into the patient Intramuscularly or

O

0 intradermally) to induce an ant-tumor response.

Vaccinia virus is used, for example, as a vector to express nudeotide sequences that encode the peptides of the 00 invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and N thereby elicits a host immune response. Vacdnia vectors and methods useful in Immunization protocols are described in, Se.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et Sal., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the 00 peptides of the invention, e.g. adeno and adeno-assocated virus vectors, relroviral vectors, Salmonella typhi vectors, Sdetoxified anthrax toxin vectors, and the like, will be apparent to those skilled In the art from the description herein.

Thus, gene delivery systems are used to deliver a 24P4C12-related nucleic acid molecule. In one embodiment the fulllength human 24P4C12 cDNA is employed. In another embodiment, 24P4C12 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or anxbody epitopes are employed.

Ex Vivo Vaccines Various ex vivo strategies can also be employed to generate an immune response. One approach Involves the use of antigen presenting cells (APCs) such as dendritic calls (DC) to present 24P4C12 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly spedalized antigen presenting cells.

In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I dinical trial to stimulate prostate cancer patients' Immune systems (Tjoa et 1996, Prostate 28:65- 69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 24P4C12 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 24P4C12 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 24P4C12 protein. Yet another embodiment involves engineering the overexpression of a 24P4C12 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-assodated virus, DNA transfection (Ribas et el., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp.

Med. 186:1177-1182). Cells that express 24P4C12 can also be engineered to express immune modulators, such as GM- CSF, and used as immunizing agents.

24P4C12 as a Tarmet for Antibody-based Therapy 24P4C12 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, complement and ADCC mediated killing as well as the use of intrabodies). Because 24P4C12 is expressed by cancer cells of various lineages relative to corresponding normal cels, systemic administration of 24P4C12-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies spedfically reactive with domains of 24P4C12 are useful to treat 24P4C12-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

00 24P4C12 antibodies can be introduced into a patient such that the antibody binds to 24P4C12 and modulates a Sfunction, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells andlor inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of

F

24P4C12, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of 0 tumor angiogenesis factor profiles, andlor apoptosis.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 24P4C12 sequence shown in Figure 2 or Figure 3. In addition, skilled 00 artisans understand that It Is routine to conjugate antibodies to cytotoxic agents (see, Slevers at at. Blood 93:11 3678- IO 3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antbodies specific for a molecule expressed by that cell 24P4C12), the cytotoxic agent will exert its known biological C effect cytotoxicity) on those cells.

00 A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in 0the art In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent an anti- 24P4C12 antibody) that binds to a marker 24P4C12) expressed, accessible to binding or localized on the cell surfaces.

A typical embodiment is a method of delivering a cytotoxic andlor therapeutic agent to a cell expressing 24P4C12.

comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 24P4C12 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenteraly to said individual a pharmaceutical composition comprising a therapeuticaly effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent Cancer immunotherapy using anti-24P4C12 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Men et at., 1998, Crit Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et aL, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et aL, 1994, Cancer Res. 54:6160-6166; Velders at aL, 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clln. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y9t or l 1 3 to ant-CD20 antibodies Zevalin IDEC Pharmaceuticals Corp. or Bexxar

M

Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptinm (trastuzumab) with paditaxel (Genentech. Inc.). The antibodies can be conjugated to a therapeutic agent To treat prostate cancer, for example, 24P4C12 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin Mylotarg"

M

Wyeth-Ayerst. Madison, NJ. a recombinant humanized lgG4 kappa antibody conjugated to antitumor antibiotic caicheamicin) or a maytansinoid taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see US Patent 5,416,064).

Although 24P4C12 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not 00 Stolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al.

(International J. of Ono. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

L) Although 24P4C12 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly Sappropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the Invention is Indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not 00 tolerate the toxidty of the chemotherapeuic agent very well.

s Cancer patients can be evaluated for the presence and level of 24P4C12 expression, preferably using immunohlstochemical assessments of tumor tissue, quantitative 24P4C12 imaging, or other techniques that reliably indicate rthe presence and degree of 24P4C12 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is Spreferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

0 Anti-24P4C12 monodonal antibodies that treat prostate and other cancers indude those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-24P4C12 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxidty (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-24P4C12 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 24P4C12. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis.

The mechanism(s) by which a particular anti-24P4C12 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monodonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from drculation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of Immune complexes which, potentially, can cause renal failure. Accordingly, preferred monodonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specfically to the target 24P4C12 antigen with high affinity but exhibit low or no antigenidty in the patient Therapeutic methods of the invention contemplate the administration of single anti-24P4C12 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti- 24P4C12 mAbs can be admipistered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators IL-2, GM-CSF), surgery or radiation. The anti- 24P4C12 mAbs are administered in their "naked' or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-24P4C12 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradennal, and the like. Treatment generally involves repeated administration of the anti-24P4C12 antibody preparation, via an acceptable route of administration such as intravenous Injection typically at a dose in the range of about 0.1, .2, 00 0 1,2, 3,4, 5,6, 7, 8, 9, 10, 15,20, or 25mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

o Based on clinical experience with the Herceptinm mAb in the treatment of metastatic breast cancer, an Initial r) loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 24P4C12 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose Is administered as 00 a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art various factors can influence the ideal dose 0 regimen in a particular case. Such factors Indude, for example, the binding affinity and half life of the Ab or mAbs used, the NK degree of 24P4C12 expression in the patient, the extent of circulating shed 24P4C12 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient SOptionally, patients should be evaluated for the levels of 24P4C12 in a given sample the levels of circulating 00 24P4C12 antigen and/or 24P4C12 expressing cells) in order to assist In the determination of the most effective dosing Sregimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

SAnt-idiotypic anti-24P4C12 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 24P4C12-related protein. In particular, the generation of anti-idiotyplc antibodies Is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-24P4C12 antibodies that mimic an epitope on a 24P4C12-4elated protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et 1995, J.

Clin. Invest 96:334-342; Hertyn et ef., 1996, Cancer Immunol. Immunother. 43.65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

XC.) 24P4C12 as a Target for Cellular Immune Responses Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the Invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the daimed peptides. A peptide can be present in a vacdne individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an Immune response. The composition can be a naturaly occurring region of an antigen or can be prepared, recombinantly or by chemical synthesis.

Carriers thad can be used with vaccines of the invention are well known in the art, and include, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino adds such as poly L-lysine, poly L-glutamlc acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant.

Adjuvants such as Incomplete Freunds adjuvant aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the Invention to lipids, such as tipalmitoyl-Sgycerycysteinlysery- serine (PsCSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanlnecontaining (CpG) oligonudeotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000)) 00 Upon immunization with a peptide composition in accordance with the invention, via Injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cels that express or overexpress 24P4C12 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

O In some embodiments, i may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative 00 embodiment of such a composition comprises a dass I and/or class II epitope in accordance with the invention, along with a NO cross reactive HTL epitope such as PADRE m (Epimmune, San Diego, CA) molecule (described in U.S. Patent Number 5,736,142).

N A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells as a 00 Svehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell 0mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vacdne compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs In vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic adds such as a minigene. It is preferred that each of the following princples be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence In the native antigen from which the epitopes are derived.

Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this indudes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2) Epltopes are selected that have the requisite binding affinity established to be correlated with immunogenicity. for HLA Class I an ICso of 500 nM or less, often 200 nM or less; and for Class II an ICa of 1000 nM or less.

Sufficient supermotif bearing-peptides, or a sufficient array of alele-spedfic motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

When selecting epitopes from cancer-related antigens it Is often useful to select analogs because the patient may have developed tolerance to the native epitope.

Of particular relevance are epitopes referred to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providng a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a mult-epitopic sequence, 00 such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it cdoes not have pathological or other deleterious biological properties.

0 If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino add residues can, for example, be introduced to avoid junctional epitopes (an epilope recognized by the immune system, not 0 present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate deavage between N epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient O may generate an immune response to that non-native epitope. Of particular concem is a junctional epitope that is a "dominant epltope.' A dominant epitope may lead to such a zealous response that immune responses to other epitopes are Sdiminished or suppressed.

00 Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA dass I binding peptide or the entire 9-mer core of a dass II binding peptide be conserved In a designated percentage of the sequences evaluated for a specific protein antigen.

XC.1. Minigene Vaccines A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic adds encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for Inclusion in a minigene are preferably selected according to the guidelines set forth In the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka ef aL, J. Immunol. 162:3915-3925,1999; An, L. and Whitton, J. L, J. Wrol. 71:2292, 1997; Thomson, S. A. et J. Immunol. 157:822, 1996; Whitton, J. L et J. Virl.

67:348,1993; Hanke, R. et al, Vaccine 16:426,1998. For example, a multi-epitope DNA plasmid encoding supermotifand/or motif-bearing epitopes derived 24P4C12, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 24P4C12 (see Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-lranslocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The Immunogenidty of a multi-epltopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vdvo can be correlated with the In vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: generate a CTL response and that the Induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino add sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be Incorporated into the minigene design. Examples of amino add sequences that can be reverse translated and induded in the minigene sequence include: HLA class I epitopes, HLA dass II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmlc reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

00 SThe minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus C strands of the minigene. Overapping oligonudeotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonuceotides can be joined, for r example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired 00 expression vector.

0 Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream doning site for 00 minigene insertion; a polyadenylation signal for efficient transcription termination; an E coli origin of replication; and an E.

N coli selectable marker ampicillin or kanamycn resistance). Numerous promoters can be used for this purpose, the Shuman cytomegalovirus (hCMV) promoter. See, U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter 0sequences.

00 Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring Introns could be Incorporated Into the transcribed region of the minigene. The indusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques.

The orientation and DNA sequence of the minigene, as well as all other elements induded in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenidty of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance Immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed indude cytokines IL-2, IL-12, GM- CSF), cytokine-indudng molecules LelF), costimulatory molecules, or for HTL responses, pan-DR binding proteins

(PADRE

T M Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA dass II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules TGF-) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. col, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation In shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bloseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution oflyophilized DNA In sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA," is currently being used for intramuscular (IM) administration in dinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic ripids, glycolipids, and fusogenic liposomes can 00 also be used in the formulation (see, as described by WO 93/24640; Mannino Gould-Fogerite, BioTechniques 6(7): O 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et al., Proc. Nat'lAcad Sci. USA 84:7413(1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

0 Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final

O

00 formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A O plasmid expressing green fluorescent protein (GFP) can be co-trensfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (sCr) labeled and used as target cells for N epitope-specfic CTL lines; cytolysis, detected by s"Cr release, indicates both production of, and HLA presentation of, 00 0 minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to Sassess HTL activity.

In vive immunogenicty is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product The dose and route of administration are formulation dependent IM for DNA In PBS, Intraperitoneal for lipid-complexed DNA). Twenty-one days after Immunization, splenocytes are harvested and restimulated for one week In the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 5 sCr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epilopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in viv induction of CTLs.

Immunogenidty of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic adds can be administered using ballistic delivery as described, for Instance, In U.S.

Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, an expression construct encoding epttopes of the invention can be incorporated into a viral vector such as vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides Vaccine compositions comprising CTL peptides of the invention can be modified, analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenidty.

For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, Ala, Gly, or other neutral spacers of nonpolar amino adds or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

0 genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class If molecules. Examples of such amino acid bind many HIA Class 11 molecules include sequences from antigens such as tetanus toxold at positions 83"83 (QYIKANSKFIGITE; SEO ID NO: 29), PlaSModium talciparUrn crcumsporOZOite (CS) protein at positions 378-398 (DIEKKIAI<MEKASSVFNV'NS; SEQ ID NO: 30), and Straptococcus l8kD protein at positions 00 116-131 (GAVDSILGGVATYGMA; SEQ ID NO: 31). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, It Is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely 00 HLA-restricted fashion, using amino acid sequences not found in nature (see, PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes PADRE70, Epimmune, Inc., San Diego, CA) are designed, most preferably, to bind most lILA-DR (human HI-A d=s 11) molecules. For instance, a pan-DR-binding epitope peptide having cK1 the formula: AKXVMAWTLKAMA (SEQ ID NO: 32), where W' is either cyclohexyalanine, phenylalanine, or tyrosine, and a 00 is either D-alanine or L-alanlne, has been found to bind to most H LA-DR alleles, and to, stimulate the response of T helper lymphocytes from most individuals, regardless of their HIA type. An alternative of a pan-DR binding epitope comprises all natural amino acids and can be provided in te form of nucleic acids that encode the epitope.

HTL peptide epitopes; can also be modified to alter their biologicall properties. For example, they can be modified to Include D-amino acids to increase their resistance to proteases and thus extend their serum hagf ife, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to Increase their biological activity. For example, a T helper peptide can be conjugated to one or more palrrutic acid chains at either the amino or carboxyt termini.

X.C.3 Combinations of CTIL Peptldes with T Cell Priming Agents In some embodiments it may be desirable to include In the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Upids have been identified as agents capable of priming Cr1L i viva. For example, palmitic adid residues can be attached to the E-and ct- amino groups of a lysine residue and then linked, via one or more linking residues such as Gly, Gly-Glyr-, Ser, Ser-Ser, or the rike, to an immunogenic peptide.

The lip4dated peptide can then be administered either directly In a micelle or partcle, incorporated Into a liposome, or emulsified in an a~uvant, incomplete Freund's adjuvant In a preferred embodiment, a particularly effective Immunogenic composition comprises patitic acid attached toec- and oL- amino groups of Lys, which is attached via linkage, Ser-Ser. to the amino tenminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteis, such as tripalmitoyl-Sglycurycysteinlysery- serine (P3CSS) can be used to primne vlrus specific CTL Mhen covalently attached to an appropriate peptide (wee, Dares, ef al., Nature 342:561, 1989). Peptides of the invention can be coupled 1615aCS, for example, and the lipopeptidle administered to an Individual to prime specifically an Immune response to the target antigen. Moreover, because the Induction of neutralizing antibodies can also be primed with PsCSS-conpigatedl epitopes, two such compositions can be combined to mnore effectively elicit both humoral and cellmediated responses.

XC.4. Vaccine Composions Comprising DC PulsedwIth CTL andlor lITL Peptides An embodiment of a vaccine composition In accordance with the invention comprises ex vivo administration of a cocktal of epitope-bearing peptides to PBMC, or Isolated DC therefrom, from the patients blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietinm (Pharmnacia-Monsanto, St. Louis, MO) or GM-CSFIIL-4.

After putslng the DC with peplides and prior to reinfusion Into patients, the DC are washed to remove unbound peptides. In this embodiment a vaccine comprises peptide-pulsed DCs which present the pulsed peptide elpitlopes; coniptexed with HI-A molecules on their surfaces.

0 The DC can be pulsed ex vive with a cocktail of peptides, some of which stimulate CTL responses to 24P4C12.

Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be C included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which Sexpresses or overexpresses 24P4C12.

00 X.D. Adoptive Immunotherapy Antigenic 24P4C12-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will 00 not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTLor HTL N responses to a particular antigen are induced by incubating in tissue culture the patients, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are 00 activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell a tumor cel). Transfected dendritic cells may also be used as C antigen presenting cells.

X.E. Administration of Vaccnes for Therapeutic or Prophvlactic Purposes Pharmaceutical and vaccine compositions of the Invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 24P4C12. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as 'therapeutically effective dose.' Amounts effective for this use win depend on, the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 24P4C12. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 24P4C12-assodated cancer.

This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, mlnigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patients health status. For example, in a patient with a tumor that expresses 24P4C12, a vaccine comprising 24P4C12-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than altemative embodiments.

It Is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the Invention.

The dosage for an Initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient Boosting dosages of between about pg to about 50,000 pg of peptide pursuant to a boosting regimen over weeks to months may be administered depending O0 0 upon the patients response and condition as determined by measuring the specific activity of CTL and HTL obtained from 0the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasla, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known In the art SIn certain embodiments, the peptides and compositions of the present invention are employed In serious disease 00 states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the Invention, It is possible and may be fell desirable by the treating physician to administer substantial excesses of these peptide compositions 00 relative to these stated dosage amounts.

N The vaccine compositions of the Invention can also be used purely as prophylactic agents. Generally the dosage Sfor an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typically range 00 from about 500 pg to about 50,000 pg per 70 klogram patient This is followed by boosting dosages of between about pg to about 50,000 pg of peptide administered at defined intervals from about four weeks to six months after the initial rK administration of vaccine. The immunogencity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patients blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, Intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrer, preferably an aqueous carrier.

A variety of aqueous carriers may be used, water, buffered water, 0.8% saline, 0.3% glyine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophllized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonidty adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine deate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, from less than about usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included In a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volumelquantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, Remington's Pharmaceutical Sciences, 170 Edition, A Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial Immunization can be from about 1 to about 50,000 pg. generaly 100-5,000 pg, for a 70 kg patent For example, for nucleic adds an initial immunization may be performed using an expression vector in the form of naked nucleic add administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nuleic acid (0.1 to 1000 pg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowipox virus administered at adose of 5-10 7 to 5x10 s pfu.

I

00 For antibodies, a treatment generally involves repeated administration of the anti-24P4C12 antibody preparation, Svia an acceptable route of administration such as intravenous injection typically at a dose in the range of about 0.1 to 03 about 10 mg/kg body weight In general, doses in the range of 10-500 mg mAb per week are effective and wel tolerated.

d) Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 24P4C12 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill 00 in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the Immunogenldty of a substance, the degree of 24P4C12 expression in the patient, the extent of circulating shed 24P4C12 antigen, the desired steady-state concentration level, frequency of treatment,

OO

CN and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as NO well as the health status of a particular patient Non-limiting preferred human unit doses are, for example, 500pg 1mg, 1mg 50mg, 50mg 100mg, 100mg 200mg, 200mg 300mg, 400mg 500mg, 500mg 600mg, 600mg 700mg, 700mg 800mg, 800mg 900mg, 900mg lg, or 1mg 700mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body 00 0weight, with follow on weekly doses of 1-3 mglkg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/kg body weight followed, in O two, three or four weeks by weekly doses; 0.5 -10mg/kg body weight, followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400mg m 2 of body area weekly, 1-600mg m 2 of body area weekly, 225-400mg m 2 of body area weekly these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11,12 or more weeks.

In one embodiment, human unit dose forms of polynudeotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynudeotide, molecular weight of the polynudeotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynudeotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70,80, 90,100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit greater than the lower limit, of about 60, 80, 100, 200, 300,400, 500, 750, 1000, 1500, 2000,3000, 4000,5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mglkg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg. 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of adminlstration may require higher doses of polynudeotide compared to more direct applicaton to the nudeotide to diseased tissue, as do polynucdeotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that, provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 10 to about 5 x 1010 cells.

A dose may also about 108 cells/m 2 to about 1010 cells/m 2 or about 106 cells/m 2 to about 108 cellsmh 2 Proteins(s) of the invention, and/or nudeic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphold tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Lposomes indude emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monodonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peplide of the invention 00 0 can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use Sin accordance with the invention are formed from standard veside-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by 0U consideration of, liposome size, acid lability and stability of the Ilposomes In the blood stream. A variety of methods are 0 available for preparing liposomes, as described in, Szoka, el Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S.

00 Patent Nos. 4,235,871, 4,501,728,4,837,028, and 5,019,369.

For targeting cells of the immune system, a figand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome N suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according IN to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid cariers may be used which include, for example, Spharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is Sformed by incorporating any of the normally employed exdpients, such as those carriers previously listed, and generally of active ingredient, that Is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peplides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01 by weight, preferably about The surfactant must, of course, be nontoxic, and preferably soluble in the propellant Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant A carrier can also be included, as desired, as with, lecithin for intranasal delivery.

XI.) Diagnostic and Proqnostic Embodiments of 24P4C12.

As disclosed herein, 24P4C12 polynudeotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HT1) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and thernpeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, both its spedfic pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled 'Expression analysis of 24P4C12 In normal tissues, and patient specimens').

24P4C12 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, Merrill et J. Urol. 163(2): 503-5120 (2000); Polascik et J. Urol. Aug; 162(2)293-306 (1999) and Fortier et el, J. Nat Cancer Inst 91(19): 1635- 1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchlnsky at at., Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto ela/., Cancer Detect Prey 2000;24(1):1-12). Therefore, this disdosure of 24P4C12 polynudeotides and polypeptides (as well as 24P4C12 polynudeotide probes and anti-24P4C12 antibodies used to Identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic essays directed to examining conditions associated with cancer.

Typical embodiments of dagnostic methods which utilize the 24P4C12 polynudeotides, polypeplides, reactive T cells and antibodies are analogous to those methods from wellestablished diagnostic assays, which employ, e.g, PSA polynudeotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynudeotides are used as probes 00 (for example in Northern analysis; see, Sharief e al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the Slevel of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 24P4C12 polynucleotides described herein can be utilized in the same way to detect 24P4C12 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies 00 specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, Alanen et Pathol. Res. Pract 192(3):233-7 (1996)), the 24P4C12 polypeptides described herein can be utilized to 00 N- generate antibodies for use in detecting 24P4C12 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung 0 or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for 00 0the presence of cells expressing 24P4C12 polynucleotides and/or polypeptides can be used to provide evidence of Smetastasis. For example, when a biological sample from tissue that does not normally contain 24P4C12-expressing cells (lymph node) is found to contain 24P4C12-expressing cells such as the 24P4C12 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Altematively 24P4C12 polynucleotides and/or polypeplides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 24P4C12 or express 24P4C12 at a different level are found to express 24P4C12 or have an increased expression of 24P4C12 (see, the 24P4C12 expression In the cancers listed in Table I and in patient samples etc. shown In the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (In addition to 24P4C12) such as PSA, PSCA etc. (see, Alanen et al., Pathol. Res. Pract 192(3): 233- 237 (1996)).

Just as PSA polynudeotide fragments and polynudeotide variants are employed by skilled artisans for use in methods of monitoring PSA. 24P4C12 polynudeotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleoides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynudeotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynudeotide fragments that can be used as primers in order to amplify different portions of a polynuceotide of interest or to optimize amplification reactions (see, Caetano-Anolles, G.

Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled "Expression analysis of 24P4C12 In normal tissues, and patient spedmens,' where a 24P4C12 polynudeotide fragment is used as a probe to show the expression of 24P4C12 RNAs in cancer cells. In addition, variant polynudeotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, Sawai et Fetal Dlagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).

Polynudeotide fragments and variants are useful in this context where they are capable of binding to a target polynudeotide sequence a 24P4C12 polynudeotide shown in Figure 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specificaly binds to that epitope are used in methods of monitoring PSA. 24P4C12 polypeptlde fragments and potypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems 0 such as fusion proteins being used by practitioners (see, Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel ef al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an C antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, U.S. Patent No.

S5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 00 24P4C 12 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skil in the art based on motifs available in the art Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence a 00 24P4C12 polypeptide shown In Figure 3).

N As shown herein, the 24P4C12 polynudeotides and polypeptides (as well as the 24P4C12 polynudeotide probes 0 and anti-24P4C12 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful In diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 00 24P4C12 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so C1 successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 24P4C12 polynucleotides and polypeptides (as well as the 24P4C12 polynucleotide probes and anti- 24P4C12 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 24P4C12 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 24P4C12 gene maps (see the Example entitled 'Chromosomal Mapping of 24P4C12" below). Moreover, in addition to their use in diagnostic assays, the 24P4C12-related proteins and polynudeotides disclosed herein have other utilities such as their use In the forensic analysis of tissues of unknown origin (see, Takahama K Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).

Additionally, 24P4C12-related proteins or polynudeoides of the invention can be used to treat a pathologic condition characterized by the over-expression of 24P4C12. For example, the amino add or nudeic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to a 24P4C12 antigen. Antibodies or other molecules that react with 24P4C12 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefil )XI.1 Inhibition of 24P4C12 Protein Function The invention includes various methods and compositions for inhibiting the binding of 24P4C12 to its binding partner or its association with other protein(s) as well as methods for inhibiting 24P4C12 function.

XIIA.) Inhibition of 24P4C12 With Intracellular Antibodies In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 24P4C12 are introduced into 24P4C12 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti- 24P4C12 antibody is expressed Intracellulary, binds to 24P4C12 protein, and thereby Inhibits its function. Methods for engineering such intracllular single chain antibodies are well known. Such intracellular antibodies, also known as 'intrabodies', are spedfically targeted to a particular compartment within the cell, providing control over where the inhibitory 00 Sactivity of the treatment is focused. This technology has been successfully applied In the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant 0 cell surface receptors (see, Richardson et al, 1995, Proc. Natl. Acad. Sd. USA 92: 3137-3141; Beerli el al., 1994. J.

SBiol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy and light chain Joined by a flexible linker po pdypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known Intracellular trafficking signals are engineered into recombinant polynudeotide vectors encoding such single chain antibodies in order to target precisely the intrabody to 00 Sthe desired intracellular compartment. For example, Intrabodies targeted to the endoplasmic reticulum (ER) are engineered Sto Incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif.

Intrabodies intended to exert activity in the nucleus are engineered to indude a nuclear localization signal. Upid moieties are Sjoined to intrabodies In order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, Sthereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 24P4C12 in the nucleus, thereby preventing its activity within the nudeus. Nuclear targeting signals are engineered into such 24P4C12 intrabodies in order to achieve the desired targeting. Such 24P4C12 intrabodies are designed to bind specifically to a particular 24P4C12 domain. In another embodiment, cytosolic intrabodles that specifically bind to a 24P4C12 protein are used to prevent 24P4C12 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nudeus preventing 24P4C12 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Patent No. 5,919,652 issued 6 July 1999).

XII.B.) Inhibition of 24P4C12 with Recombinant Proteins In another approach,'recombinant molecules bind to 24P4C12 and thereby Inhibit 24P4C12 function. For example, these recombinant molecules prevent or inhibit 24P4C12 from accessingbinding to its binding partner(s) or assodating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 24P4C12 specific antibody molecule. In a particular embodiment, the 24P4C12 binding domain of a 24P4C12 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 24P4C12 ligand binding domains inked to the Fc portion of a human IgG, such as human IgGI. Such IgG portion can contain, for example, the CH2 and C3 domains and the hinge region, but not the C1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 24P4C12, whereby the dimeric fusion protein specifically binds to 24P4C12 and blocks 24P4C12 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of 24P4C12 Transcription or Translation The present invention also comprises various methods and compositions for inhibiting the transcription of the 24P4C12 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 24P4C12 mRNA Into protein.

00 0 In one approach, a method of inhibiting the transcription of the 24P4C12 gene comprises contacting the 24P4C12 gene with a 24P4C12 antisense polynudeotide. In another approach, a method of inhibiting 24P4C12 mRNA translation comprises contacting a 24P4C12 mRNA with an antisense polynucleotide. In another approach, a 24P4C12 specific Sribozyme is used to deave a 24P4C12 message, thereby inhibiting translation. Such antisense and rbozyme based 0 methods can also be directed to the regulatory regions of the 24P4C12 gene, such as 24P4C12 promoter and/or enhancer 0 elements. Similarly, proteins capable of inhibiting a 24P4C12 gene transcription factor are used to inhibit 24P4C12 mRNA transcription. The various polynudeotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is wel known in the art N Other factors that inhibit the transcription of 24P4C12 by interfering with 24P4C12 transcriptional activation are Salso useful to treat cancers expressing 24P4C12. Similary, factors that interfere with 24P4C12 processing are useful to treat 0cancers that express 24P4C12. Cancer treatment methods utilizing such factors are also within the scope of the invention.

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00 0 XII.D.) General Considerations for Therapeutic Strategies Gene transfer and gene therapy technologies can be used to deliver therapeutic polynudeotide molecules to tumor cells synthesizing 24P4C12 antisense, ribozyme, polynudeotides encoding Intrabodes and other 24P4C12 Inhibitory molecules).

A number of gene therapy approaches are known in the art Recombinant vectors encoding 24P4C12 anfsense polynudeotides, ribozymes, factors capable of interfering with 24P4C12 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vtro and in vivo assay systems. In vitro assays that evaluate therapeutic activity indude cell growth assays, soft agar assays and other assays hdicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will Inhibit the binding of 24P4C12 to a binding partner, etc.

In vivo, the effect of a 24P4C12therapeutic composition can be evaluated in a suitable animal model. For example, xenogenlc prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Mein et 1997, Nature Medicine 3:402-408). For example, PCT Patent Application W098/16628 and U.S. Patent 6,107.540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vim assays that evaluate the promotion of apoptosis are useful In evaluating therapeutic compositions. In one embodiment xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers Indude any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard 00 0 pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally.

SRemington's Pharmaceutical Sciences 16# Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic D composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, Sparenteral, Intraperttoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred 0 0 formulation for intravenous injection comprises the therapeutic composition In a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under 00 vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or In sterile Swater prior to injection.

a Dosages and administration protocols for the treatment of cancers using the foregoing methods wil vary with the C method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

00 0 XIII.1 Identification. Characterization and Use of Modulators of 24P4C12 Methods to Identify and Use Modulators in one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having Identified differentially expressed genes important in a particular state; screens are performed to dentify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to Identify modulators that alter a biological function of the expression product of a differentialy expressed gene.

Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product In addition, screens are done for genes that are induced In response to a candidate agent After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to Identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed In normal tissue or cancer tissue, but are expressed In agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agenttreated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.

Modulator-related Identification and Screening Assays: Gene Expression-related Assays Proteins, nucleic acids, and antibodies of the Invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cels containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a 'gene expression profile," expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent Davis, GF, etal, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986- 94,1996).

r SThe cancer proteins, antibodies, nucleic acds, modified proteins and cells containing the native or modified cancer 0 proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This Is done on a gene itself or by evaluating the effect of drug candidates on a "gene expression profile" or biological function. In Sone embodiment expression profiles are used, preferably in conjunction with high throughput screening techniques to allow OO monitoring after treatment with a candidate agent, see Ziokamik, supra.

A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds 00 are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. "Modulation" in IN this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the Soriginal change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably more preferably 100-300%. and in some embodiments 300-1000% or greater. Thus, If a gene exhibits a 4-fold 00 increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound Sis often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, as an upregulated target in further analyses.

The amount of gene expression is monitored using nucleic add probes and the quantification of gene expression levels, or, altematively, a gene product itself is monitored, through the use of antibodies to the cancer protein and standard Immunoassays. Proteomics and separation techniques also alow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression In one embodiment, gene expression monitoring, an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of Figure 2. In this embodiment cancer nucleic add probes are attached to blochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.

Expression monitoring Is performed to identify compounds that modify the expression of one or more cancerassociated sequences, a polynuceotide sequence set out In Figure 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or Interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.

In one embodiment high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries' are then screened in one or more assays to identify those library members (particular chemical species or subcasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional lead compounds," as compounds for screening, or as therapeutics.

In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptlde or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound') with some desirable property or activity, inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

00 0 As noted above, gene expression monitoring is conveniently used to test candidate modulators protein, nuceic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the 0 sample containing a target sequence to be analyzed is, added to a blochlp.

SIf required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification andlor amplification such as PCR performed as 0 appropriate. For example, an in vitro transcription with labels covalently attached to the nudeotides is performed. Generally, the nucleic adds are labeled with biotin-FITC or PE, or with cy3 or The target sequence can be labeled with, a fluorescent a chemiluminescent, a chemical, or a radioactive Ssignal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that Sbinds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or 00 biotin which specifically binds to streptavidin. For the example of biotin, the streplavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise 'sandwich assays', which Include the use of multiple probes, as is generally outlined in U.S. Patent Nos. 5, 681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697. In this embodiment, in general, the target nucleic add is prepared as outined above, and then added to the biochip comprising a plurality of nucleic add probes, under conditions that allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention, Including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Patent No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, In different orders, with preferred embodiments outlined below. In addition, the reaction may Include a variety of other reagents. These indude salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease Inhibitors, nudease Inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target The assay data are analyzed to determine the expression levels of Individual genes, and changes in expression levels as between states, forming a gene expression profile.

Biological Activity-related Assays The invention provides methods identify or screen fora compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a ca comprising a cancer protein of the Invention. The cells contain a recombinant nucleic add that encodes a cancer protein of the invention.

In another embodiment, a library of candidate agents is tested on a plurality of cells.

00 O In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of Sphysiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, Spharmacological agents including chemotherapeutics, radiation, carcnogenics, or other cells cell-cell contacts). In another example, the determinations are made at different stages of the cell cyde process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or 00 Interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to Identify critical structural features of the compound.

In one embodiment, a method of modulating Inhibiting) cancer cell division is provided; the method 00 Scomprises administration of a cancer modulator. In another embodiment a method of modulating inhibiting) cancer is I provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.

SIn one embodiment a method for modulating the status of a cell that expresses a gene of the invention Is provided.

As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, Ssenescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.

High Throughput Screening to Identify Modulators The assays to Identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or Inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and Inhibition or enhancement of polypeptide activity.

In one embodiment, modulators evaluated In high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being espedaly preferred. Particulady useful test compound will be directed to the dass of proteins to which the target belongs, substrates for enzymes, or ligands and receptors.

Use of Soft Aaar Growth and Colony Formation to Identify and Characterize Modulators Normal cells require a solid substrate to attach and grow. When cels are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assays are described in Freshnay, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev at al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density LUmitation to Identify and Characterize Modulators Normal cells typically grow in a flat and organized pattern In cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities In disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in 00 foci. Alternatively, labeling index with (H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect Ssame. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a Snormal phenotype and become contact inhibited and would grow to a lower density.

In this assay, labeling index with 3 H)-thymidine at saturation density is a preferred method of measuring density 00 limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours Sat saturation density in non-limiting medium conditions. The percentage of cells labeling with 3 )-thymidine is determined by incorporated cpm.

00 Contact Independent growth is used to identify modulators of cancer sequences, which had led to abnormal N cellular prolferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the Scells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators 00 Transformed cells have lower serum dependence than their normal counterparts (see, Temin. J. Natl. Cancer Inst 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of CN various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.

Use of Tumor-specific Marker Levels to Identify and Characterize Modulators Tumor cells release an increased amount of certain factors (hereinafter "tumor specific markers') than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses.in Cancer, pp. 178-184 (Mihich 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, Folkman, Anglogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Bio. Chem. 249:42954305 (1974); Strickland Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich 1985); Freshney, Anticancer Res. 5:111-130 (1985).

For example, tumor specific marker levels are monitored in methods to Identify and characterize compounds that modulate cancer-associated sequences of the invention.

Invasiveness Into Matrioel to Identify and Characterize Modulators The degree of invasiveness into Matbigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used.

Briefly, the level of invasion of host cels is measured by using filters coated with Matrigel or some other extracellular matrix constituent Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 12sl and counting the radioactivity on the distal side of the filter or bottom of the dish. See, Freshney (1984), supra.

Evaluation of Tumor Growth In Viv to Identify and Characterize Modulators 00 O Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms.

CTransgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., Smammals such as mice, are made, In which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out Stransgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in 00 the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer 0gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, by exposure to carcinogens.

00 To prepare transgenic chimeric animals, mice, a DNA construct is introduced into the nuclei of embryonic C stem cells. Cells containing the newly engineered genetic lesion are injected Into a host mouse embryo, which Is reimplanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice 00 .containing the introduced genetic lesion (see, Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived 0according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et al., SManipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocardnomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, (1987).

Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic 'nude" mouse (see, Glovanella et al, J. Natl. Cancer Inst 52:921 (1974)), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J.

Cancer 41:52 (1980)) can be used as a host Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically.

Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Students T test) are said to have inhibited growth.

In Vitro Assays to Identify and Characterize Modulators Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA.

The level of protein is measured using immunoassays such as Westem blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, using PCR, LCR, or hybridization assays, e. Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, fluorescently or radioactively labeled nuclec acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.

Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as ludferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell.

After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis GF, supra; Gonzalez, J. Negulescu, P. Curt.

Opin. BiotechnoL 1998: 9:624).

00 0 As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a K particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the f gene or the gene product itself is performed.

P) In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the 0 expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.

(N

IN Binding Assays to Identify and Characterize Modulators SIn binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally 0 used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the 0amount and/or location of protein. Altematively, cels comprising the cancer proteins are used in the assays.

SThus, the methods comprise combining a cancer protein of the Invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.

Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support The support can, be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.

Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon

M

etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as It is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not stericaly block either the Ilgand binding site or activation sequence when attaching the protein to the support, direct binding to "sticky or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or Ilgand/blnding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.

Once a cancer protein of the Invention is bound to the support, and a test compound is added to the assay.

SAlternatively, the candidate binding agent Is bound to the support and the cancer protein of the Invention is then added.

Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.

Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

00 A determination of binding of the lest compound (ligand, binding agent, modulator, etc.) to a cancer protein of the Sinvention can be done in a number of ways. The test compound can be labeled, and binding determined directly, by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound L) a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various Sblocking and washing steps can be utilized as appropriate.

00 In certain embodiments, only one of the components Is labeled, a protein of the invention or ligands labeled.

Alternatively, more than one component is labeled with different labels, 11 2 5 for the proteins and a fluorophor for the compound. Proximity reagents, quenching or energy transfer reagents are also useful.

00 IO Compeitive Binding to Identify and Characterize Modulators In one embodiment, the binding of the "test compound' is determined by competitive binding assay with a C1 "competitor." The competitor is a binding moiety that binds to the target molecule a cancer protein of the invention).

00 SCompetitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, Sthe competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 400C.

Incubation periods are typically optimized, to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound Is labeled, the presence of the label on the support indicates displacement In an altemative embodiment the test compound is added first with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.

Accordingly, the competitive binding methods comprise differential screening to Identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples Indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is Scapable of binding to the cancer protein.

Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used In rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins.

Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce theactivity of such proteins.

00 Positive controls and negative controls can be used in the assays. Preferably control and test samples are C performed In at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to Sallow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the 00 samples can be counted in a scintillation counter to determine the amount of bound compound.

A va r ety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific 00 or background Interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, C nudease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.

00 Use of Polynudeotides to Down-regulate or Inhibit a Protein of the Invention.

SPolynucleotide modulators of cancer can be Introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91104753. Suitable ligand-bindlng molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to Its corresponding molecule or receptor, or block entry of the sense or antisense oligonudeotide or its conjugated version into the cell. Aternatively, a polynudeotide modulator of cancer can be Introduced into a cell containing thelarget nuceic add sequence, by formation of a polynudeotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment Inhibitory and Antisense Nudeotides In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynudeotlde or inhibitory small nudear RNA (snRNA), a nucleic acd complementary to, and which can preferably hybridize specifically to, a coding mRNA nudeic acid sequence, a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynuceoltde to the mRNA reduces the translation and/or stability of the mRNA.

In the context of this invention, antisense polynudeotides can comprise naturally occurring nudeotides, or synthetic species formed from naturally occurring subunits or their dose homologs. Antisense polynudeotldes may also have altered sugar moeties or Inter-sugar linkages. Exemplary among these are the phosphorothloate and other sulfur containing species which are known for use in the art Analogs are comprised by this invention so long as they function effectively to hybridize with nudeotides of the Invention. See, Isis Pharmaceuticals, Cartsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynudeotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, induding Applied Biosystems. The preparation of other otgonudeotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art Anisense molecules as used herein Include antisense or sense oligonudeotides. Sense olgonudeotides can, be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonudeotide comprise a single stranded nucleic add sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonudeotides, according to the present invention, comprise a fragment generally at least about 12 nudeotides, preferably from about 12 to 30 nudeotides. The ability to derive

I

00 San antisense or a sense oligonucleolide, based upon a cDNA sequence encoding a given protein is described in, Stein C1 &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

^Q Ribozvmes SIn addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer- 00 assodated nuceotide sequences. A ribozyme is an RNA molecule that catalytically deaves other RNA molecules. Different 0 kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, Castanotto et al., Adv. In Pharmacology 25: 289-317 (1994) for a general review of the 00 properties of different ribozymes).

N The general features of hairpin ribozymes are described, in Hampel et al., Nuc. Adds Res. 18:299-304 S(1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Methods of preparing are well known to Sthose of skill in the art (see, WO 94126877; Ojwang et al., Proc. Natl. Acad. Sd. USA 90:6340-344 (1993); Yamada et 00 al, Human Gene Therapy 1:3945 (1994); Leavitt et al., Proc. Nall. Acad Sd. USA 92:699- 703 (1995); Leavitt et al., Human SGene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205:121-126 (1994)).

Use of Modulators in Phenotypic Screening In one embodiment a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By "administration' or 'contacting' herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic add encoding a proteinaceous agent a peptide) Is put Into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accompished, PCT US97/101019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue Is screened for agents that modulate, induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway Is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker As outlined above, screens are done to assess genes or gene products. That Is, having Identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays.

For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularizatlon, hormone release, transcriptional changes to both known and uncharacterized genetic markers by Northem blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNP.

00 SMethods of Identifying Characterizing Cancer-associated Sequences Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or Svariant cancer genes are determined. In one embodiment, the invention provides methods for Identifying cells containing 00 variant cancer genes, determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a call. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, determining all or part of the sequence of at least one gene of the invention in the 00 individual. This is generally done in at least one tissue of the individual, a tissue set forth in Table I, and may include Sthe evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, a wild-type gene to determine the presence of family Smembers, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the 0 sequence of a known cancer gene to determine If any differences exist. This is done using any number of known homology 0programs, such as BLAST, Bestfil, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes.

Information such as chromosomal localization finds use in providing a diagnosis or prognosis In particular when chromosomal abnonnalities such as translocations, and the like are identified in the cancer gene locus.

XIV,) KitslArticles of Manufacture For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a Figure 2-related protein or a Figure 2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic add, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a bloti-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino add sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

The kit of the invention wi typically comprise the container described above and one or more other containers comprising materials desrable from a commercial and user standpoint Including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.

A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a diagnostic or laboratory application, and can also indicate directions for either in vie or in vito use, such.as those described herein. Directions and or other information can also be Inclded on an Insert(s) or label(s) which s Included with or on the kit The terms kit and 'article of manufacture" can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small mdeule(s), nucleic add sequence(s), and/or antibody(s), materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth In Table I is provided. The article of 00 manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, ibottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

The container can hold amino add sequence(s), small molecule(s), nudeic add sequence(s), and/or antibody(s), in one embodiment the container holds a polynuceotide for use in examining the mRNA expression profile of a cell,, together with 0 reagents used for this purpose.

0The container can altematively hold a composition which is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or 0 a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an N antibody capable of spedfically binding 24P4C12 and modulating the function of 24P4C12.

The label can be on or associated with the container. A label a can be on a container when letters, numbers or Sother characters forming the label are molded or etched into the container Itself; a label can be associated with a container 0 when it is present within a receptacle or carrier that also holds the container, as a package insert The label can SIndicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers solution andlordextrose solution. It can further include other materials desirable from a commercial and user standpoint induding other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with Indications and/or Instructions for use.

EXAMPLES:

Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to rimit the scope of the invention.

Example 1: SSH-Generated Isolation of cDNA Fragment of the 24P4C12 Gene Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cONA from the LAPC-9 AD prostate cancer xenograft. The gene 24P4C12 was derived from an LAPC-9 AD minus benign prostatic hyperplasla experiment.

The 24P4C12 SSH cDNA of 160 bp is listed in Figure 1. The full length 24P4C12 cDNAs and ORFs are described in.Figure 2 with the protein sequences listed in Figure 3.

Materials and Methods Human Tissues: The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, PA).

mRNA for some normal tissues were purchased from Clontech, Palo Alto, CA.

RNA Isolation: Tissues were homogenized In Trizol reagent (Ufe Technologies, Gibco BRL) using 10 ml g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis 260/280 nm) and analyzed by gel electrophoresis.

Oliponucdeotides: The following HPLC purified oligonudeotides were used.

DPNCDN (cDNA synthesis primer): 5'TTTGATCAAGCTTo3' (SEQ ID NO: 33) 00 Adaptor 1: 5'CTAArACGACrCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO, 34) (SEQ ID NO: 00 5'GTMTACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3 (SEQ ID NO: 36) 00 3'CGGCTCCTAG5' (SEQ ID NO, 37) PCR primer 1: 5'CTATACGACTCACTATAGGGC3' (SEQ ID NQr 38) 00 Nested Primer (NPW1 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 39) Nested pimer (NP)2: 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: Supression Subtractive Hybridization: Suppression Subtractive Hybridization (SSH) was used to identify cDNAs; corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cONA from prostate cancer and normal tissues.

The gene 24P4C12 sequence was derived from LAPC-4AD) prostate cancer xenograft minus begnin prostatic hyperplasla cDNA subtraction. The SSH DNA sequence (Figure 1) was identified.

The cDNA derived from a pool of normal tissues and benign prostatichyperplasla was used as the source of the 'driver' cONA, while the cDNA from LAPC-4AD xenograft was used as the source of the 'tester' cONA. Double stranded cONAs corresponding to tester and driver cONAs were synthesized from 2 pig of poly(A)+ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Sedect cDNA Subtraction 1it and 1 ng of oligonucleotide DPNCDN as primier. Firstand second-strand synthesis were carried out as described In the Kit's user manual protocol (CLONTECH Protocol No. PT1 117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn 11 for 3 is at 37CC. Digested cONA was extracted with phenoUdiloroform and ethanol precipitated.

Driver cONA was generated by combining In a 1:1 ratio Dpn 11 digested cONA from the relevant tissue source (see above) with a mix of digested cDNAs; derived from the nine normal fissues: stomach, skeletal muscle, lung, brain, liver, kidney.

pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1Idp of Dpn 11 digested cDNA from the relevant tissue source (see above) (400 ng) In 5 1 of water. The diluted cDNA (2 0.,160 ng) was then ligated to 2 IA of Adaptor 1 and Adaptor 2 (10 ply 1 in separate ligation reactions, in a liotal volume of 10 p1 at 1600 ovemight, using 400 u of T4 DNA figase (CLONTECH). Ligation was terminated with 10p of 0.2 M EDIA and heating at 720C for 5 min.

The first hybridization was performed by adding 1.5 td (600 ng) of driver cDN1A to each of two tubes containing 1.5 P1 ng) Adaptor 1- and Adaptor 2- figated tester cONA. In a final volume of 4 Id, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98C for 1.5 minutes, and then were allowed to hybridize for 8 hirs at W8C. The two hybidizationis were then mixed together with an additional I Id of fresh denatured driver eflNA and were allowed to hybridize 00 O ovemighl at 68C. The second hybridization was then diluted in 200 pl of 20 mM Hepes, pH 8.3,50 mM NaCI, 0.2 mM EDTA, heated at 70oC for 7 min. and stored at -200C.

SPCR Amplification. Cloning and Seouencina of Gene Fragments Generated from SSH: STo amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 pi of the diluted final hybridization mix was added to 1 pi of PCR primer 1 (10 pM), 0.5 pJ dNTP mix (10 pM), 2.5 pJ 00 x reaction buffer (CLONTECH) and 0.5 pI 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 pI. PCR 1 was conducted using the following conditions: 75C for 5 min., 940C for 25 sec., then 27 cycles of 940C for 10 sec, 66oC for 30 sec, 72C for 1.5 min. Five separate primary PCR reactions were performed for each experiment The products were pooled and Sdiluted 1:10 with water. For the secondary PCR reaction, 1 p from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 pM) were used instead of PCR primer 1. PCR 2 was 0performed using 10-12 cycles of 94oC for 10 sec, 68C for 30 sec, and 72PC for 1.5 minutes. The PCR products were analyzed 00 using 2% agarose gel electrophoresis.

The PCR products were inserted Into pCR2.1 using the TIA vector cloning kit (Invitrogen). Transformed E. coi were c- subjected to blue/white and mpicillin selection. White colonies were picked and arrayed Into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ul of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format Plasmid DNA was prepared, sequenced, and subjected to nudeic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis: First strand cONAs can be generated from 1 pg of mRNA with digo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an Incubation for 50 min at 42 C with reverse transcriptase followed by RNAse H treatment at 37oC for 20 min. After completing the reaction, the volume can be Increased to 200 pi with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

Normalization of the first strand cONAs from multiple tissues was performed by using the primers 5atatccgccgctgtcgtcgacaa3' (SEQ ID NO: 41) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 42) to ampify p-actin. First strand cDNA (5 pl) were amplified in a total volume of 50 pl containing 0.4 pM primers, 02 pM each dNTPs, 1XPCR buffer (Clontech, 10 mM Trs-HCL, 1.5 mM MgCI, 50 mM KCI, pH8.3) and IX Klenaq DNA polymerase (Contech). Five pd of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94oC for 15 sec, followed by a 18, 20, and 22 cycles of 94C for 15, 65C for 2 min, 72°C for 5 sec. A final extension at 720C was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. p-actin bands from multiple tissues were compared by visual inspection.

Dilution factors for the first strand cDNAs were calculated to result in equal p-actin band intensities in all tissues after 22 cydes of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cydes of PCR.

To determine expression levels of the 24P4C12 gene, 5 pi of normalized first strand cDNA were analyzed by PCR using 26, and 30 cydes of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 24P4C12 SSH sequence and are listed below: 24P4C12.1 AGATGAGGAGGAGGACAAAGGTG 3' (SEQ ID NO: 43) 00 24P4C12.2 ACTGCTGGGAGGAGTACCGAGTG 3' (SEQ ID NO: 44) Example 2: Isolation of Full Length 24P4C12 Encoding cDNA 0 The 24P4C12 SSH cDNA sequence was derived from a substraction consisting of LAPC-4AD xenograft minus benign Sprostatic hyperplasla. The SSH cONA sequence (Figure 1) was designated 24P4C12.

The isolated gene fragment of 160 bp encodes a putative open reading frame (ORF) of 53 amino adds and exhibits 00 significant homology to an EST derived from a colon tumor library. Two larger cDNA dones were obtained by gene trapper N experiments, GTE9 and GTF8. The ORF revealed a significant homology to the mouse gene NG22 and the C.elegans gene CEESB82F. NG22 was recently identified as one of many ORFs within a genomic BAC done that encompasses the MHC dass III Sin the mouse genome. Both NG22 and CEESB82F appear to be genes that contain 12 transmembrane domains. This suggests OO that the gene encoding 24P4C12 contains 12 transmembrane domains and is the human homologue of mouse NG22 and C.

Selegans CEESB82F. Functional studies in Ce. elegans may reveal the biological role of these homologs. If 24P4C12 is a cell surface marker, then it may have an application as a potential imaging reagent and/or therapeutic target in prostate cancer.

The 24P4C12 v.1 of 2587 bp codes for a protein of 710 amino acids (Figure 2 and Figure Other variants of 24P4C12 were also identified and these are listed in Figures 2 and 3.24P4C12 v.1, v.3, v.5 and v.6 proteins are 710 amino acids in length and differ from each other by one amino acid as shown in Figure 11. 24P4C12 v.2 and v.4 code for the same protein as 24P4C12 v.1. 24P4C12 v.7, v.8 and v.9 are alternative splice variants and code for proteins of 598, 722 and 712 amino acids in length, respectively.

Example 3: Chromosomal Mapping of 24P4C12 Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available Induding fluorescent in s|tu hybridization (FISH), humanlhamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the CorieD Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic dones (NCBI, Bethesda, Maryland). 24P4C12 maps to chromosome 6p21.3 using 24P4C12 sequence and the NCBI BLAST tool located on the World Wide Web at (.ncbi.nlm.nih.gov/genome/seqipage.cgi?F=HsBlasthtml&&ORG=Hs).

Example 4: Expression Analysis of 24P4C12 Expression analysis by RT-PCR demonstrated that 24P4C12 is strongly expressed in prostate and ovary cancer patient specmens (Figure 14). First strand cDNA was generated from vital pool 1 (kidney, liver and lung), vital pool 2 (colon, pancreas and stomach), a pool of prostate cancer xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, ovary cancer pool, breast cancer pool, and cancer metastasis pool.

Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and 30 cycles of ampification. Results show strong expression of 24P4C12 in prostate cancer pool and ovary cancer pool.

Expression was also detected in prostate cancer xenografts, bladder cancer pool, kidney cancer pool, colon cancer pool, breast cancer pool, cancer metastasis pool, vital pool 1, and vital pool 2.

Extensive northern blot analysis of 24P4C12 in multiple human normal tissues is shown in Figure 15. Two multiple tissue northern blots (Clontech) both with 2 pg of mRNAAane were probed with the 24P4C12 SSH sequence. Expression of 24P4C12 was detected in prostate, kidney and colon. Lower expression is detected in pancreas, lung and placenta amongst all 16 normal tissues tested.

00 0 Expression of 24P4C12 was tested in prostate cancer xenografts and cell lines. RNA was extracted from a panel of cell lines and prostate cancer xenografts (PrEC, LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, PC-3, DU145, TsuPr, and LAPC- 4CL). Northern blot with 10 pg of total RNAlane was probed with 24P4C12 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The 24P4C12 transcript was detected In LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, and LAPC-4

SCL

00 Expression of 24P4C12 in patient cancer specimens and human normal tissues is shown in Figure 16. RNA was extracted from a pool of prostate cancer specimens, bladder cancer specimens, colon cancer specimens, ovary cancer specimens, breast cancer specimens and cancer metastasis specimens, as well as from normal prostate normal bladder normal kidney 00 and normal colon Northern blot with 10 pg of total RNAnane was probed with 24P4C12 SSH sequence. Size standards in kilobases (kb) are indicated on the side. Strong expression of 24P4C12 transcript was detected in the patient cancer pool Sspecimens, and in normal prostate but not in the other normal tissues tested.

SExpression of 24P4C12 was also detected In individual prostate cancer patient specimens (Figure 17). RNA was 0 extracted from normal prostate prostate cancer patient tumors and their matched normal adjacent tissues (Nat).

Northern blots with 10 pg of total RNA were probed with the 24P4C12 SSH fragment Size standards in kilobases are on the side. Results show expression of 24P4C12 in normal prostate and all prostate patient tumors tested.

Expression of 24P4C12 In colon cancer patient specimens is shown in figure 18. RNA was extracted from colon cancer cell lines (CL: Colo 205, LoVo, and SK-CO-), normal colon colon cancer patent tumors and their matched normal adjacent tissues (Nat). Northern blots with 10 pg of total RNA were probed with the 24P4C12 SSH fragment. Size standards in kilobases are on the side. Results show expression of 24P4C12 in normal colon and all colon patient tumors tested. Expression was detected In the cell lines Colo 205 and SK-CO-, but not in LoVo.

Figure 20 displays expression results of 24P4C12 In lung cancer patient specimens. Ma was extracted from lung cancer cell lines (CL CALU-1, A427, NCI-H82, NCI-H146), normal lung lung cancer patient tumors and their matched normal adjacent tissues (Nat). Northern blots with 10 pg of total RNA were probed with the 24P4C12 SSH fragment Size standards in kilobases are on the side. Results show expression of 24P4C12 In lung patient tumors tested, but not in normal lung. Expression was also detected in CALU-1, but not in the other cell lines A427, NCI-H82, and NCI- H146.

24P4C12 was assayed in a panel of human stomach and breast cancers and their respective matched normal tissues on RNA dot blots. 24P4C12 expression was seen in both stomach and breast cancers. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may Indicate that these tissues are not fully normal and that 24P4C12 may be expressed in early stage tumors.

The level of expression of 24P4C12 was analyzed and quantitated in a panel of patient cancer tissues. First strand cDNA was prepared from a panel of ovary patient cancer specimens uterus patient cancer specimens prostate cancer specimens bladder cancer patient specimens lung cancer patient specimens pancreas cancer patient specimens colon cancer specimens and kidney cancer spedmens Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and 30 cycles of amplification.

Samples were run on an agarose get and PCR products were quantitated using the Alphalmager software. Expression was recorded as absent, low, medium or strong. Results show expression of 24P4C12 in the majority of patient cancer specimens tested, 73.3% of ovary patient cancer specimens, 83.3% of uterus patient cancer specimens, 95.0% of prostate cancer specimens, 61.1% of bladder cancer patient specimens, 80.6% of lung cancer patient specimens, 87.5% of pancreas cancer patient specimens, 87.5% of colon cancer specimens, 68.4% of dear cell renal carcinoma, 100% of papillary renal cell carcinoma.

00 O The restricted expression of 24P4C12 in normal tissues and the expression detected in prostate cancer, ovary cancer, Sbladder cancer, colon cancer, lung cancer pancreas cancer, uterus cancer, kidney cancer, stomach cancer and breast Scancer suggest that 24P4C12 is a potential therapeutic target and a diagnostic marker for human cancers.

0 Example 5: Transcript Variants of 24P4C12 Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points.

00 Splice variants are mRNA variants spliced differently from the same transcript In eukaryotes, when a mulli-exon gene Is lN transcribed from genomic DNA, the initial RNA Is spliced to produce functional mRNA, which has only exons and is used for Stranslation into an amino add sequence. Accordingly, a given gene can have zero to many alternative transcripts and each Stranscript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different 00 coding andlor non-coding or 3' end) portions, from the original transaipt Transcript variants can code for similar or 0different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in Sthe same tissue at the same time, or In different tissues at the same time, or in the same tissue at different times, or in different tissues at different limes. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are Identified by full-length doning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into dusters which show direct or indirect identity with each other. Second, ESTs in the same duster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence Is a potential splice variant for that gene. Even when a variant is Identified that is not a full-length done, that portion of the variant is very useful for antigen generation and for further doning of the full-length splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs indude FgenesH (A Salamov and V. Solovyev, 'Ab nitio gene finding in Drosophila genomic DNA,' Genome Research. 2000 April; 10(4):516-22); Grail (URL at compbio.oml.gov/Grail-bin/EmptyGralForm) and GenScan (URL at genes.mitedu/GENSCAN.htil). For a general discussion of splice variant identification protocols see., Southan, A genomic perspective on human proteases, FEBS Lett. 2001 Jun 8; 498(2-3):214-8; de Souza, et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Nat Acad Sd U S A 2000 Nov 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant a variety of techniques are available In the art, such as full-length doning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see Proteomic Validation: Brennan, et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug 17;1433(1-2):321-6; Ferranti P, et el., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothellal growth factor (VEGF) splice variants by LghtCyder technology, Clin Chem. 2001 Apr47(4):654-60; Jia, et al., Discovery of new human betadefensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation: Brigle, et el., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Blophys Acta. 1997 Aug 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated In cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well.

00 0 Disclosed herein Is that 24P4C12 has a particular expression profile related to cancer. Altemative transcripts and splice Svariants of 24P4C12 may also be involved in cancers in the same or different tissues, thus serving as tumor-assodated markers/antigens.

The exon composition of the original transcript, designated as 24P4C12 v.1, is shown in Table LI. Using the full- Slength gene and EST sequences, three transcript variants were identified, designated as 24P4C12 v.7, v.8 and v.9.

00 Compared with 24P4C12 v.1, transcript variant 24P4C12 v.7 has spliced out exons 10 and 11 from variant 24P4C12 v.1, as shown In Figure 12. Variant 24P4C12 v.8 inserted 36 bp in between 1931 and 1932 of variant 24P4C12 v.1 and variant 24P4C12 v.9 replaced with 36 bp the segment 1136-1163 of variant 24P4C12 v.1. Theoretically, each different combination 00 of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant. Figure 12 shows the schematic alignment of exons of the four transcript variants.

STables LII through LXIII are set forth on a variant by variant basis. Tables UI, LVI, and LX show nudeolide cK sequences of the transcript variant Tables LllI, LVII, and LXI show the alignment of the transcript variant with the nucleic 00 acd sequence of 24P4C12 v.1. Tables LIV, LVIII, and LXII lay out the amino acid translation of the transcript variant for the identified reading frame orientation. Tables LV, LIX, and LXIII display alignments of the amino add sequence encoded by

C

the splice variant with that of 24P4C12 v.1.

Example 6: Single Nucleotide Polvmorphisms of 24P4C12 A Single Nudeotide Polymorphism (SNP) is a single base pair variation in a nudeotide sequence at a specific location. At any given point of the genome, there are four possible nudeotide base pairs: ArT, C/G, GIC and T/A. Genotype refers to the specific base pair sequence of one or more locations In the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors Including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (caled haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals Nowotny, J.

M. Kwon and A. M. Goate, "SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct 11(5):637-641; M.

Pirmohamed and B. K. Park, 'Genetic susceptibility to adverse drug reactions,' Trends Phannacol. Sd. 2001 Jun; 22(6):298- 305; J. H. Riley, C. J. Allan, E. Lai and A. Roses,* The use of single nudeotide polymorphisms in the isolation of common disease genes,' Pharmacogenomics. 2000 Feb; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The predictive power of haplotypes in clinical response,' Pharmacogenomics. 2000 feb; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods Bean, "The promising voyage of SNP target discovery," Am. COn. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res. 1998 Jul; 8(7):691-697; M. M. She. 'Enabling large-scale pharmacogenelic studies by high-throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data In public and private databases, one can discover SNPs by comparing sequences using computer programs Gu, L Hillier and P. Y. Kwok, "Single nuceotide polymorphism hunting in cyberspace,' Hum. Mutat 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct 00 sequendng and high throughput microarrays Y. Kwok, "Methods for genotyping single nucleotide polymorphisms," Annu.

Rev. Genomics Hum. Genet 2001; 2:235-258; M. Kokoris, K. Dix, K Moynlhan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol. Diagn. 2000 Dec; 5(4):329-340).

Using the methods described above, five SNPs were identified in the original transcript, 24P4C12 v.1, at positions 542 l 564 818 981(AIG) and 1312 The transcripts or proteins with alternative alleles were designated as 00 variants 24P4C12 v.2, v.3, v.4, v.5 and v.6, respectively. Figure 10 shows the schematic alignment of the SNP variants.

Figure 11 shows the schematic alignment of protein variants, corresponding to nudeotide variants. Nudeotide variants that code for the same amino acid sequence as variant 1 are not shown in Figure 11. These alleles of the SNPs, though shown 00 separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 24P4C12 v.7) that contains the sequence context of the SNPs.

SExample 7: Production of Recombinant 24P4C12 In Prokaryotic Systems 00 To express recombinant 24P4C12 and 24P4C12 variants in prokaryotic cells, the full or partial length 24P4C12 O and 24P4C12 variant cDNA sequences are doned into any one of a variety of expression vectors known in the art The full CK length cDNA, or any 8, 9,10,11,12, 13, 14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 24P4C12. variants, or analogs thereof are used.

A. In vitro transcription and translation constructs: gCIE To generate 24P4C12 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 24P4C12 cDNA. The pCRII vector has Sp6.

and T7 promoters flanking the insert to drive the transcription of 24P4C12 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the ceil and tissue expression of 24P4C12 at the RNA level. Transcribed 24P4C12 RNA representing the cDNA amino acid coding region of the 24P4C12 gene is used in in vitro translation systems such as the TnT M Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize 24P4C12 protein.

B. Bacterial Constructs: pGEX Constructs; To generate recombinant 24P4C12 proteins in bacteria that are fused to the Glutathione Stransferase (GST) protein, all or parts of the 24P4C12 cDNA or variants are cloned into the GST- fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 24P4C12 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxylterminus. The GST and 6X HIs tags permit purification of the recombinant fusion protein from Induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, of the open reading frame (ORF). A proteolytic deavage site, such as the PreSdsslon T recognition site in pGEX-6P-1, may be employed such that it permits deavage of the GST tag from 24P4C12-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 24P4C12 proteins that are fused to maltose-binding protein (MBP), all or parts of the 24P4C12 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverty, MA). These constructs allow controlled expression of recombinant 24P4C12 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl, terminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with ant-MBP and ant-His antibodies. The 6X His epitope tag is generated by adding 6 histidine codons to the 3' clonng primer. A Factor Xa recognition site permits cleavage of the pMAL 00 O tag from 24P4C12. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

T pET Constructs: To express 24P4C12 in bacterial cels, all or parts of the 24P4C12 cDNA protein coding sequence are doned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 24P4C12 protein in bacteria with and without fusion to proteins that enhance solubility, such as SNusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag m that aid purification and detection of the recombinant protein. For example, constructs are made utiizing pET NusA fusion system 43.1 such that regions of the 24P4C12 protein are expressed as amino-terminal fusions to NusA.

N C. Yeast Constructs: pESC Constructs: To express 24P4C12 in the yeast species Saccharomyces cerevislae for generation of recombinant protein and functional studies, all or parts of the 24P4C12 cONA protein coding sequence are cloned into the 0 pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La 00 Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag T or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 24P4C12. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed In eukaryotic cells.

ESP Constructs: To express 24P4C12 in the yeast species Saccharomycespombe, all or parts of the 24P4C12 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 24P4C12 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A FlagT epitope tag allows detection of the recombinant protein with anti- Fag m antibody.

Example 8: Production of Recombinant 24P4C12 In Higher Eukarvotic Systems A. Mammalian Constructs: To express recombinant 24P4C12 in eukaryotic cells, the ful or partial length 24P4C12 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art One or more of the following regions of 24P4C12 are expressed In these constructs, amino acids 1 to 710, or any 8, 9,10, 11, 12,13,14,15,16,17,18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30 or more contiguous amino acds from 24P4C12 v.1 through v.6; amino adds 1 to 598, or any 8, 9, 10, 11, 12,13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23,24, 25,26, 27,28,29,30 or more contiguous amino acids from 24P4C12 v.7.

amino acids 1 to 722, or any 8,9,10,11, 12, 13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 24P4C12 v.8, amino adds 1 to 712 or any 8, 9, 10, 11, 12, 13,14,15, 16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 24P4C12 v.9, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells.

Transfected 293T cell lysates can be probed with the anti-24P4C12 polyclonal serum, described herein.

pcDNA3.1/MycHis Constructs; To express 24P4C12 in mammalian cells, a 24P4C12 ORF, or portions thereof, of 24P4C12 with a consensus Kozak translation initiation site was doned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the cartoxy-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 orgin for episomal replication and simple vector rescue In cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin 00 O resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Figure 24 demonstrates C expression of 24P4C12 from the pcDNA3.1/MycHis construct in transiently transfected 293T cells.

apcDNA4/HisMax Constructs: To express 24P4C12 in mammalian cells, a 24P4C12 ORF, or portions thereof, of r 24P4C12 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression Is driven from the 00 cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpressm and six 0histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the 00 SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeodn N resistance gene allows for selection of mammalian cells expressing the protein and the ampidllin resistance gene and ColE1 I origin permits selection and maintenance of the plasmid in E. coii.

SpcDNA3.1CT-GFP-TOPO Construct: To express 24P4C12 in mammalian cells and to allow detection of the 00 recombinant proteins using fluorescence, a 24P4C 12 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned Into pcDNA3.11CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus S(CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyt-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein and the ampicillin resistance gene and CoE1 origin permits selection and maintenance of the plasmid in E. coil. Additional constructs with an aminoterminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 24P4C12 protein.

A 24P4C12 ORF, or portions thereof, were cloned into pTag-5. This vector Is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 24P4C12 protein with an amino-terminal IgGi signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 24P4C12 protein were optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 24P4C12 proteins.

Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

Figure 26 shows expression of 24P4C12 from two different pTag5 constructs.

PAPtag: A 24P4C12 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 24P4C12 protein while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an aminoterminal IgGK signal sequence is fused to the amino-terminus of a 24P4C12 protein. The resulting recombinant 24P4C12 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 24P4C12 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyt-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E coi.

PsecFc: A 24P4C12 ORF, or portions thereof, Is also cloned into psecFc. The psecFc vector was assembled by doning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgGi Fc fusion at the carboxyl-terminus of the 24P4C12 proteins, while fusing the IgGK signal sequence to N-terminus. 24P4C12 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 24P4C12 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as 00 Simmunogens or to Identify proteins such as Ilgands or receptors that Interact with 24P4C12 protein. Protein expression is Sdriven from the CMV promoter. The hygromydn resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid In E coli.

DSRa Constructs: To generate mammalian cell lines that express 24P4C12 constitutively, 24P4C12 ORF, or 00 portions thereof, of 24P4C12 were cloned into pSRa constructs. Amphotropic and ecotropic retroviruses were generated by Otransfection of pSRa constructs into the 293T-10A1 packaging line or co-transfection of pSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovtrus is used to Infect a variety of 00 mammalian celllines, resulting in the Integration of the cloned gene, 24P4C12, into the host cell-lines. Protein expression is N driven from a long terminal repeat (LTR). The Neomycin resistance gene present In the vector allows for selection of Smammalian cells that express the protein, and the ampidllin resistance gene and ColE1 origin permit selection and maintenance of the plasmld In E. cofl. The retroviral vectors can thereafter be used for Infection and generation of various 00 cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells. Figure 23 shows RNA expression of 24P4C12 driven from the 24P4C12pSRa construct in stably transduced PC3, 3T3 and 300.19 cells. Figure 25 shows 24P4C12 protein CK. expression in PC3 cells stably transduced with 24P4C12pSRa construct.

Additional pSRa constructs are made that fuse an epitope tag such as the FLAG T tag to the carboxyl-terminus of 24P4C12 sequences to allow detection using anti-Flag antibodies. For example, the FLAG T sequence 5' gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 45) is added to cloning primer at the 3' end of the ORF. Additional pSRa constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full-length 24P4C12 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 24P4C12.

High virus titer leading to high level expression of 24P4C12 is achieved In viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 24P4C12 coding sequences or fragments thereof are amplified by PCR and subdoned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturers instructions to generate adenoviral vectors. Altematively, 24P4C12 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 24P4C12 in mammalian cells, coding sequences of 24P4C12, or portions thereof, are doned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 24P4C12. These vectors are thereafter used to control expression of 24P4C12 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems To generate recombinant 24P4C12 proteins in a baculovirus expression system, 24P4C12 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus.

Specifically, pBlueBac-24P4C12 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) Into SF9 (Spodoptera fnrglperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supematant and purified by plaque assay.

Recombinant 24P4C12 protein is then generated by infection of HighFive insect cells (Invilrogen) with purified baculovirus. Recombinant 24P4C12 protein can be detected using antl-24P4C12 or anti-His-tag antibody. 24P4C12 protein can be purified and used in various cell-based assays or as immunogen to generate polydonal and monodonal antibodies specific for 24P4C12.

00 SExample 9: Antlaenlcltv Profiles and Secondary Structure r Figures 5-9 depict graphically five amino acid profiles of the 24P4C12 variant 1, assessment available by Saccessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy n molecular biology server.

OO These profiles: Figure 5, Hydrophilicity, (Hopp Woods KR., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828); Figure 6, Hydropathidty, (Kyte Doolittle 1982. J. Mol. Biol. 157:105-132); Figure 7, Percentage Accessible Residues (Janin 1979 Nature 277:491-492); Figure 8, Average Flexibility, (Bhaskaran and Ponnuswamy 1988.

00 Int J. Pept Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, Roux B. 1987 Protein Engineering 1:289-294); and N optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 24P4C12 protein. Each of the above amino acid profiles of 24P4C12 were generated using the following ProtScale 0 parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 00 3) amino add profile values normalized to lie between 0 and 1.

SHydrophilidty (Figure Hydropathidty (Figure 6) and Percentage Accessible Residues (Figure 7) profiles were Sused to determine stretches of hydrophilic amino adds values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathidty profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.

Average Flexibility (Figure 8) and Beta-tum (Figure 9) profiles determine stretches of amino adds values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained In secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.

Antigenic sequences of the 24P4C12 protein and of the variant proteins indicated, by the profiles set forth in Figure 5, Figure 6, Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-24P4C12 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 11, 12,13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino adds, or the corresponding nucleic acids that encode them, from the 24P4C12 protein variants isted in Figures 2 and 3. In particular, peplide immunogens of the invention can comprise, a peptide region of at least 5 amino acds of Figures 2 and 3 in any whole number increment that indudes an amino add position having a value greater than 0.5 In the Hydrophllidty profile of Figure 5; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathidty profile of Figure 6; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; a peptide region of at least 5 amino adds of Figures 2 and 3 in any whole number increment that includes an amino add position having a value greater than 0.5 in the Average Flexibility profile on Figure 8; and, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number Increment that includes an amino add position having a value greater than 0.5 in the Beta-turn profile of Figure 9. Peptide immunogens of the invention can also comprise nudeic adds that encode any of the forgoing.

Al immunogens of the invention, peptide or nucleic add, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excpient compatible with human physiology.

The secondary structure of 24P4C12 variant 1, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the respective primary amino add sequences using the HNN Hierarchical Neural Network method (Guermeur, 1997, httpl/pbiibcp.frigi-ninnpsa_automat.pl?pagenpsa_nn.html), 00 accessed from the ExPasy molecular biology server (http//www.expasy.ch/tools/). The analysis indicates that 24P4C12 variant 1 is composed of 53.94% alpha helix, 9.44% extended strand, and 36.62% random coil (Figure 13a).

0 Analysis for the potential presence of transmembrane domains in 24P4C12 variants were carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server (http://www.expasy.chtools).

Shown graphically are the results of analysis of variant 1 depicting the presence and location of 10 transmembrane domains using the TMpred program (Figure 13b) and TMHMM program (Figure 13c). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table L.

Example 10: Generation of 24P4C12 Polvclonal Antibodles

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N Polyconal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent IN and, if desired, an adjuvant Typically, the immunizing agent andlor adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 24P4C12 protein, computer 00 algorithms are employed in design of immunogens that, based on amino add sequence analysis contain characteristics of 0being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled S'Antigenicity Profiles'. Such regions would be predicted to be hydrophilc, flexible, in beta-tum conformations, and be exposed on the surface of the protein (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9 for amino acid profiles that indicate such regions of 24P4C12 and variants).

For example, 24P4C12 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-tur regions of 24P4C12 variant proteins are used as antigens to generate polyconal antibodies in New Zealand White rabbits.

For example, such regions include, but are not limited to, amino adds 1-34, amino adds 118-135, amino adds 194-224, amino adds 280-290, and amino acids 690-710, of 24P4C12 variants 1. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 1-14 of 24P4C12 variant 1 was conjugated to KLH and used to immunize a rabbit This antiserum exhibited a high tiler to the peptide (>10,000) and recognized 24P4C12 in transfected 293T cells by Western blot and flow cytometry (Figure 24) and in stable recombinant PC3 cells by Western blot and immunohistochemistry (Figure 25). Alternatively the immunizing agent may indude al or portions of the 24P4C12 variant proteins, analogs or fusion proteins thereof. For example, the 24P4C12 variant 1 amino add sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known In the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding amino acids 379-453, encompassing the third predicted extracellular loop of variant 1, is produced, purified, and used as immrunogen. Other recombinant bacterial fusion proteins that may be employed indude maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 24P4C12 In Prokaryotic Systems' and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Unsley, Brady, Umes, Grosmaire, L, Damle, and Ledbetter, L(1991) J.Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the Example entitled "Production of Recombinant 24P4C12 in Eukaryotic Systems'), and retains post-translational modifications such as glycosylations found in native protein. In two embodiments, the predicted 1st and third extracellular loops of variant 1, amino adds 59-227 and 379-453 respectively, were each doned into the Tag5 mammalian secretion vector and expressed in 293T cells (Figure 26). Each recombinant protein is then purified by metal chelate chromatography from tissue culture 00 Ssupematants andlor lysates of 293T cells stably expressing the recombinant vector. The purified Tag5 24P4C12 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Upid A, synthetic trehalose dicorynomycolate).

00 In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 pg, typically 100-200 g, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 pg, typically 100-200 pg, of the immunogen in incomplete Freund's adjuvant 00 (IFA). Test bleeds are taken approximately 7-10 days following each Immunization and used to monitor the titer of the sO antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with a KLH- CN conjugated peptide encoding amino acids 1-14 of variant 1, the full-length 24P4C12 variant 1 cDNA is doned into pCDNA S3.1 myc-his or retroviral expression vectors (Invitrogen, see the Example entitled "Production of Recombinant 24P4C12 in 0Eukaryotic Systems'). After transfection of the constructs into 293T cells or transduction of PC3 with 24P4C12 retrovlrus, cell lysates are probed with the anti-24P4C12 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 24P4C12 protein using the Western blot technique. As shown in Figures 24 and 25 the antiserum spedfically recognizes 24P4C12 protein in 293T and PC3 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry, and immunohistochemistry (Figure 25) and immunopredpitation against 293T and other recombinant 24P4C12-expressing cells to determine specific recognition of native protein. Western blot, Immunoprecipitation, fluorescent microscopy, immunohistochemistry and flow cytometric techniques using cells that endogenously express 24P4C12 are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 24P4C12 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irelevant fusion protein. For example, antiserum derived from a GST- 24P4C12 fusion protein encoding amino adds 379453 of variant 1 is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-fusion protein also encoding amino acids 379-453 covalentiy coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11: Generation of 24P4C12 Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 24P4C12 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 24P4C12 variants, for example those that would disrupt the interaction with Igands and substrates or disrupt its biological activity. Immunogens for generation of such mAbs include those designed to encode or contain the entire 24P4C12 protein variant sequence, regions of the 24P4C12 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, and the Example entitled "Antigenicity Profiles'). Immunogens indude peptides, recombinant bacterial proteins, and mammalian expressed Tag proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 24P4C12 variant, such as 293T-24P4C12 variant I or 300.19.24P4C12 variant Imurine Pre-B cells, are used to immunize mice.

00 O To generate mAbs to a 24P4C12 variant mice are first immunized intraperitoneally (IP) with, typically, 10-50 pg of C protein immunogen or 107 24P4C12-expressing cells mixed In complete Freund's adjuvant Mice are then subsequently C immunized IP every 2-4 weeks with, typically, 10-50 pg of protein immunogen or 107 cells mixed in incomplete Freund's Sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In one embodiment, mice were immunized as above 00 with 300.19-24P4C12 cells In complete and then incomplete Freund's adjuvant, and subsequently sacrificed and the spleens 0 harvested and used for fusion and hybridoma generation. As is can be seen in Figure 27, 2 hybridomas were generated whose antibodies specifically recognize 24P4C12 protein expressed In 293T cells by flow cytometry. In addition to the above 00 protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian N expression vector encoding a 24P4C12 variant sequence Is used to immunize mice by direct injection of the plasmid DNA.

SIn one embodiment, a Tag5 mammalian secretion vector encoding amino acids 59-227 of the variant 1 sequence (Figure 26) Swas used to immunize mice. Subsequent booster immunizations are then carried out with the purified protein. In another 00 example, the same amino acids are cloned into an Fc-fusion secretion vector in which the 24P4C12 variant 1 sequence is Sfused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or Cl murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins as above and with cells expressing the respective 24P4C12 variant During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, Immunopredpitation, fluorescence microscopy, immunohistochemistry, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, Harlow and Lane, 1988).

In one embodiment for generating 24P4C12 variant 8 spedfic monoclonal antibodies, a peptide encoding amino adds 643-654 (RNPITPTGHVFQ) (SEQ ID NO: 46) of 24P4C12 variant 8 is synthesized, coupled to KLH and used as immunogen. Balb C mice are initially immunized intraperitoneally with 25 pg of the KLH-24P4C12 variant 8 peptide mixed in complete Freund's adjuvant Mice are subsequently immunized every two weeks with 25 pg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the free peptide determines the reactivity of serum from immunized mice. Reactivity and spedficity of serum to full length 24P4C12 variant 8 protein is monitored by Westem blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 24P4C12 variant 8 cDNA compared to cells Iransfected with the other 24P4C12 variants (see the Example entitled "Production of Recombinant 24P4C12 in Eukaryotic Systems'). Other recombinant 24P4C12 variant 8-expressing cells or cels endogenously expressing 24P4C12 variant 8 are also used. Mice showing the strongest specific reactivity to 24P4C12 variant 8 are rested and given a final injection of antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988).

Supematants from HAT selected growth wells are screened by ELISA Western blot immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 24P4C12 variant 8-specfic antibody-produdng clones. A similar strategy is also used to derive 24P4C12 variant 9-specific antibodies using a peptide encompassing amino acids 379-388 (PLPTQPATLG) (SEQ ID NO: 47).

The binding affinity of a 24P4C12 monodonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 24P4C12 monoclonal antibodies.preferred for diagnostic or therapeutic use, as appredated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt Quant Elect 23:1; Morton and Myszka, 1998, Methods In Enzymology 295: 268) to 00 monitor bimolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, 2 dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 12: HLA Class I and Class II Binding Assays HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed

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protocols PCT publications WO 94/20127 and WO 94103205; Sidney et at, Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et at., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC 00 molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 'l-radiolabeled probe

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ri peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary eperiments, each MHC preparation is titered in the 0 presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 00 20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA 0concentrations.

Since under these conditions [label]<[HLA] and ICso(HLA, the measured ICso values are reasonable approximations of the true Ko values. Peptide inhibitors are typically tested at concentrations ranging from 120 ipgml to 1.2 ngfml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC5o of a positive control for inhibition by the ICso for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back Into ICso nM values by dividing the ICso nM of the positive controls for inhibition by the relative binding of the peptide of interest This method of data compilation Is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotif andlor HLA motif-bearing peptides (see Table IV).

Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Eptopes HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise .multiple HLA. supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and Sconfirmation of supermotif- and motif-bearing epitopes for the Inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer searches and algorithms for Identification of supermotif and/or motif-bearing eiltooes The searches performed to identify the motif-bearing peptide sequences in the Example entitled 'Antigenidty Profiles' and Tables VIII-XXI and XXII-XUX employ the protein sequence data from the gene product of 24P4C12 set forth in Figures 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows.

'AD translated 24P4C12 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art In view of known motif/supermolif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-. A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to spedfic HLA-Class I or Class II molecules. These polynomial algorithms account for the Impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: O 00 'AG=avxa2xa3 xa where ay is a coefficient which represents the effect of the presence of a given amino add at a given position (i) along the sequence of a peptlde of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other independent binding of individual side-chains). When residue j occurs at C position i in the peptide, it is assumed to contribute a constant amount i to the free energy of binding of the peptide 00 irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described In Gulukota et al., J. Mol. Biol.

267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363- 00 3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding IN (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate ofj4 For Class SII peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure.

CTo calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide 00 are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 suoertpe cross-reactive peptides Protein sequences from 24P4C12 are scanned utilizing motif identification software, to identify 9- 10- and 11mer sequences containing the HLA-A2-supermotit main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A'0201 molecules in vito (HLA-A'0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A'0202, A*0203, A'0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA- A2 supertype molecules.

Selection of HLA-A3 supermotif-bearino epitopes The 24P4C12 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A'0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of 500 nM, often 200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles A'3101, A'3301, and A'6801) to Identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 supermotif bearing eDitopes The 24P4C12 protein(s) scanned above is also analyzed for the presence of 9- 10-, or 11-mer peptides with the HLA-87-supermotif. Corresponding peplides are synthesized and tested for binding to HLA-8B0702 the molecule encoded by the most common B7-supertype allele the prototype 87 supertype allele). Peptides binding 8*0702 with ICso of :500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules B*3501, B5101, B'5301, and 8*5401). Peptides capable of binding to three or more of the five B7supertype alleles tested are thereby identified.

00 Selection of Al and A24 motif-bearing evitones To further increase population coverage, HIA-Al and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 24P4C12 protein can also be performed to identify HIA-AIl- and A24-rnotif-contalning sequences.

High affinity andior cross-reacive binding epitopes that bear other motif and/or supermotfs are identified using 00 analogous methodology.

Example 14: Confirmation of Inimuno-ienlclt 00 Cross-reactive canddate CTIL A2-supermnoti-bearing peptides that are identified as described herein are selected N ~to confirm In vft lmmunogenicty. Confirmation is performed using the following methodofogy.

Taroet Cell Lines for Cellular Screening: The .221A2.1 cell line, produced by transferring the HLA-A2.l gene into the HLA-A, -C null mutant human B- 00 lymphoblastoid cell fine 721.221. is used as the peptide-loaded target to measure activity of IILA-A2.l-restricted Cm. This cell Olne is grown In RPMI-16840 medium supplemented with antibiotics, sodim pyruvate, nonessential amino acids and CK1 (vlv) heat inactivated FCS. Cells that express an antigen of interest or transfectants comprising the gene encoding the antigen of Interest can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures: Generation of Dendrftic Cells PBMCs are thawed in RPMI with 30 pgtml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essenial amino acids, sodium pyruvate, Lglutamine and penllnlstreptomycin). The monocytes are purified by plating 10 x 106 PBMCfweI in a 6-well plate. After 2 hours at 37C, the non-adherent cells are removed by gently shaking the plates and aspirating the superniatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 nglmld of GM-CSF and 1,000 UlmI of 11-4 are then added to each well. TNFat Is added to the D~s on day 6 at 75 ngiml and the cells are used for Ct Induction cultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-oells; are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads@i M-450) and the detacha-bead@ reagent Typically about 200-250x105 PBMO are processed to obtai n 24x006 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed In RPMI with 30pgWm DNAse, washed once with PBS containing 1% human AS serumn and resuspended In PBS/l% AB serum at a concentration of 20x10Wcls~ml. The magnetic beads are washed 3 times with PBSJAB serum, added to the cells (l4Ojjl beads/2OxlO 5 cells) and incubated for 1 hour at 40C with continuous mixing. The beads and cells are washed Ux with PBSIAB serum to remove the nonadherent cells and resuspended at 100A06 cellstimI (based on the original cell number) In PBSIAB serum containing 110Oplml detacha-bead@ reagent and 30 pgh/iI DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/ONAse to collect the CDB4 T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40jidm of peptide at a cell oonoentration of 1-2x10 6 m1 in the presence of 3p~ghnl Rz- mla'oglobulln for 4 hours at 200C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up Induion culwu 0.25 ml cytokine-generated DC (at 1105 cellstnil) are co-cultured with 0.2Snil of CDB+ T-cells (at 20l08 cell/ml) In each well of a 48-well plate in the presence of 10 nglml of 11-7. ReoombInant human IL-1 0 is added the next day at a final concentration of 10 ngknl and rhuman I1-2 Is added 48 hours later at i D lU/mi.

.Rasimufation of the Induction cufture with peptide-pulsed adherent cogs: Seven and fourteen days after the prinary Induction, the cells are restimulated with peptide-pulsed aierent cells. The PBM~s are thawed and washed twice 00 with RPMI and ONAse. The cells are resuspended at 5x10 6 cells/mi and irradiated at -4200 rads. The PBMCs are plated at 2x10 6 in 0.5 ml complete medium per well and incubated for 2 hours at 37 0 C. The plates are washed twice with RPMI by Stapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10pgiml of peptide in the Spresence of 3 pg/ml 82 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37 0 C. Peptide solution from each well is Saspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) 00 and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 501Ulml (Tsai et al., Critical Reviews in Immunology 00 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 5 1 Cr release assay. In some N experiments the cultures are assayed for peptide-specific recognition in the in situ IFNy ELISA at the time of the second 0restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays Sfor a side-by-side comparison.

00 Measurement of CTL vtic activity by s 5 Cr release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) s'Cr release assay by Nassaying individual wells at a single E-T. Peptide-pulsed targets are prepared by incubating the cells with 10pgIml peptide overnight at 37 0

C.

Adherent target cells are removed from culture flasks with typsin-EDTA. Target cells are labeled with 200pCi of Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37C. Labeled target cells are resuspended at 10 6 per ml and diluted 1:10 with K562 cells at a concentration of 3.3x10/ml (an NK-sensitive erythroblastoma cell line used to reduce nonspecific lysis). Target cells (100 pl) and effectors (100pl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37 0 C. At that time, 100 pl of supernatant are collected from each well and percent lysis is determined according to the formula: [(cpm of the test sample- cpm of the spontaneous s'Cr release samp!e)/(cpm of the maximal 5 "Cr release samplecpm of the spontaneous 5 sCr release sample)] x 100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is,defined as one In which the specific lysis (sample- background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In situ Measurement of Human IFNy Production as an Indicator of Peptide-specific and Endogenous Recognition Immulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4 tg/ml 0.1M NaHCO3, pH8.2) 'overnight at 4°C. The plates are washed with Ca 2 Mg 2 -free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 pilwell) and targets (100 pl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of lx10 6 cells/m. The plates are incubated for 48 hours at 37*C with 5% CO2.

Recombinant human IFN-gamma Is added to the standard wells starting at 400 pg or 120pg/100 mlcroliter/well and the plate incubated for two hours at 37"C. The plates are washed and 100 .i of biotinylated mouse anti-human IFNgamma monodonal antibody (2 microgramfml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates Incubated for one hour at room temperature. The plates are then washed 6x with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microlitertweU 1M H3PO4 and read at OD450. A culture is considered positive If It measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

0 Those cultures that demonstrate specific lytic activity against peplide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, Ux10 4 CD8. cells are added to a T25 flask containing the following: 10x0 6 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2010 5 Irradiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (anti-CD3) at 3Ong per rn! in RPMI- 640 containing 10% (vlv human AB serum, non-essential amino acids, 0socf urn pyruvate, 25pM 2-mercaptoethanol, 1-glutamine and peniciflinlstreptomycin. Recombinant human 11-2 is added 24 -hours later at a final concentration of 200lU/ml and every three days thereafter with fresh media at 501U/mI. The cells are split If the cell concentration exceeds 1xlO 6 lml and the cultures are assayed between days 13 and 15 at Vl ratios of 30, 00 '3 and 1:1 in the 5 1 Cr release assay or at lxlO 6 Imld In the in situ IFNy assay using the same targets as before the expansion.

INDCultures are expanded in the absence of antl.CD3+ as follows. Those cultures that demonstrate specific lyllc activity against peptide and endlogenous targets are selected and 5x10' CD8* cells are added to a T25 flask containing the ri following: 106 autotogous PBMC per ml which have been peptide-pulsed with 10 4agml peplde for two hours at 3MC and 00 Irradiated (4,200 rad), 2x10 5 irradiated (8,000 rad) EBV-transformed cells per nml RPMI-1640 containing 10%(vlv) human AB serum, non-essential AA, sodium pyruvate, 25mM 2-ME, 1-glutamine and gentamicin.

r-K1Immuno-genicity of A2 supermotif-bearina nepticdes A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptidespecific Ct in normal individuals. In this analysis, a peptide is typically considered to be an epitope If it Induces peptidespecific CTI-s In at least indivduals, and preferably, also recognizes t1he endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 24P4C1 2. Briefly, P8MCs are isolated from patients,' re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A'031A1 I immunooenicitv HLA-A3 supermotif-bearing cross-reactive binding peptIdes are also evaluated for trnimunogenlcity using methodology analogous for tt used to evaluate the imnmogenicdty of the HLA-A2 supermotif peptides.

Evaluation of B7 lmmunogenldtv Immunogenicity screening of the B7-supertype cross-reactive binding peptidles Identified as set forth herein are confirmed in a manner analogous to the confirmation of A2-and A31-supermotif-bearing peptides.

Peptides bearing other supermotifslmotifs, HIA-Al, HLA-A24 etc. are also confirmed using similar methodology lExamnle 15: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs HLA motifs and supermotifs (comprising primary andor secondary residues) are useful In the identifiction and preparation of hi~lhy cross-reacive native peptidaes, as demonstrated herein. Moreover, the definition of HIA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence whi can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HIA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HIA molecules.

Examples of analoglng peptides to exhibit modulated binding affinity are set forth In this example.' Arialoolno at Primary Anchor Residues Peplide engineering strategies are implemented to further Increase Mhe cross-ractivity of the epitopes. For example, the main anchrs of A2-supermoif-bearing peptides are altered, for example, to Introduce a preferred L, 1, V. or M at position 2, and I or V at the C-terminus.

00 To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A'0201 binding capacity is maintained, for A2-supertype cross-reactivity.

C Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

4 The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the 00 capacity of the parent wild type (WT) peptide to bind at least weakly, bind at an ICso of 5000nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have Increased immunogenicity and cross- 00 reactivity by T cells specific for the parent epitope (see, Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue at al., N Proc. Natl. Acad. Sci. USA 92:8166,1995).

In the cellular screening of these peptide analogs, it is important to confinn that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cels that endogenously express the epitope.

00 Anaooinq of HLA-A3 and B7-supermotif-bearinq peptides C- Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 and A'11 (prototype A3 supertype alleles). Those peptides that demonstrate 500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. 87 supermotif-bearing peptides are, for example, engineered to possess a preferred residue I, L, or F) at the C-terminal primary anchor posltion, as demonstrated by Sidney et al. Immunol. 157:3480-3490,1996).

Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally Important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when .possible, targets that endogenously express the epitope.

Analooina at Secondary Anchor Residues Moreover, HLA supermotifs are of value In engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 Is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 24P4C12expressing tumors.

Other analoainq strategies 00 O Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with a- Samino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the D peptide structurally so as to reduce binding capacity. Substitution of a-amino butyric acid for cysteine not only alleviates this

L

problem, but has been shown to improve binding and crossbinding capabilities In some Instances (see, the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley Sons, England, 1999).

Thus, by the use of single amino a c d substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.

00 C\1 Example 16: Identification and confirmation of 24P4C12-derved sequences with HLA-DR binding motifs SPeptide epitopes bearing an HLA dass II supermotif or motif are identified and confirmed as outlined below using Smethodology similar to that described for HLA Class I peptides.

0 Selection of HLA-DR-supermotif-bearino eptooes.

0 To identify 24P4C12-derived, HLA class II HTL epitopes, a 24P4C12 antigen is analyzed for the presence of Ssequences bearing an HLA-DR-motif or supermoltif. Specifically, 15-mer sequences are selected comprising a DRsupermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino adds total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood t al., J. Immunol.

160:3363-3373,1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors.

Using allele-specific selection tables (see, Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 24P4C12-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7.

Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 p1, DR2w2 p2, OR6w19, and DR9 molecules In secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5wt 1, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 24P4C12-derived peptides found to bind common HLA-DR alleles are of particular interest Selection of DR3 motif peptides Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be 'assayed for their DR3 binding capacity. However, in view of the binding specifdcty of the DR3 motif, peptides binding only to 'DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently Identify peptides that bind DR3, target 24P4C12 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1pM or better, less than 1 pM. Peptides are found that meet this binding criterion and qualify as HLA dass II high affinity binders.

DR3 binding epitopes identified in this manner are induded In vaccine compositions with DR supermotif-bearing peptide epitopes.

00 0 Similarly to the case of HLA dass I motif-bearing peptides, the class II motif-bearing peptides are analoged to Simprove affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Examole 17; Immunoaenlcltv of 24P4C12-derlved HTL epttones 00 This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenidty of HTL epitopes are confirmed ina manner analogous to the determination of immunogenidty of 0 CTL epitopes, by assessing the ability to stimulate HTL responses andlor by using appropriate transgenic mouse models.

O Immunogenicty is determined by screening for: in vitro primary induction using normal PBMC or recall responses from patients who have 24P4C12-expressing tumors.

00

(O

Example 18: Calculation of phenotvpic frequencies of HLA-supertypes In various ethnic backgrounds to determine breadth of population coveraqe This example illustrates the assessment of the breadth of population coverage of a vacdne composition comprised of multiple epitopes comprising multiple supermotifs andlor motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1-(SQRT(1af)) (see, Sidney et al., Human Immunol. 45:79-93,1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1-(-Cgf)j.

Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by Inter-loc combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered total=A+B'(1-A)). Confirmed members of the A3-like supertype are A3, All, A31, A'3301, and A'6801. Although the A3-lke supertype may also include A34, A66, and A'7401, these alleles were not induded in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A'0202, A*0203, A'0204, A*0205, A'0206, A0207, A*6802, and A'6901. Finally, the B7-like supertype-confirmed alleles are: B7, B'3501-03, B51, B'5301, B'5401, B*5501-2, B*5601, B*6701, and 8*7801 .(potentially also B'1401, B'3504-06, B84201, and B'5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the Al and A24 motifs. On average, Al is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnctties when Al and A24 are combined with the coverage of the A2-, A3- and 87-supertype alleles is see, Table IV An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Immunogenidty studies in humans Bertoni at al., J. Cin. Invest 100:503,1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld etal., J Immunol. 159:1648,1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in Identifying candidate epitopes for indusion In a vaccine that is Immunogenic in a diverse population.

00 SWith a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, Swhich is known in the art (see Osbome, M.J. and Rubinstein, A. "A course in game theory" MIT Press, 1994), can be Sused to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred 00 Spercentage is 90%. A more preferred percentage is Example 19: CTL Recognition Of Endoaenouslv Processed Antiaens After Priming N This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as I described herein recognize endogenously synthesized, native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 0.supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An Sadditional six days later, these cell lines are tested for cytotoxic activity on '5Cr labeled Jurkat-A2.1/K target cells in the absence or presence of peptide, and also tested on 51 Cr labeled target cells bearing the endogenously synthesized antigen, I.e. cells that are stably transfected with 24P4C12 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 24P4C12 antigen. The choice of transgenlc mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201 1 Kb transgenic mice, several other transgenic mouse models including mice with human All, which may also be used to evaluate A3 epitopes, and B7 aleles have been characterized and others transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA- DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 20: Activity Of CTL-HTL Conlugated Epitopes In Transeenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 24P4C12-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 24P4C12-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenidty can be achieved by Inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise -an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptldes may be lipldated, if desired.

Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J.

Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are ransgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lpldated CTUHTL conjugate, in DMSO/saline, or If the peptide composition is a polypeptide, In PBS or Incomplete Freund's AdjuvanL Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPSactivated lymphoblasts coated with peptide.

Cell ines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/KI chimeric gene Vitiello at al., J. Exp. Med. 173:1007,1991) 00 In vro CTL activation: One week after priming, spleen cells (30x10 6 cells/flask) are co-cultured at 37°C with Ssyngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x10 6 cells/flask) in 10 ml of culture medium/T25 flask.

After six days, effector cells are harvested and assayed for cytotoxic activity.

L) Assay for cytotoxic activity: Target cells (1.0 to 1.5x10 6 are incubated at 37*C in the presence of 200 pl of sCr.

SAfter 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a Sconcentration of 1 pg/ml. For the assay, 104 s 1 CrJabeled target cells are added to different concentrations of effector cells (final volume of 200 pl) in U-bottom 96-well plates. After a six hour incubation period at 37'C, a 0.1 ml aliquot of supematant Is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The 00 percent specific lysis is determined by the formula: percent specific release 100 x (experimental release spontaneous Srelease)/(maxmum release spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, 5.Cr release data is expressed as lytic units/10 6 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour s 5 Cr release assay. To obtain specific lytic 00 Sunits/106, the lytic units/10 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence Sof peptide. For example, if 30% s'Cr release is obtained at the effector target ratio of 50:1 5x10 s effector cells for 10,000 targets) in the absence of peptide and 5:1 5x10 4 effector cells for 10,000 targets) in the presence of peptide, the spedfic lytic units would be: [(1/50,000)-(1/500,000)] x 10s 18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the Immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled "Confirmation of Immunogencity.' Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21: Selection of CTL and HTL epitopes for Inclusion in a 24P4C12-speclfic vaccine.

This example ilustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nuceic acid sequence, either single or one or more sequences (Le., minigene) that encodes peplide(s), or can be single and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality of epitopes for Inclusion in a vaccine compositon.

Each of the following principles is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responses that are correlated with 24P4C12 clearance. The number of epitopes used depends on observations of patients who spontaneously cear 24P4C12. For example, If it has been observed that patients who spontaneously dear 24P4C12-expressing cells generate an immune response to at least three epltopes from 24P4C12 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an ICso of 500 nM or less for an HLA class I molecule, or for dass II, an ICso of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dct.nih.gov.

In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

00 When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the Ssmallest peptide possible that encompasses the epitopes of interest The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine L) composition Is selected because it has maximal number of epitopes contained within the sequence, it has a high concentration of epitopes. Epitopes may be nested or overlapping frame shifted relative to one another). For example, OO with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino add peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via deavage from the native source. Altematively, an analog can be made of 00 this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-feactivity and/or O binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system N processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif- Sbearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 24P4C12, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of ep'topes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or dears cells that bear or overexpress 24P4C12 Example 22: Construction of "Minigene" Multi-Epitope DNA PlasmiMs This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmld typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes andlor DR3 epitopes. HLA dass Isupermotif or motif-bearing peptide epltopes derived 24P4C12, are selected such that multiple supermotifsknotifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 24P4C12 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally Include sequences that direct the HTL epitopes to the endoplasmic reticulum.

For example, the li protein may be fused to one or more HTL epilopes as described in the art, wherein the CLIP sequence of the i protein is removed and replaced with an HLA cass II epitope sequence so that HLA dass II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA dass II molecules.

This example Illustrates the methods to be used for construction of a minigene-bearing expression plasmld. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus muine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disdosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc.His vector.

00 Overlapping oligonudeotides that can, for example, average about 70 nudeotides in length with 15 nudeotide K overlaps, are synthesized and HPLC-purified. The oligonudeotides encode the selected peptide epitopes as well as 0 appropriate linker nudeotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping ollgonudeotidesn three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95*C for 15 sec, annealing temperature below the Slowest calculated Tm of each primer pair) for 30 sec, and 72*C for 1 min.

For example, a minigene Is prepared as follows. For a first PCR reaction, 5 pg of each of two oligonucleotides are 00 annealed and extended: In an example using eight oligonucleotides, Le., four pairs of primers, oligonudeotides 1+2, 3+4, C 5+6, and 7+8 are combined in 100 p1 reactions containing Pfu polymerase buffer (1x= 10 mM KCL, 10 mM (NH4)2SO4, SmM Tris-chloride, pH 8.75,2 mM MgS04, 0.1% Triton X-100, 100 pg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu Spolymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and 00 the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cydes. Half of the two reactions are then mixed, and cycles of annealing and extension carried out before flanking primers are added to amplify the full length product The full- C length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequendng.

Example 23: The Plasmid Construct and the Degree to Which It Induces Immunogenlcity.

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity' and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of eptope-HLA dass I complexes on the cell surface.

Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, Sijts of al., J.

Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of pepttde-HLA dass I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, Kageyama et al., J. Immunol. 154:567-576, 1995).

Altematively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capadty of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are immunized intramuscularly with 100 pg of naked cDNA. As a means of comparing the level of CTLs Induced by cONA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from Immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity In a 'Cr release assay. The results Indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenidty of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supennotif peptide epitopes as does the polyepitoplc peptide vacdne. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate Immune responses directed toward the provided epitopes.

00 O To confirm the capacity of a dass II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for rthose epitopes that cross react with the appropriate mouse MHC molecule, I-A-restricted mice, for example, are immunized S intramuscularly with 100 pg of plasmid DNA. As a means of comparing the level of HTLs Induced by DNA Immunization, a T group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.

00 CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective O compositions (peptides encoded in the minigene). The HTL response is measured using a H-thymidine incorporation proliferation assay, (see, Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL 00 response, thus demonstrating the in viv immunogenicity of the minigene.

N DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in Scombination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein Bamett at al., Aids Res. and Human Retrovinuses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinla, for 00 example, expressing a minigene or DNA encoding the complete protein of interest (see, Hanke et al., Vaccine 16:439- O 445,1998; Sedegah et Pmc. Natl. Acad. Sci USA 95:7648-53,1998; Hanke and McMichael, Immunol. Letters 66:177- N 181, 1999; and Robinson eta., Nature Med. 5:52634, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2 1 /A transgenic mice are immunized IM with 100 pg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearng peptide. After an incubation period (ranging from 3- 9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are Immunized with 100 pg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an EUSPOT assay.

Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccnia, then assayed for peptide-specific activity in an alpha, beta andlor gamma IFN ELISA.

It is found that the minlgene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL Induction by HLA-A3 or HLA-B7 motif or supermoif epitopes. The use of prime boost protocols in humans is described below in the Example entitled "Induction of CTL Responses Using a Prime Boost Protocol." Example 24: Pentlde Compositions for Prophvlactic Uses Vaccine compositions of the present invention can be used to prevent 24P4C12 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic add comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to Individuals at risk for a 24P4C12-assodated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant such as Incomplete Freunds Adjuvant The dose of peptide for the initial immunization is from about 1 to about 50,000 pg, generally 100-5,000 pg, for a 70 kg patient The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient by techniques that determine the presence of epitopespecific CTL populations In a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 24P4C12-assodated disease.

Alternatively, a composition typically comprising transfecting agents is used for the administration of a nudeic addbased vaccine in accordance with methodologies known in the art and disclosed herein.

00 SExample 25: Polvepitopic Vaccine Compositions Derived from Native 24P4C12 Sequences SA native 24P4C12 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify "relatively short" regions of the polyprotein that comprise multiple epitopes. The relatively short' regions are preferably less in length than an entire native antigen. This relatively short sequence that 00 contains multiple distinct or overlapping, "nested' epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The 'relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids 00 in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is N0 selected because it has maximal number of epitopes contained within the sequence, it has a high concentration of 0 epitopes. As noted herein, epitope motifs may be nested or overlapping frame shifted relative to one another). For Sexample, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present In a 10 amino acid peptide.

00 Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 24P4C12 antigen and at least one CK HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity andlor binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possiblity that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motifbearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 24P4C12, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic add vaccine compositions.

Related to this embodiment computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.

Example 26: Polvepitopic Vaccine Compositions from Multiple Antigens The 24P4C12 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-assocated antigens, to create a vaccine composition that Is useful for the prevention or treatment of cancer that expresses 24P4C12 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that Incorporates multiple epitopes from 24P4C12 as well as tumor-associated antigens that are often expressed with a target cancer associated with 24P4C12 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27: Use of peptides to evaluate an Immune response Peptides of the invention may be used to analyze an Immune response for the presence of specific antibodies, CTL or HTL directed to 24P4C12 Such an analysis can be performed in a manner described by Ogg et aL, Science 279.2103-2106. 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

00 In this example highly sensitive human leukocyte antigen tetrameric complexes (tetramers") are used for a cross- Ssectional analysis of, for example, 24P4C12 HLA-A'0201-specific CTL frequences from HLA A'0201-positive individuals at C different stages of disease or following immunization comprising a 24P4C12 peptide containing an A'0201 motif. Tetrameric U) complexes are synthesized as described (Musey et al, N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and p2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy 0 chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, p2-microglobulin, and peptide are refolded by dilution. The refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin 00 N (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a S1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer- 0phycoerythrin.

0 For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes Sand resuspended in 50 pl of cold phosphate-buffered saline. Tricolor analysis is performed with the tetramer-phycoerythrin, along with anti-C08-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodles on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers indude both A'0201-negative individuals and A'0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readly indicating the extent of immune response to the 24P4C12 epitope, and thus the status of exposure to 24P4C12, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28: Use of Peptlde Epitapes to Evaluate Recall Responses The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 24P4C12-associated disease or who have been vaccinated with a 24P4C12 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 24P4C12 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St Louis, MO), washed three times In HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50Uhnl), streptomycin (50 pg/mi), and Hepes (10mM) containing heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 pglml to each well and HBV core 128-140 epitope Is added at 1 pg/ml to each well as a source of T cel help during the first week of stimulation.

In the microculture format, 4 x 10 5 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 pl/well of complete RPMI. On days 3 and 10,100 pl of complete RPMI and 20 U/ml final concentration of riL-2 are added to each wel. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rlL-2 and 10 Irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific stCr release, based on comparison with nondiseased control subjects as previously described (Rehermann, et at., Nature Med.

2:1104,1108, 1996; Rehermann et aL, J. Cln. Invest. 97:1655-1665,1996; and Rehermann et al. J. Clin. Invest. 98:1432- 1440,1996).

1 00 O Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, MA) or established from the pool of patients as described (Guilhot, et J. Viol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogenetc HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of Sthe invention at 10 pM, and labeled with 100 pCi of 5 'Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well s 5 Cr release assay using U-bottomed 96 well plates

OO

Scontaining 3,000 targets/well. Stimulated PBMC are tested at effectorltarget ratios of 20-50:1 on day 14. Percent Scytotoxicity is determined from the formula: 100 x [(experimental release-spontaneous release)/maximum releasespontaneous release)l. Maximum release is determined by lysis of targets by detergent Triton X-100; Sigma Chemical 0 Co., St Louis, MO). Spontaneous release is <25% of maximum release for all experiments.

00 0The results of such an analysis indicate the extent to which HLA-resricted CTL populations have been stimulated 0 by previous exposure to 24P4C12 or a 24P4C12 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5x10 5 cells/well and are stimulated with 10 pg/ml synthetic peptide of the invention, whole 24P4C12 antigen, or PHA. Cels are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 1OUknl IL-2. Two days later, 1 pCi 3 H-thymidine Is added to each well and Incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3

H-

thymldlne Incorporation In the presence of antigen divided by the H-thymldine Incorporation in the absence of antigen.

Example 29: Induction Of Specific CTL Response In Humans A human dinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows: A total of about 27 individuals are enrolled and divided into 3 groups: Group 1: 3 subjects are injected with placebo and 6 subjects are injected with 5 pg of peptide composition; Group II: 3 subjects are injected with placebo and 6 subjects are Injected with 50 pg peptide composition; Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 pg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenlcity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed In terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection.

Peripheral blood mononudear cells are isolated from fresh heparinlzed blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

00 0 Example 30: Phase II Trials In Patients Expressing 24P4C12 2 Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 24P4C12. The main objectives of the trial are to determine an effective dose and regimen for Inducing CTLs In cancer patients that express 24P4C12, to establish the safety of Inducing a CTL and HTL response in 0 these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, 0e.g., by the reduction andlor shrinking of lesions. Such a study is designed, for example, as follows: The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation 00 protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot CN of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-assodated adverse effects (severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and 0 .the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 24P4C12.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 24P4C12associated disease.

Example 31: Induction of CTL Responses Using a Prime Boost Protocol A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled "The Plasmid Construct and the Degree to Which It Induces Immunogenidty," can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccne, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids" In the form of naked nudeic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nudeic acid (0.1 to 1000 ug) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowipox virus administered at a dose of 5-10' to 5x10i pfu. An altemative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine.

Peripheral blood mononudear cells are Isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 24P4C12 is generated.

Example 32: Administration of Vaccine Compositlons Using Dendritic Cells (DC) Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professionar APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response In vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the Invention. The dendritic cells are infused back into the patient to elcit CTL and HTL responses in vio. The induced CTL 00 and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 24P4C12 protein from which the Sepitopes in the vaccine are derived.

For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom.

A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin T (Monsanto, St Louis, MO) or GM- CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound 00 peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997).

O

00 Although 2-50 x 100 DC per patient are typically administered, larger number of DC, such as 10' or 108 can also be provided.

Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For Sexample, PBMC generated after treatment with an agent such as Progenipoietin" are injected into patients without 00 purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell Sdoses injected into patients Is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin T m mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 10 s DC, then the patient will be injected with a total of 2.5 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenlpoletin T is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex vive activation of CTUHTL responses Alternatively, ex vivo CTL or HTL responses to 24P4C12 antigens can be induced by incubating, In tissue culture, the patients, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused Into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, tumor cells.

Example 33: An Alternative Method of Identifying and Confirming Motif-Bearing Peptides Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cels express only a single type of HLA molecule.

These cells can be transfected with nucleic adds that express the antigen of Interest, e.g. 24P4C12. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild add conditions and their amino add sequence determined, by mass spectral analysis Kubo ef ae., J.

Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an altemative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, they can then be transfected with nucleic acids that encode 24P4C12 to isolate peptides corresponding to 24P4C12 that have been presented on the cell surface. Peptides obtained from such an analysis will bear molif(s) that correspond to binding to the single HLA allele that is expressed In the celL 00 As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the Scell.

00 SExample 34: Complementary Polvnucleotldes Sequences complementary to the 24P4C12-encoding sequences, or any parts thereof, are used to detect, 00 decrease, or inhibit expression of naturally occurring 24P4C12. Although use of oligonucleotides comprising from about Sto 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.

Appropriate oligonudeotides are designed using, OLIGO 4.06 software (National Biosciences) and the coding sequence Sof 24P4C12. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and 00 used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is Sdesigned to prevent ribosomal binding to a 24P4C12-encoding transcript Example 35: Purification of Naturally-occurrina or Recombinant 24P4C12 Using 24P4C12-Specific Antibodies Naturally occurring or recombinant 24P4C12 Is substantially purified by immunoaffinity chromatography using antibodies specific for 24P4C12. An immunoaffinity column is constructed by covalently coupling anti-24P4C12 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmada Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 24P4C12 are passed over the immunoaffinity column, and the column Is washed under conditions that allow the preferential absoibance of 24P4C12 high ionic strength buffers in the presence of detergent).

The column is eluted under conditions that disrupt antibody/24P4C12 binding a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 36: Identification of Molecules Which Interact with 24P4C12 24P4C12, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent (See, Bolton et a. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 24P4C12, washed, and any wells with labeled 24P4C12 complex are assayed. Data obtained using different concentrations of 24P4C12 are used to calculate values for the number, affinity, and association of 24P4C12 with the candidate molecules.

Example 37: In Vivo Assay for 24P4C12 Tumor Growth Promotion The effect of the 24P4C12 protein on tumor cell growth is evaluated in viv by evaluating tumor development and growth of cels expressing or lacking 24P4C12. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of either 3T3, prostate, colon, ovary, lung, or bladder cancer cell lines PC3, Caco, PA-1, CaLu or J82 cells) containing tkNeo empty vector or 24P4C12. At least two strategies may be used: Constitutive 24P4C12 expression under regulation of a promoter, such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterdogous mammalian promoters, the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and Regulated expression under control of an Inducible vector system, such as ecdysone, tetracydine, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper 00 measurement at the appearance of palpable tumors and followed over Urns to determine if 24P4C12-expressing cells grow at a faster rate and whether tumors produced by 24P74C1 2-expressing ceils demonstrate characteristics of altered aggressiveness enhanced metastasis, vascularizaton, reduced responsiveness to chemotherapeutic drugs). As shown in figure 31 and Figure 32, 24P4C12 has a profound effect on tumor growth in SCID mice. The prostate cancer cells PG3 and PC3-24P4C1 2 were injected subcutaneously In the right flank of SCID mice. Tumor growth was evaluated by calipier 00 measurements. An Increase in tumor growth was observed in PC3-24P4C12 tumors within 47 days of injection (fig 31). In addition, subcutaneous injection of 3T3-24P4C12 Induced tumor formation in SCID mice (Figure 32). This finding is 00 significant as control 3T3 cells fail to form tumors, Indicating that NNWC1 has several tumor enhancing capabilities.

N including transformation, as well as tumor Initi ation and promotion.

Example 38: 24P4C12 Monoclonal Antibody-medlated Inhibition of Prostate Tumors In Vivo.

CK1The significant expression of 24P4C12 in cancer tissues, together with Its restrictlive exression In normal tissues 00 and cell surface localization, make 24P4IC12 a good target for antibody thierapy. Similarly, 24P4C12 is a targe! for T cellbased immunotherapy. Thus, the therapeutic efficacy of anti-24P4C12 mAbs In human prostate cancer xenogaft mouse models is evaluated by using recombinant cell lines such as PC3-24P4CI2, and 3T3-24P4C12 (see, Kaighn, et al., Invest Urol, 1979. 17(1): p. 16-23), as well as human prostate xenograft models such as L.APC9 (Safran et at, Proc Nat! Aced Sci U S A. 2001, 98:2658). Similarly, anti-24P34C12 mAbs are evaluated in xenogralt models of human bladder cancer colon cancer, ovarian cancer or lung cancer using recombinant cell lines such as .182-20P4C112, Caco-24P34C12, PA- 24P4IC1 or CaLu-24P4Cl 2, respectively.

Antibody efficacy on tumor growth and metastasis formation is studied, in a mouse orthotopic bladder cancer xenograft model, and a mouse prostate cancer xenograft model. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art Anti-24P4C1 2 mAbs inhibit. formation of prostate and bladder xenografts. Anti-24P54C12 mAbs also retard the growth of established orthotoplc tumors and prolonged survival of tumnor-bearlng mice. These results indicate the utility of anti24P4IC12 mAbs in the treatment of local and advanced stages of prostate, colon, ovarian, lung and bladder cancer. (See, Saifran, el al., PNAS 10:1073-1078 or www.pnas.orgloglldoI1l. 1073/pnas.05 1624698).

Admninistration of the anti24P4C12 mAbs led to retardation of established orthotopic tumor growth and Inhibition cf metastass to distant sites, resultiIn a significant prolongation in the survival of tumnor-bearing mice. These studies indicate that 24P34C1 2 as an attractive target for Immunotharapy and demonstrate the therapeutic potential of anti-24P4C1 2 mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 24P4C12 monoclonal antibodies are ,effective to inhibit the growth of human prostate, colon, ovarian, luing and bladder cancer tumor xenografts grown in SClD mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor Inhibition using multiple unconjugated 24P4C12 mAbs Materials and Methods 24P4C1 2 Monoclonal Antibodies: Monoclonal antibodies are raised against 24P54C1 2 as desabed! in the Example entitled 'Generation of 24P4C1 2 Monoclonal Antibodies (mAbs)." The antibodies am characterized by ELISA, Western blot FACS, and immunoprecipitaion for their capacity to bind 24P4C12. Epitope mapping data for the anti-24P4C1 2 mAbs, as determined by EUSA and Western analysis, recognize epitops on the 24P4C12 protein. tmmunohlstochemical analysis of prostate cancer tissues and cells with these antibodies is performed, 00 The monocdonal antibodies are purified from ascites or hybridoma tissue culture supematants by Protein-G O Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are performed by a Bradford assay (Blo-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or Sorthotopic injections of SCABER, J82, A498, 769P, CaOvi or PA1 tumor xenografts.

00 Cell Lines The prostate, colon, ovarian, lung and bladder cancer carcinoma cell lines,, Caco, PA-1, CaLu or J82 cells as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in media supplemented with L-glutamine 00 .and 10% FBS.

SPC3-24P4C12, Caco-24P4C12. PA-24P4C12, CaLu-24P4C12 or J82-24P4C12 cells and 3T3-24P4C12 cell O populations are generated by retroviral gene transfer as described in Hubert, et al., Proc Nati Acad Sci U S A, 1999.

C96(25): 14523.

SXenograft Mouse Models.

Subcutaneous tumors are generated by injection of 1 x 10 cancer cells mixed at a 1:1 dilution with Matrigel S(Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell Injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoconal antibody that recognizes an irrelevant antigen not expressed in human cells. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as: Length x Width x Height Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic Injections are performed under anesthesia by using ketamine/xylazine. For bladder orthotopic studies, an incision is made through the abdomen to expose the bladder, and tumor cells (5 x 10 5 mixed with Matrigel are injected into the bladder wall in a 10-pl volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure BTA levels. For prostate orthopotic models, an incision Is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. Tumor cells e.g.

LAPC-9 cells (5 x 10 s mixed with Mabigel are injected into the prostate in a 10-pl volume (Yoshida Yet al, Anticancer Res.

1998,18:327: Ahn et al, Tumour Biol. 2001, 22:146). To monitor tumor growth, blood is collected on a weekly basis measuring PSA levels. Similar procedures are followed for lung and ovarian xenografl models. The mice are segregated Into groups for the appropriate treatments, with anti-24P4C12 or control mAbs being injected I.p.

Anti-24P4C12 mAbs Inhibit Growth of 24P4C12-Expressino Xenograft-Cancer Tuors The effect of anti-24P4C12 mAbs on tumor formation is tested on the growth and progression of bladder, and prostate cancer xenografts using PC3.24P4C12, Caco-24P4C12, PA-24P4C12, CaLu-24P4C12 or J82-24P4C12 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate, colon, ovary, lung and bladder, respectively, results in a local tumor growth, development of metastasis In distal sites, deterioration of mouse health, and subsequent death (Saffran, et al., PNAS supra; Fu, X, et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, J Cell Biochem, 1994. 56(1): p. The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, tumor cells are Injected into the mouse organs, and 2 days later, the mice are segregated into two groups and treated with either a) 200-500pg, of anti-24P4C12 Ab, or b) PBS three times per week for two to live weeks.

A major advantage of the orthotopic cancer models is the ability to study the development of metastases.

Formation of metastasis in mice bearing established ortholopic tumors is studies by IHC analysis on lung sections using an 00 O antibody against a tumor-specific cell-surface protein such as antl-CK20 for bladder cancer, anti-STEAP-1 for prostate O cancer models (Ln S et al, Cancer Detect Prev. 2001;25:202; Saffran, et a, PNAS supra).

SMice bearing established orthotopic tumors are administered 1000g injections of either anti-24P4C12 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of 0 metastasis formation In mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the 0presence of tumor cels by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-24P4C12 antibodies on initiation and progression of 00 prostate and kidney cancer in xenograft mouse models. Anti-24P4C12 antibodies inhibit tumor formation of tumors as well N as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-24P4C12 mAbs demonstrate a dramatic inhibitory effect on the spread of local bladder and prostate tumor to distal sites, even in the 0presence of a large tumor burden. Thus, anti-24P4C12 mAbs are efficacious on major clinically relevant end points (tumor 0 growth), prolongation of survival, and health.

0O Example 39: Therapeutic and Diagnostic use of Antil24P4C12 Antibodies in Humans.

Anti-24P4C12 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic andlor therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-24P4C12 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 24P4C12 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic andlor prognostic indicator. Anti-24P4C12 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-24P4C12 mAb specifically binds to carcinoma cells. Thus, anti-24P4C12 antibodies are used in diagnostic whole body imaging applications, such as radiolmmunosdntigraphy and radioimmunotherapy, (see, Potamianos et al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 24P4C12. Shedding or release of an extracellular domain of 24P4C12 Into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. Hepatology 27:563-568 (1998)), allows diagnostic detection of 24P4C12 by anti-24P4C12 antibodies in serum andlor urine samples from suspect patients.

Anti-24P4C12 antibodies that specifically bind 24P4C12 are used in therapeutic applications for the treatment of cancers that express 24P4C12. Anti-24P4C12 antibodies are used as an unconjugated modality and as conjugated form In which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radiolsotopes. In preclinical studies, unconjugated and conjugated antl-24P4C12 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, kidney cancer models AGS-K3 and AGS-K6, (see, the Example entitled "24P4C12 Monodonal Antibody-mediated Inhibition of :Bladder and Lung Tumors In Vivo). Either conjugated and unconjugated anti-24P4C12 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described In following Examples.

Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Antl.24P4C12 Antibodies In vi Antibodies are used in accordance with the present invention which recognize an epitope on 24P4C12, and are used in the treatment of certain tumors such as those listed In Table I. Based upon a number of factors, including 24P4C12 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

00 0 Adjunctive therapy: In adjunctive therapy, patients are treated with anti-24P4C12 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as _Q those listed in Table I, are treated under standard protocols by the addition anti-24P4C12 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-24P4C12 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycn (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (predinical).

0 SII.) Monotherapy: In connection with the use of the anti-24P4C12 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some Sdisease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

00 III.) Imaging Agent Through binding a radionudide iodine or yttrium (I131, Yo) to anti-24P4C12 Santibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesIons of cells expressing 24P4C12. In connection with the use of the anti-24P4C12 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains andlor returns.

In one embodiment a In)-24P4C12 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 24P4C12 (by analogy see, Dlvgl etal. J NatL Cancer Inst. 83.97-104 (1991)).

Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified Dose and Route of Administration As appreciated by those of ordinary skil in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-24P4C12 antibodies can be administered with doses in the range of 5 to 400 mg/m 2 with the lower doses used, in connection with safety studies. The affinity of anti-24P4C12 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skil in the art for determining analogous dose regimens. Further, anti-24P4C12 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower dearance; accordingly, dosing in patients with such fully human anti-24P4C12 antibodies can be lower, perhaps In the range of 50 to 300 mgn 2 and still remain efficadous. Dosing In mg/m2, as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and Is a convenient dosing measurement that is designed to include patients of al sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-24P4C12 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct. other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that Is appropriate for regional perfuslon. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term dearance of the antibody.

Cinical Develooment Plan (CDP) Overview. The COP follows and develops treatments of anti-24P4C12 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent Trials initially demonstrate safety and thereafter confirm efficacy In repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-24P4C12 antibodies. As will 00 0 be appreciated, one criteria that can be utilized In connection with enrollment of patients is 24P4C12 expression levels in their tumors as determined by biopsy SAs with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to cytokine release syndrome, hypotension, fever, shaking, chills; (ii) the development of an immunogenlc response to the material 00 development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxidty to 0normal cells that express 24P4C12. Standard tests and follow-up are utilized to monitor each of these safety concerns.

Anti-24P4C12 antibodies are found to be safe upon human administration.

00 C1 Example 41: Human Clinical Trial Adiunctive Therapy with Human Antl-24P4C12 Antibody and Chemotherapeutlc SAgent 0A phase I human cinical trial is initiated to assess the safety of six intravenous doses of a human anti-24P4C12 00antibody In connection with the treatment of a solid tumor, a cancer of a tissue listed in Table I. In the study, the safety 0of single doses of anti-24P4C12 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: dsplatin, topotecan, doxorubidn, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-24P4C12 antibody with dosage of antibody escalating from approximately about 25 mg/ 2 to about 275 mg/m over the course of the treatment in accordance with the following schedule: DayO Day7 Day 14 Day21 Day 28 mAb Dose 25 75 125 175 225 275 mghn 2 mg/m 2 mg/m z mg/m g/ mg /m 2 mg/ 2 Chemotherapy 4 (standard dose) Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i cytokine release syndrome, hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material development of human antibodies by the patient to the human antibody therapeutic,.or HAHA response); and, (ii) toxicity to normal cells that express 24P4C12. Standard tests and folow-up are utilized to monitor each of these safety concerns. Patients are also assessed for dinical outcome, and particularly reduction In tumor mass as evidenced by MRI or other imaging.

The anti-24P4C12 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42: Human Clinical Trial: Monotherapv with Human Antl.24P4Ci2 Antibody Anti-24P4C12 antbodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such tial Is accomplished, and entails the same safety and outcome analyses, to the abovedescribed adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-24P4C12 antibodies.

Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-24P4C12 Antibody Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human cinical trial is conducted concerning the use of anti-24P4C12 antibodies as a diagnostic imaging agent The protocol is 00 designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 C(1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44: Homoloav Comparison of 24P4C12 to Known Sequences 00 The 24P4C12 protein of Figure 3 has 710 amino acids with calculated molecular weight of 79.3 kDa, and pl of 8.9.

0 Several variants of 24P4C12 have been identified, including 4 SNPs (namely v.1, v.3, v.5, v.6) and 3 splice variants (namely v.7, v.8 and v.9) (figures 10 and 11). 24P4C12 variants v.3, v.5, and v.6 differ from 24P4C12 v.1 by 1 amino acid each, at aa 00 positions 187, 326 and 436, respectively. Variant v.7 carries a deletion of 111 aa long starting at aa 237, while variant v.8 N and v.9 contain insertons at aa 642 and 378, respectively. The 24P4C12 protein exhibits homology to a previously cloned human gene, namely NG22 also known as chorine transporter-like protein 4 (gi 14249468). It shows 99% identity and 99% Ohomology to the CTL4 protein over the length of that protein (Figure 24P4C12 is a multi-transmembrane protein, 00 predicted to carry 10, 11 or 13 transmembrane domains. Blolnfonnatic analysis Indicates that the 24P4C12 protein localizes Sto the plasma membrane with some endoplasmic reticulum localization (see Table Recent evidence indicates that the C 24P4C12 protein is a 10 transmembrane protein that localizes to the cell surface (ORegan S et al PNAS 2000, 97:1835).

Choline as an essential component of cell membranes that plays an important role in cell integrity, growth and survival of normal and tumor cells. Choline accumulates at increased concentration in tumor cels relative to their normal counterparts and as such constitutes a tool for the detection of cancer cells by magnetic resonance imaging (Kurhanewicz J et al, J Magn Reson Imaging. 2002.). In addition to its role in maintaining membrane integrity, choline mediates signal transduction event from the membrane to the nucleus (Spiegel S, Milstien S. J Membr Biol. 1995,146:225). Choline metabolites include sphingosylphosphorylcholine and lysophosphatidylcholine, both of which activate G-protein coupled receptors (Xu F et al Blochim Blophys Acta 2002,1582:81). In addition, choline results in the activation of kinase pathways including Raf-i (Lee M, Han SS, Cell Signal 2002,14:373.). Choline also plays a role in regulating DNA methylation and regulation of gene expression. For example, choline methabolites regulate the expression of cytokines and chemokines essential for tumor growth (Schwartz BM et al, Gynecol Oncol. 2001, 81:291; Denda A et al, Cardnogenesis. 2002, 23:245).

Due to its effect on cell signaling and gene expression, choline controls cell growth and survival (Holmes-McNary MQet al, J Biol Chem. 2001, 276:41197; Albright et al, FASEB 1996,10:510). Choline deficiency results in cel death, apoptosis and transformation, while accumulation of choline is associated with tumor growth (Zelsel S et al, Carcinogenesis 1997, 18:731).

-Acordingly, when 24P4C12 functions as a regulator of tumor formation, cell proliferation, invasion or cell signaling, 24P4C12 is used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Examole 45: Identification and Confirmation of Potential Silnal Transduction Pathways Many mammalian proteins have been reported to interact with signaling molecules and to participate In regulating signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, choline have been reported to activate MAK cascades as wel as G proteins, and been associated with the DAG and ceramide and sphingophosphorylcholine signaling pathway (Cummings et al, above). In addition, choline transmit its signals by regulating choline-kinase and phospholipase activity, .:-resulting in enhance tumorigenic effect (Ramirez et al, Onoogene. 2002, 21:4317; Lucas et al, Oncogene. 2001, 20:1110; Chung T et al, Cell Signal. 2000, 12:279).

Using immunoprecipitation and Westem blotting techniques, proteins are identified that associate with 24P4C12 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 24P4C12, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenicsurvival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003; J. Cell Biol. 1997,138:913). Using Western blotting and other techniques, the ability of 00 24134C12 to regulate these pathways is confirmed. Cells expressing or lacing 24P4C1 2 are either left untreated or stimulated with cytokines androgen and anti-integrin antibodies. Cell lysates are analyzed using anti-hospho-spedfic r-K1 antibodies (Cell Signaling. Santa Cruz Biotechnology) in order to detect phosphorylatlon and regulation of ERK. p3B, AKT, P13K, PLC and other signaling molecules.

To confirm that 24P4IC12 directly or indirectly activates known signal trantsduction pathways in cells, luciferase (luc) 00 based transcriptional reporter assays are carried out In cells expressing individual genes. These trascriptioanal reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription facors, signal transduction pathways, and 00 activation stimuli are listed below.

1 NFkB-luc, NFkB/Rel; lk-kinaseSAPK; growthlapoptosls/stress 2. SRE-luc, SRFITCFIELKI; MAPK/SAPK; growth/differentiation 00 3. AP-1-luc, FOSJUIN; MAPKJSAKIPKC; groWthapoptoslslstress 4. ARE-luc, androgen receptor, steroidsMAK; growthldifferenflationapoptosis p53-tuc, p53; SAPK; growth/differentiationlapopfosis 6. CRE-tuc, CREB/ATF2; PKA/p38; growthlapoptosislstress 7. TCF-luc, TCFILef; &-ctenin, Adhesionlinvaslon Gone-mediated effects can be assayed in cells showing rnRNA expression. Luciferase reporter plasmids can be Introduced by lipid-mediated transfection (TFX.50, Promega). Luciferase activity, an Indicator of relative transcriptional activity, is measured by incubation of cell extracts with tuciferi substrate and luminescence of the reaction Is monitored in a lulminonieter.

Signaling pathways actvated by 24P4C1 2 are mapped and used for the identification and validation of therapeutic targets. When 24P4C12 is involved in call signaling, it is used as target for diagnostic, prognostic, preventative andfor therapeutic purposes.

Examols 46: 24P4C12 Functions as a Chollne transporter Sequence and homology analysis of 24P4C12 Indicate that 24P4C12 carries a transport domain and that 24P4C12 functons as a cholIne transporter. In order to confirm that 24P4C12 transports choline, primary and tumor cells, indludeing prostate, colon, bladder and lung lines, are grown In the presence and absence of 3 H--choline. Radioactive choline uptake is measured by counting incorporated counts per minutes (cpm). Parental 24P4C1 2 negative cells are compared to 24P4C12expressing cells using this and similar assays. Similarly, parental and 24P4C1 2-expressing cells can be compared for chollne content using NMR spectroscpy. These assay systems can be used to identify small molecules and antibodies that interfere with choline uptake and/or with the functon of 24P4C12.

Thus, compounds and small molecules designed to Inhibit 24P4C1 2 function and downstream signaling events are used for therapeutic diagnostic, prognostic and/or preventative purposes.

Examile 47: Regulation of Transcription The cell surface localization of 24P4C12 and its ability to regulate DNA methylation Indicate that it is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confimed, e.g..

by studying gene expression In cells expressing or lacing 24P4C12. For this purpose, two tye of experiments are performed.

00 0 In the first set of experiments, RNA from parental and 24P4C12-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as Scells treated with FBS, pheromones, or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art The differentially expressed genes are then mapped to biological pathways (Chen Ketal. Thyroid. 2001. 11:41.).

0O In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs Including: NFkB-luc, SRE-luc, ELK-luc, ARE-luc, p53-luc, and CRE-luc.

These transcripional reporters contain consensus binding sites for known transcription factors that lie downstream of well- 00 N characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulatorsof pathway activation.

Thus, 24P4C12 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative Sandlor therapeutic purposes.

00 SExample 48: Involvement in Tumor Proaression The 24P4C12 gene can contribute to the growth of cancer cells. The role of 24P4C12 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, and bladder cell lines, as well as NIH 3T3 cells engineered to stably express 24P4C12. Parental cells lacking 24P4C12 and cells expressing 24P4C12 are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, et al., Prostate 2000;44:61, Johnson DE, Ochieng J, Evans SL Anticancer Drugs. 1996, 7:288). Such a study was performed on prostate cancer cells and the results are shown in figure 28. The growth of parental PC3 and PC3-24P4C12 cells was compared in low and 10% FBS. Expression of 24P4C12 Imparted a growth advantage to PC3 cells grown in 10% FBS. Similarly, expression of 24P4C12 in NIH-3T3 cells enhances the proliferation of these cells relative to control 3T3-neo cells. The effect of 24P4C12 can also be observed on cell cycle progression. Control and 24P4C12-expressing cells are grown in low serum ovemight and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU and propidium Iodide incorporation by FACS analysis.

To confirm the role of 24P4C12 in the transformation process, its effect In colony forming assays is investigated.

Parental NIH-3T3 cells lacking 24P4C12 are compared to NIH-3T3 cells expressing 24P4C12, using a soft agar assay under stringent and more permissive conditions (Song Z at al. Cancer Res. 2000;60:6730).

To confirm the role of 24P4C12 in Invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Control cells, Including prostate, and bladder cell lines lacking 24P4C12 are compared to cells expressing 24P4C12. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane analog the Transwel Insert) and used In the assay. Invasion Is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.

24P4C12 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 24P4C12 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol.

1988, 136:247). In short cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the Gi, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 24P4C12, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cydoheximide. Cells 00 are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 24P4C12 can play a critical role in regulating tumor progression and tumor load.

CK1 When 24P4C12 plays a role in cell growth, transformation, Invasion or apoptosis, it is used as a target for Sdiagnostic, prognostic, preventative and/or therapeutic purposes.

00 Example 49: Involvement In Angioqenesis Angiogenesis or new capillary blood vessel fomnalion is necessary for tumor growth (Hanahan D, Folkman J. Cell.

1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effectof phsophodieseterase inhibitors on 00 endothelial cells, 24P4C12 plays a role in angiogenesis (DeFouw L et al, Microvasc Res 2001,62:263). Several assays Ni have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothefial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 24P4C12 SIn angiogenesis, enhancement or inhibition, is confirmed.

00 For example, endothelial cells engineered to express 24P4C12 are evaluated using tube formation and Sproliferation assays. The effect of 24P4C12 is also confirmed In animal models in vivo. For example, cells either expressing K or lacking 24P4C12 are implanted subcutaneously in immunocompromised mice. Endothellal cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 24P4C12 affects angiogenesis and it is used as a target for diagnostic, prognostic, preventative andlor therapeutic purposes.

Example 50: Involvement In Adhesion Cell adhesion plays a critical role in tissue colonization and metastasis. The presence of leucine rich and cysteine rich motifs in 24P4C12 is Indicative of its role in cell adhesion. To confirm that 24P4C12 plays a role in cell adhesion, control cells lacking 24P4C12 are compared to cels expressing 24P4C12, using techniques previously described (see, Haleret al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst 1998,90:118). Briefly, in one embodiment, cels labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. This experimental system can be used to identify proteins, antibodies andlor small molecules that modulate cell adhesion to extracellular matrix and cel-cell interaction. Since cell adhesion plays a critical role in tumor growth, progression, and, colonization, the gene involved in this process can serves as a diagnostic, preventative and therapeutic modality.

Example 51: Detection of 24P4C12 protein In cancer patient specimens To determine the expression of 24P4C12 protein, specimens were obtained from various cancer patients and stained using an affinity purified polydonal rabbit antibody raised against the peptide encoding amino acids 1-14 of 24P4C12 variant 1 and conjugated to KLH (See, Example 10: Generation of 24P4C12 Polyclonal Antibodies.) This antiserum exibited a high titer to the peptide (>10,000) and recognized 24P4C12 in transfected 293T cells by Westem blot and flow cytometry (Figure 24) and in stable recombinant PC3 cells by Western blot and immunohistochemlstry (Figure 25). Formalin fixed, paraffin embedded tissues were cut into 4 micron sections and mounted on glass slides. The sections were dewaxed, rehydrated and treated with antigen retrieval solution (0.1M Tris, pH10) at high temperature. Sections were then incubated in potydonal rabbit anti-24P4C12 antibody for 3 hours. The slides were washed three times in buffer and further incubated with DAKO EnVision+T peroxidase-conjugated goat anti-rabbit immunoglobuln secondary antibody (DAKO Corporation, Carpenteria, CA) for 1 hour. The sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxyin, and analyzed by bright field microscopy. The results showed expression of 24P4C12 in cancer patients' tissue (Figures 29 and 30). Tissue from prostate cancer patients showed expression of 24P4C12 in the 00 ri tumor cells and in the prostate epithelium of tissue normal adjacent to tumor (Figure 29).

d Generally, expression of 24P4C12 was high in all prostate tumors and was expressed mainly T around the cell membrane indicating that 24PC12 is membrane associated in prostate tissues.

00 0 All of the prostate samples tested were positive for 24P4C12. Other tumors that were positive for 24P4C12 included heart, skeletal muscle, liver, brain, spinal cord, skin, adrenal, lymph 00 node, spleen, salivary gland, small intestine and placenta. None demonstrated any expression (N of 24P4C12 by immunohistochemistry. Normal adjacent to tumor tissues were also suited to Sdetermine the presence of 24P4C12 protein by immunohistochemistry. These included, breast, 00 lung, colon, ileum, bladder, kidney and pancreas. In some of the tissues from these organs 00 there was weak expression of 24P4C12. this expression may relate to the fact that the samples were not truly normal and may indicate a precancerous change. The ability to identify malignancy in tissue that has not undergone obvious morphological changes is an important diagnostic modality for cancerous and precancerous conditions.

These results indicate that 24P4C12 is a target for diagnostic, prophylactic, prognostic and therapeutic applications in cancer.

Throughout this application, various website data contact, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web).

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

TABLES:

TABLE 1I: Tissues that Express 24P4C12: a. Maignant Tissues i Prostate Blad der Kidney Lung Colon Ovary Breast Uterus Stomach TABLE 11: Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu Ioucine S Ser senne Y Tyr tyrosine C Cys cysteinve W Trp btophan P Pro proline H His histidlne 0 Gln glutainine R Arg arkiline Ilie isoleucine M met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A AJa alanine D Asp aspartic acid E Glu glutamic acdd G I Gly gfydne 00 TABLE III: Am~ino Acid Substitution Matrix Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL 00 ikp.unibe.chlmanual1sun162.htlfll A C D E~ F G H I KL M N P Q R S T V W Y.

40-2 -1 -2 0-2-1-1 -1 1-2 1-1- 1 10 0 -3-2 A 9 -3 -4 -2 -3 -3 -1 -3 1 -1 -3 -3 -3 -3 11 -1 -2 -2 C 00 6 2 1-1 -3 1-4-3 1-1 0 -2 0-1 -3 -4-3D N1 5-3 -2 0 -3 1 -3 -2 0-1 2 0 0-1 -2 -3 -2 E 6 -3-1 0 -3 0 0 -3 -4-3 -3-2 -2 -1 1 3 F 6 -2 -4-2 -4-3 0 -2-2 -2 0 -2 -3 -2-3 G 8 -3 1-3-2 1 -2 0 0 1-2-3 -2 2 H 4~ 4-3 2 1 -3-3 -3 -3-2-1 3 -3 1I 00 5 -2-1 0-1 1 2 0-1 -2 -3-2 K 4 2 -3-3 -2-2 -2-1 1 -2-1I L -2 -2 0 -1-1-1 1 -1 -11M 6~ 6-2 0 0 1 0 -3 -4-2 N 7 -1 -2 -1 -1 -2 -4 -3 P 1 0-1 -2 -2 -1Q -1 -1 -3 -3 -2 R 4 1 -2 -3-2 S 0 -2 -2 T 4 -3 1 V 11 2 W 7 Y TABLE IV: HLA Class till MotiflSuperMOtifiS TABLE IV HIA Class I SupermotifsMotlfs SUPERMOTIF POSITION POSITION POSITION 2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Al TIL VMS

FWY

A2 LIVMATQ lVMATL A3 VSMATUJ

RK

A24 YFWIVLMT FIYWtM B7 P

VILFMWYA

B27 RHK _________FYLWMIVA B44 ED

___FWYLUMVA

ATS _________FWYLIVMA B62 01VA4P _________FWYMIVLA MOTIFS Al TSM y Al IDEAS V A21 LMVQ1AT

VLIMAT

A3 LMVISATFCGD All VrMLISAGNCDF

KRYH

A24 YFWM

FLIW

A13101 MVTAIJS

RK

P'3301 MVALFIST

RK

A6801 AVTMSLI

RJ(

8*0702 P

LMFWYAIV

B'3501 p

LMFWYIVA

B51 P ________UVFWYAM 135301 P IMFWYALVI B-5401 P IATIVLMFW J Bolded residues are preferred, italicized residues are less preferred: A peptlde is considered motl-beaing If It has pimary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE IV HLA Class 11 Supermnotif 1 6 9 W F. Y, V,L A, V1.L, P,C, S, T A, V,1, L, C,S, T,M. Y TABLE IV HLA Class 11 Motifs MOTIFS 1l*anchorl1 2 3 4 5 1 *achor 6 7 8 9 DR4 preferred FMYLJVW M T I VSTCPALIM MH MH deleterious W R WDE DRI preferred MFLIVWY' PAMQ VMATSPLJC M AVM deletenous C CH- FD) CWD GDE D DR7 preferred MFLJVWV M W A IVM~SACTPL M IV delterious C G GIRD N G PMMOTIFS I*anchori1 2 3 1'anchor 4 5 1lawachor 6 Moiff a preferred UIVMFY D MWfi b preferred LIVMFAY DNOEST KRH DR Supemiotif MFLJVWY

VMSTACPLI

Italicized residues indicate less preferre or tolerated" residues TABLE IV HLA Class I Supermotifls POSITION: 1 2 3 4 5 6 7 8 C-terbus

SUPER-

MOTIS

Al 11*Anchor 1 ILVMS

FWY

A2 r-Anchor 1*Aco UVMATQ

UIVMAT

A3 Preferred 1 Anchor YFW YFW YFW P I* Anch VSMATI (Y15) RK deleterious DE (315); DE P (5/5)(45 A24 191 Anchor YFWIVLMT FP[iLM B7 Preferred F'W(5/5) 1 jAnho FWY FWY *Anchor LIMI P (415) (315) VILFMWYA deleterious DE DE G QIN DE P(515); (415) 627 11 AnchorJAc RHK FYI WMI VA B44 11.iichr1 Anchor ED

FWYLIMVA

B58 I nhr1 Anco ATS FWYLIVM4 852 1 0 n"1Anchr QLjvmP

FWYMIVLA

Italiized residues indicate less preleffed or Woerated' residues TABLE IV HLA Class I MOWiS POSITION 1 2 3 4 5 6 7 8 9 ctermninus or C-terininus Al preferred GFYW J*Anctror DEA YEW 9-nmer STM deleterious DE RHKLIVMP A Al preferred GRHK AS§TCUVM I 9 Anchor GSTC 9-mer

DEAS

deleterious A RHKDEPYFW DE Al preferred YFW I Anchor DEAQN A

STM

mer deleterious GP RHKGUVM DE Al preferred YFW STCLIVM I.A A

DEAS

mer deleterious RHK RHKDEPYFW AZ 1 preferred YFW l*Andi YFW STC 9-mer LMIVQAT deleteriou DEP DERKH POSITION:.1 3 4 A2Z1 preferred AYFW PoAnclior LAIM G ID- LMIVQAT mer deleterious DEP DE RKHA A3 preferred RHK l*Anctior YFW PRHK LMV1SATFCGD W deletenous DEP DE All preferred A loAnchor YFW YFW VrLMISAGNtCD

F

deleterious DEP A24 preferred YFWRHK J1Al STC 9-met YFWM deleteriou DEG DE G A24 Preferred 1*Ancho

YFWM

mner Deleterious GDE ON A310 Preferred 1*1K J2!A12! YFW P

MVTAUS

Deleterious DEP DE A330 Prfre opnc YFW 1 MVALFIST DeleteriousGP

DE

A680 Preferred YFWSTC J£hcti 1 AVTMSIJ deleterious GP DEG B070 Preferred RHKFWY 1&Anc RHK 2 P deleterios DEQNP DEP DE.

P DEON YFW P*Andao

Y

G A ASTC LIVM DE l*Ancho

Y

PON 1*1K PG GP YFWON PASTC GDE P I Anchior

Y

RHK ONA RHKY'FW 1*1K A YFW PG G YEW l 0 Anchor P G PRHK QN YEW A P 1 Andio

VLMAT

RKI4 DERKH 5 6 7 8 9 C- Terminus G FYWL 1*Anchor vim VLIMAT P RKH DERK RXH

H

YEA YFW P P*Anchor

KYRHFA

A YEW YEW P ilnm KR YH A G YFW YEW I :&nc

ELIW

QINP DERH G AQN

K

YFWP P l'Anchor FuIW RHK DE A QN DEA YEW YFW AP VAnDor

RK

ADE DE DE DE AYFW fl

RK

YFWUIV YEW P rlnm M

RK

RHK *A RHK 1*1K 1*1K. PA £'Aon]

LMFWYAJ

V

DE GDE ON DE POSMON 1 2 3 4 5 6 7 8 9 Cterminus or C-tem~nus Al preferred GFYW I *Anchor DEA YFW P DEON YFW l Archor 9-er STM y deleterious DE RHKLIVMP A G A Al preferred GRHK ASTCLIVM J*Ancho GSTC ASTC LIVM DE Vj&c 9-mer DEAS Y deleterious A RHKDEPYFW DE PQN RHK PG GP B350 Preferred FWYLIM j~I*nh FWY FWY I I P

LMFWY/V

A

deleterious AGP G G 651 Preferred UVMFWY' 1*Anh FWft STC FWY G FWY jIfln3h P

UVFWYA

A4 deleterious AGPOER DE G DEON GDE

HKSTC

B530 preferred LIVMVFWY £'Anh FWY STC FIW UIVMFW FWx' £*Ar~ 1 P Y IMFWYAL

V

deleterious AGPQN G RHKQN DE B540 preferred FWY~ 1*nh FWYUVM UIVM ALIVM FWYA 1VAndio 1P P ATIVLMF

WY

deleterious GPQNDE GDESTC RHKDE DE QNDGE DE TABLE IV umma~y of HLAsupertypes all phenotypic frequencies of HLA-superlypes in different ethnic populations Specificity Pheno requcncy pe oition 2 Terminusiaucasian A Black apa nes spani e 7 V 3.2 .5.1 .1 3.0 K9.3 LMVST 7.5 '2.1 5.8 27 311 .2 LMVT MVT 5.8 '9.0 2.4 5.9 3.0 2 4 VI.L 3.9 8.9 .6 0.1 8.3 0.0 J) LIMV 3.0 1.2 2.9 9.1 9.0 1 (LVMS) 7.1 1 6.1 1.8 4.7 6.3 5.2 27 HK .4 6.1 3.3 i3.9 5. 4 62 LOVMP WY(M 2.6 .8 .5 5. 11 81 58 TS 11 0.0 5. .6 .0A.

TABLE IV Cacuated touaton coverag afforded by different HLA-supertop combinations HLA-supertypes Phenotypic frequency aucasian A Blacks Jpanese Inese isanic erawe 6.1 87.5 A4 .3 6.2 19A3 and B7 .5 8.1 100.0 .5 .4 .3 A3, 87. A24, .9 9.6 100.0 .8 9.9 .8 nd Al ,A3,137, A24.

.Al. 827. B62, nd B 58 otiIndicate the residues defining supeutype specificites. The motifs "nororate residues determined on the basis of ublished data to be recognized by multiple alleles within the supertype. Residues Wthin brackets are additional residues (so predicted to be tolerated by multiple alleles within the supertype.

Table V: Frequently Occurring Motifs Nm vrg. Description eWntial Function ame ~~den 4udeic acid-binding protein functions as ranscription factor, nuclear location *fC21-2 Zinc finger, C2H-2 type 7obable Cytochrome b(N- netbrane bound oxidase, generate cyobromeL bN 6% termlnal)/b6/peIB speroxide lomains are one hundred amino acids ong and Include a conserved lo19% Immunoglobulin domain ntradomain disulfide bond.

anem repeats of about 40 residues, )ach containing a Trp-Asp motif.

-unction In signal transduction and 18% WDdomain, G-beta repa xtam interaction iay function In targeting signaling 02 PDZ domain wlcles to sub-membranoujs sites JI 8% Leucine Rich Repeat Mort sequence mothf Involved in _________tei-protein interactions mnsved catalytic core common to oth serlnelthreonine and tyrosine rotein kinases containing an ATP kinase 23% Prtein kinase domain Inding site and a cataytic site PH 16% PH domain A~l th oskeleton EGF 3% 1GF-tike domain bond proteins or in secreted proteins Reverse transaiptase RNA-dependent DNA Rvt 49% olymerase) 'ytoplasmic; protein, associates integral Ak 25% k repeat 'nmbrane proteins to the cytoskeleton NADH- iembrane associated. Involved in biquinonelplastoquinone xoton translocatlon across the xiddoredgql 32% (complex various chains iembrane cium-binding domain, consists of a12 sidue loop flanked on both sides by a Ilhand 24% EF hand 12 residue alpha-helical domain letroviral aspartyl partyl or acid proteases, centered on Rvp 79% )rotease cataytc aspartyl residue !xtracellular structural proteins Involved hlx -n formation of connective tissue. The :lagen triple hlxrepeat ieuence consists of the G-X-Y and the Cllagen. 42% 20 Cois ypeptide chains forms a triple helixt t=ed In the extrawelular ligand- Wing region of receptors and is about o amino acid residues long withi two *of cysteines involved in disulfide Fn3 0% ibronectin type Ill domain nds ven hydrophobic transmembrane eglons, with the N-temininus located transmembrane receptor xtracellulaly while the C-terminus Is 19% Ertodopsin family) a lasmic. Signal thirough G proteins Table VI: Motifs and Post-translational Modifications of -24P4C12 N-glywosylation site 29 -32 NRSC (SEQ ID NO: 48) 69 -72 NSTG (SEQ IDNG. 49) 158 NMTV (-SEQ ID NO, 197 -200 NDTT (SEQ IDNO,. 51) 298- 301 NLSA (SEQ ID NO: 52) 393- 396 NISS (SEQ I13 NO: 53) 405 -408 NTSC (SEQ ID NO, 54) 416 -419 NSSC (SEO ID NO: 678- 681 NGSL (SEQ ID NO: 56) Protein kinase C phosphorylation site 22 -24 SIR~ 218 -220 SvK 430 -432 SsK 494 -496 TIR 573 -575 SaK 619-621 SgR Casein kinase 11 phosphorylation site 31 34 SCID (SEQ ID NO: 57) 102- 105 SVAE (SEQ ID NO: 58) 119 -122 SCPE (SEQ ID NO, 59) 135 -138 1VGE (SEQ ID NO, 304 -307 MVE (SEQID NO, 01) Tyrosine kinase phosphorylation site 6 13 RDEDDEAY (SEQ ID NO: 62) N-mnyistoylation site 72 -77 GAYCGM (SEQ ID NO: 63) 76.-81 GMGENK (SEQ ID NO: 64) 151 156 GVPWNM (SEQ 10 NO: 207 .212 GLIOSI. (SEQ ID NO: 66) 272- 277 GIYYCW (SEQ ID NO: 67) 287 292 GASISO (SEQ ID NO, 68) 349 354 GQMMST (SEQ ID NO: 69) 449- 454 GLFWTL (SEQ ID NO: 467 -472 GAFASE (SEQ ID) NO: 71) Aniidation site 695 698 IGKK (SEQ ID NO: 72) Leucine zipper pattern 245. 266 LFILLLRLVAGPLVLVLILGVL (SEQ ID NO: 73) Cystelne-dch region 536.- 547 CIMCCFKCCLWC (SEQ ID NO: 74) Table VII: Search Peatides Variant 1, 9-siers, 10-mers, 15-mers (SEQ ID MGGKQRDEDD EAYGKPVKYD PRQVLYPRNS TGAYCGMGEN PEDPWTVGKN EFSQTVGEVF CFPWTNVTPP ALPGITNDTT VLSLLFILLL RLVAGPLVLV AYQSVQETWL AALIVLAVLE TFMVLULCIA YWAMTALYLA GLMCVFQGYS SKGLIQRSVF DIPTFPLISA FIRTLRYHTG FKCCLWCLE< FIIKFLNRNAY FGKLLVVGGV GVLSFFFFSG Z4CVDTLFLCF LEDLERNNGS

PSFRGPIKNR

KDKPYLLYFN

YTKNRNFCLP

IQQGISGLID

LILGVLGVLA

Al LLLMLI FL

TSGQPQYVLW

NLQIYGVLGL

SLAFGALILT

IMIAIYGKNF

RI PGLGKDFK

LDRPYYMSKS

SCTDVICCVL

IFSCILSSNI

GVPWNMTVI T

SLNARDISVK

YGIYYCWEEY

RQRI RIAIAL

ASNISSPGCE

EWTLNWVLAL

LVQIARVILE

CVSAKNAFI4L

SPHLNYYWLP

LLKILGKKNE

NO:

FLLFILGYIV

ISVAENGLQC

SLQQELCPSF

IFEDFAQSWY

RVLRDKGAS I

LKEASKAVGQ

KVPINTSCNP

GQCVLAGAFA

YIDHKLRGVQ

LMRNIVRVVV

IMTSIL#GAYV

APPDNKKRKK

VGIVAWLYGD

PT P0VCVSSC

LLPSAPALGR

WI LVALGVAL

SQLGFTTNLS

NHSTMFYPLV

TAHLVNSSCP

SFYWAFH1(PQ

NPVARCIMCC

LDKVTDLLLF

IASGFFSVFG

Variant 3: 9-mers GRCFPWTNITPPALPGI (SEQ ID NO: 76) 1O-mers LGRCFPWTNITPPALPGIT (SEQ ID NO: 77) PSAPALGRCFPWTNITPPALPGITNDTTI (SEQ ID NO: Variant 9-mers VLEAILLLVLIFLRQRI (SEQ ID NO: 79) 1O-mers AVLEAILLLVLIFLRQRIR (SEQ ID NO: 1 ALl VLAVLEAILLLVLI FLRQRI RIAIAL (SEQ ID NO: Variant 6: 9-mers GYSSKGLIPRSVFNLOI (SEQ ID NO: 02) rs QGYSSKGLIPRSVFNLQIY (SEQ ID NO: 83) 00 LMCVFQGYSSKGLIPRSVFNLQIYGVLGL (SEQ ID NO: 84) Variant 7 9-mers SWYWILVAVGQI2STM (SEQ ID NO: 1O-mers 00 QSWYWILVAVGQMHSTMF (SEQ ID NO: 86) rs FEDFAQSWYWILVAVGQMMSTt4FYPLVT (SEQ ID NO: 87) 00 N Variant 8 9-mers NYYWLPIMRNPITPTGHVFQTSTLGAYV (SEQ ID NO: 88) 00 LNYYWLPIMRNPITPTGHVFQTSILGAYVI (SEQ ID NO: 89) 1S-mers FRSPHLNYYWLPIMRNPITPTGAVFQTSILGAYVIASGFF (SEQ ID NO: Variant 9 9-me rs YWAITALYPLPTQPATLGYVLWASNI (SEQ ID NO: 91) 1O-mers AYWAMTALYPLPTQPATLGYVLWASNIS (SEQ ID NO: 92) LLICIAYWAMTALYPLPTOPATLGYVLWASNISSPGCE (SEQ ID NO: 93) Tables ViII XXI: Table VII-VI-HLA.A1.9mers.

24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amIno acids, and the end position for each peptide is the start position plus eight.

Start Subseuence I Sco7e 58 UYGI)PRMY 662 CVDTLFLCF F__000 77 MGENKDKPY[11250 594 VTDLLFFG 625] 698 KEAPPONK 11 318 VLEAILLM I 4.500 363 VLLLICIAY j 2.500 489 SAFIRTIRY 2.500 267 GVLAYGIYY 2.500 689 KSLLKILGK 1.500 470 ASFYWAFHK 150] 222J FEDFASWY II 32 CMVIICMI1.5 QRDEDDEAY 1.250] 121 r PEDPWIfVGK 1 t000 379 700 EAPPDNKKR 1000] 81 NAYIMIAIY 111.000] 42J KCCLWCLEK ii T DE-DDEAYGK If TUU EAYGKPVKY If i.ooo] 670 11 FLERNN 0--90 276 I CWEEYRVLR F 0900 518 ILYIDUKLI -900j 417-11 SSCPGLMCV 1 0.750 437 RSVFNLQIY 0750 NKDKPYLLY 2 LGVLGVLAY 0.625 546 WCLEKF 0.500 243 SILLFILLL 0.500 238 V/ALVLSUYF 0o.5 579 MLLMRNIVR 0.500 465 LAGAFASFY 0.500 421 GLMCVFQGY 0.500 508 ILTLVQIAR 0.500 Table VII.-V1-HLA-A1I.9mers 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acds, and the end position for each peptide is the start position plus eight Start]! Subsequence 1Score 593 KVDLFF J0.500 I Z AILLLMLIF 0.500 36 11 ICCWVF 0.500 50 VWGIVAWLY 0.500 186 NVTPPALPG 6.566 609 GVGVLSFFF 0.500 287 GASISOLGF 0500 187 VTPPALPGI 0.500 1 668 LCFI.EDLER 0500 323-1 LMLIFLR 0.500 272 ifGIYYCWEEY 310.500] 521 11 YIDHKLRGV 0.500 253 VAGPL MV 0.500 398 GCEKVPINT 0.450 560 YIMAIYGK 0AO0 338[ IA EASK 0.400 135 VGEVYK 0.400 349 GQMIASTMFY 0.375 118 SSCPEDPW 0.300 305 VQEVLAAL 110270 629 FKSPHLNYY -2501 214 1 ARDISVKIF71 0.250 702 FPPDNKKRKK 0.250 641 IMTSILGAY 0250 678 NGSLDRPY 0.250 513 QIARVILEY 0.250 483 PTFPLISAF 0250 120 CPEDPWTVG _0.225 129 KNEFSQTVG 1[W2 136 VGEVFYTKN 0225 VC) FUPSAPAL 10.200 147 FCIPGVPWN _.200 39311 NISSPGCEK 0.200 464A AGAFASF If 517r VN.EYIDHK If 1 424 CVQGYSSK 0200 394] 0.150 Table VIII-V1HLAAl-Smers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eiqht Start I Subsequence Ii Score] 133 I SQ1VGEVFY 613 LSFFFFSGR 0.150] 132 FSQTVGEVF 0.150] 488 1S.AFIRTLR 163 QQELCPSFL 0.135 199 TTIQQGISG 0125 485 FPUSAFIR 0.125 607 VGGVGV.SF 0.125 7134 QTVGEVYT 0.125 575 KNAFMLLMR 0.125 266 LGVLAYGIY 0.125 7 40 LFLLFILGY 0.125 1196 TNDTTIQQG 0.125 610 01VG2SFFFF 360 VTFVUIIC 0.125 156 MTVITSLQQ 0.125 677 NNGSLDRPY 0.125 498 HTGSLAFGA 0.125 172 LPSAPALGR 0.125 195 ITNDTTIM0 0.125 452 WTLNWVLALf 0.125 353 STMFYPLVT 0.125 443 QIYGVLGLF 0.100 543 CCLWCLEKF 0.100 207 GLDSLNAR 0100 407 SCNPTAHLV 0100 180- RCFPWrTwVT 0.100 3 TMFYPLVrF 0.100 Table VIII-W-HILA-All-9mers 24P4C12 Each pepfide is a portion of SEQ ID NOr 7; each start position Is specified, the length of peptide Is 9 amino adds, and the end position for each poptde is the startpoiinOsegt 8tart II Subequnce Score Table VIll-V84LA-AI*9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start I Subsequence H Score 18 VFQTSILGA [0003 T NPITPTGHV 0.003 1 TGHVFQTSI 0.003 9 RNPITPTGH 0.003 14 [PTGHWQTS 0.003 77f IMRNPITPT 1 5.551 3 YWLPIMRNP 0.01 16 11 GHVFOTSIL 0.001 2 rTV&PIMRN 0.000 6 I PIMRNPITP 0.000 Table VIIIV64ILAAI-Smers- 24P4C12 Each peptide Is a portion of SEQ ID NO- 13; each start position is specified, the length of peptide Is 9 arino acids, and the end position for each peptide is the startubsun sco start 11 Subsequence e: Sor Table IX-VM-LA.Almrs 24P34C12J Each peptide is a portion of SEQ NO: 3; each start position is specified, the length of peptide Is amino acids, and the end position for each peptide is the start position plus nine.

Start Subsequence IScore] 609 GVGVWSFFFF 353 STMFYPLVTF 0.0 VLGAFASFY jf O-5R7 322 .LLULRj0500 3571 CCVLFLLF 11.500 656 1WGG G LsF A00 Table IK-VSHL-A.Almers.

24P4C12 Each peplide Is a portion of SEQ ID NO: 7; each start position is specified, the length of pelptide is amino acids, and the end position for each peptide Is the start posito lus nine..

Start Score 0I ITPPALPGIT 1 250 F3 RCFPWTNrTP o050 8 1TNITPPALPG 0.013 7I WTITPPALP 0.005 1 FPWINITPPA 0001 2 GRCFPWTT 10001 I LGRCFPWTNI 0.000 6 PWTNITPPAL 0.000 4 CFPWTNITPP D0000 Table IX.VSHLA-Ai-lOmers- 24P4C12 Each peptide is a portion of SEQ ID NO: I each start position is specified, the length of pepde Is amino acds, and the end position for each peptide is the start position plus nine.

Ftart I[ Subsequence I Score 2 i VLEAJLLLVL 1[4500 F ILLL-WFLR 7 15 F4II EALLLVLIF 0.500 Liii UVLI1FLRQR [0.i00 1 VIFLRQRIR 0.100 1 7fUIVUFLRO ]0.050 1 IIAIUIVUFL ]0.050 LVL LIFLRQRI 1 0.015- Table lXV6.HLA-A-lOmers- 24P4C12 Each peptide is a portion of SEQ ID NO: 13; eact start position is specified, the length of peptide is amino acids, and the end position for each peptide is the staul position Ous nine.

Start I( Subsequence I fore 7' II GLIPRSVNL 10.500 I 2 IIGYSSKGLIPR I 6 KGLIPRSVN I o 1 I SKGUPRSV11I 0.005 3 YSSKGUPRS 1 0.003 LPRSVFLQIY 11 0.003 4 SSKGLIPRSV 0.002 9 IPRSVNLQI 0.001 1 GYSSKGLIP 0.001 8 1 LIPR N' 0.001 Table iX.V?.HLA-A-1lOmers.

24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the sW M wition ou ie Start Susuence Re [T 1 AVGQMMSTMF 0.100 r611 ILVAVGQMMS I005I I LVAVGQMMST 0.050] 81 VAVGQMMSTM 0.010 5 WILVAVGQMM 0.010 1 T QSWYWILVAV !0.003 2 tSWYWLVAVG 0.001 4 YMLVAVGQM 110-001 3 WYW(LVAVGQ FO000 able IX-V-HLA.A4l-0mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Start Subseence IrScore i] LNYYWLPIMR 110.125 13[1[ ITPTGHvFQT 0.125 21 QTSLGAYVI 0.050 18 11 HVFQTSILGA 0.050 1 1 NPITPTGHVF 0.025 19 VF2TSILGAY 12 1 PITPTGHVFQ 0.020 5 WLPIMRNPIT 0.020 4 YWLPIMRNPI 0.005 9 MRJPITPTGH 0.005 20 0 FQTSILGA .3 15 0. PTGHQTSI 3 14 TPTGHVFQTS 10.003 10 I RNPI1PTGHV 0.003 2 H N PMN .03 8 16]j TGIVFQTSIL ]00031 S17 0 GHVFQTSILG fla.1 76 I LPIMRNPITP 0.001.

8 IMRNPITPTG 0.001 7 11 PMRNPlTPT W 3 YYWLPIMRNP 0.000 Table IX.V9-HLA.A1.O1mers.

24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is IDamiino acids, and the end position for each peptide is the start position plus nine.

Start Subsequence Score [ii LPTQPAThGY 0.625 [7 ALYP[PTQPA 0.100 9 YPLPTQPATL 10.050 1511 MTALYPLPTQ 0.050 1k[ IPTQPATLGYV 0.025 [i41i AMTALYPLPT 0.025 16 11 ATLGYVLWAS 0.025 17 1 TLGYVLWASN 0.020 15 11 PATLGYVLWA 14][ QPATL'tVLW 10.005 7137( TQPATLGV 1 18 1 IGYVIWASNI. 110.003 13 1 WAMTALYPLP 1 2 ILYWAMTALYPL 0.001 F 10I PLPTQPATLG 0.001 T TALYPLPTP jF001 8 [LYPLPTQPAT 0.001 R991 GYVEWASNIS 00 AYWAMTAYP 1W Table X-VI-HLA.A02019mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start _stn plus eight Isut Subs uence Table X-VI-HLA.A0201.9ners- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is speciied, the length of peptide is 9 amino acids, and the end position for each peptide is the startpi luseht.

Start q F 4 GLWLNWV 25 709 381 322 ILLLMLIFL 774 580 LLMRNIVRV 1006.

M2O9 597 LLLFFGKU 1.

104 544 CLWCLEKFIp 2 57 598 U.FFGKLLV 82 82 1770 ELLEAAL] 86 LL YFNIFSC 0.

26 578 FMLLMRNIV P29 244 FILLRL 0 41 FLLGY 2 08 11 ILSSNIISV rl- 48- 260 1 VLILGVLGV 48 58 WLYGDPRQV 04.7 42 LLFILGYIV 68 Vl 1 VASGFFSV17.

564 AIYGKNFCV 1147 97 239 ALVLSl1FI I 4 LLWGGVGV 11%2 2 38 589 VV VMKVTDL 0 1 72' 268 VLAYGlYYC 1 Table X-VI-HLA-A0201.9mers.

24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peplide is the start position plus eig t Subsequence Score 37 456 WVLALGQCV 1035 180 537 IMCCFKCCL 66 446 GVLGLFWTL 257 LVLVILGV 3n 661 GMCVDTtFL 686 YMSKSLLKI 7.

81 177 ALGRCFPWT I7.8 211 SLNARDSV 2 1072 107 241 VLSLLF 4341. LIQRSWNL 61 353 35 VICC ii 3 5472 547 CLEKFIKFL 7 7317T AVLEIL P.2 240 LSLLFIL 79 302 YQSVQETWL 8 309 WLAALA 6 MMSTMFypI 365 j IICIAYWA 6.45 45 79 M5 ILGYIWGI .29 Tale-.IHLA-A0201-9mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

118 457 AOSWWLV 11 *61 426 FQYSG 1996 554 j LRAI [g~ 642 TI LY 9.032 164 ECSFL 8.1 25171 RLVAGPLVL38.759 501 11 SLAFGALIL ]I8.759 487 I SFRL 8.729 447 LQIYGVLGL 846 262 If ILGVIG VIA [F.-4 521 YIDHKLRGV 8.9 373 I TA 8.073 134 1 jTVEVTJ 1 759 191 ALOGTNITJ .452.

362 FVJ6.977 '200 IIjSGL E5 83 KPYLLYFNI 314 IVLAVLEAI 67 383 GOPOYVIWA6.2 M225 EA FQWYVIL 16.295 269 ISS(IGF1T 5.M4 73-RUA1 ILIILY .2

F

596 1 1 DLLFGL155 [611 [GVLSFFFFS 5.7 28 VLDGS 5.526 Fl~g 5WNTVT4 59 73-80-j ATro y15-313 F612 VLSFFFFSG J5.305 100 usNGL 4.93 158 VITSjQEL 4 9-3 .504 FGAULTLV 11.0 Table X-Vi-HLA.A0201.9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each pepfide is the start position pus eeght Et Subseuence Iscore 246 FLJLLVAI 4.7671 Table X-V3-HLA-A0201.9mors1 24341CI2 Each peptide is a portioni of SEQ ID NOr. 7; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eia t Start H Subsequence IScorel T I WThITPPAL 11.365 ITPPALPGI 0.567 2 Hf RCFPWTNIT J7o074 1821 N[EPPALPG 0.10 4E FPWTNITPP 10.0091 TNITPPALP 0.0001 Table X.V5.HLA.A0201.9mors- 24P4C12 Each peplide is a portion of SEQ ID NO- 11; each start position is specified, the legt oflpeptidelIs 9 arrdno acids, and the end post for each pepfide Is the Start Su~bseunc e c 5 _VIF 1 VLEALLLV I 5] 6 LLVFLR 1.251] 2 LEAI IVL 06.666] 7 if LLVUIFLRQ 0o.0481 Table X-VS.HLA.A0201.gmars.

24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 aino acidis, and the end position for each peptide is the [s-art Sbeuence Score L LLLF1 0.036 [37 EAIILVI IF0.025 LVLIFLROR 011.014 Table X-V6.H'LA-A0201 -9mers.

24P4C12 Each peptide Is a portion of SEQ 10 NO: 13; each startposition is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus egL 7iilaJt Subsequence rsCr 7 UPRSVFNL 6.61 6 GLIPRSVFN 0.41D 4 ifSKGUJPRSV 0.019 5 I KGLIPRSVF 10.003 F2 YSSKGLIPR 0.00 9~ PRVNI Li 5.F06 8~ IPRSVFNLQ [Il GYSSKGLIP iiQ.00 [Table X.V7-HLA.A02D1 .9mers.

24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide Is 9 armino acids, and the end position for each peptide is the tatposition plus ei-ht.

Str Subsequeco IScore 5T ILVAVGQMM I8.446 8T AVGQMMSTM 11.000 77 VAVQMMST 0.405 i SWVYILVAV 1.7 LVAVGQMMS 0,1 jTable X.V.HLA.A0201.9mers- 24P4C12 Each peptide is a portion of SEQ NO: 15; each start position is specified, the lengt of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start 11Subse uecore F-21 WYWILVAVG o YM~tLVAVGQ STable X-V8-HLA-A0201.9mers- 24P4C2! Each peptide is a portion of SEQ ID NO. 17; each starl position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

SatISubsauence IScorel 4 WL~PI]I .9 OT_ __Y 13 PTGHVFQT 069 ]iEJ THVQTSI 0.259 NPITPTG-HV 09

LPIMRNPIT

18 IJ TSGA 003 19 I I~LGY 10.010 16" GHVFQTSIL 0.6 [12 I ITPTGHVFQ 0.0 [2 YI'YWL.PIMRN 0.001 17 HIVFQTSILG 0.001 9 IIRNPITPTGH 0.0001 6 f(PimRNPITP 0.000 1 I PITPTGHvF 0.500 '14 I PTGHVFQTS 0.0 8 If MRNPITPTG 0.0001 3 11 YWLPIMRNP o1 1 I NYWYILPIMR 10.0001 'Table X-V9*HLA-A0201-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the startsition pus elht.

Start Susqec IScore 2I WAMALYPL 11.61 12 TQPATLGYV 11.59 MJI AThGY'AtWA7 F.23 f6_[ TLGYVLWAS 1128 8J1 YPLPTQPAT 0.82 9 PLTPATR 10.470 4 Jr LYLP L0.1 76 131 nGV 11.5 3 AMAYL 1~0.016 17 LGYVVLWASN 0 .004 5 1[ TALYPIPTO 10.002 1871 i GYVLWA'I 0.1 7= 1 LYPLPTQPA 10.001 -iZ9i LPTQPATrLG -0.001 1 YWAMTrALYP o .ooo 147[ PATLGYVLW 10.000 11 1 PTQPATLGY 10.000 Table XI-VI-HLA.A0201.l0mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 am~no adds, and the end position for each peptide Is the start Position Dius nine.

Start I Subsequence Score 341TMFYPLVTF 351.

85 1 UFNIFSC 1127 I969 579 M LLM R N NRV 1006.

603 KLLVVGGVGV .96 309 WLdAIVLAV 35.

00 00 00 00 Table XJ-VI.HLA-A0201 -i0ners- 24PC12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amno adds, and the end position for each peplide Is the start positon plus nine.

Start ubsuence y 28.9 56 IWLYGDRQVL2 239 ALVSLLFL 40 350 QMMSTMFYPL 62 S LLYFNIFSC I 8) 361L I L LLICLAYWAM 01 F2591 LLILGVLGV *0 33 162 LQQELCPSFL 580 LLMRNIVRVV125 94 CILSSNIISV 13 .1 5 517 VILEYIDHKL 54 FLIRNAYIMI 686 YMSKSLLKIL 44 FILGYIWGI 11 E~543 133 SQTVGEVM 438 SVFNLQIYGV 1.7 231 WILVALGVAIL99 3 1 235 ALGVAL 9 2 3 SLr 441 NQIYG4GL 6604GMCVDTLFLC 78 2 LMLIFRQR I 7 9 536 CIMCCFKCCL 1.29 Table XJ.Vi.HLA.A0201-11iners- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

start Sb uneIFSCO 315 VLAVL LL M, 1 94 448 LGLF rLNW 6.1 662 CVDTLFLCFL 64 VLYPRNSTGA 589 WLDKVTU- 596 DLLLFFGKLL 240 LVLSLLFILL .33 357

YPLVWVLLL

a4 267 GVLAYGIYYC 0r3 66 304 SVQEW&AAL 76 V.6 302 YQSVQETW .A 501 SLAFGALILT 7.14

AVLEAILLLM

S4.52 14491 45 ILGYIWGIV 55 13.05 456 WVLALGOCV 1.0 148 CLPGVPWNMT __8 108 LQCPTPQVCV Table XIVI-HLAA0201.lmm- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specfied, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

[iSw][ Subseauence Scor 478 KPQOIPTFPL 11.60 238 VALLSLF it6 312 ALIVLAVLEA 1142 459 ALGQCVAGA 1.2 571 CVSAKNAFM 1 0 563 IAIYGKNFCV 9152j 445 YGVLG.FWTL 9.141 379 LATSGQPQYV 03 327 LIFLRQRIRI 9.023 249 LLRLVAGPLV 8.986 _539 CCFKCCLWCL 8.900 517[ QRVMLEYI 510 TLVQIARVIL 457 VALGQCVLA 8.446 95 ILSSNIISVA 7.964 657 SVFGMCVDTL 7.794 I 2i1 FAQSWYWILV W.5 I FAR -M KVTL F7.309 593 KVTDLLLFFG 6.e65 368 CIAYWAMTAL 6.756 562 MiNYGMC 6.387 363 I LLICIAYW 5.929 V ILFF 318 M AILLML 5346 292 QLGFTTNLSA 4.6 314 IV[ JY EAIL 4.821 393 NISSPGCEKV 4 .6 506 ALILTLVQIA A..685 260 VLILGVLGVL 4.452 604 LLWGGVGVL 4.452 261 LILGVLGVLA 4.297 502 LAFGALILTL F4292 147 FGVPWNM 4.

able XJ.V3HLAA0201-10mer 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position i specified, the length of peptide is amino adds, and the end position for each peptide Is the start position plus nine.

Start Subsuence Score 9 R NITPPALPGI 3.299 FPWTNITPPA 1.238 1 LGRCFPWNI IO0.015 H ITPPALPGIT 0.009 7 WTNITPPALP 0.001 8 TNITPPALPG 0.000 2 I GRCFPWTNT 0.000 13 1 RCFPWrNITP 0.000 I6 PWTNITPPAL 0.000 I CFPWTNITPP 0.000 able XI-V5HLA-AD201-0ers- 24P4C02 Each peptide Is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is amIno adds, and the end position for each peptide is the start position plus nine.

Start Subse uence 1 IjAVLEALLL A1.

1AILLLLIFL 118 9 l LFLRQR 2 2 V MEALLLVL 12.92 6 0 ILLLVFLR 01.251 3 LEAILLLVLI 10.793 7 ij LVLIRQ j A78 8 LLIFQR 0.044 11 VIFLRQRIR 0.002 4 EAIU IF H0.000 abl X-V-HLA021-0mers 24P4C12 table XIW.4HLAAO0201 0mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino adds, and the end position for each peptide is the sa position plus n* e.

Start sencJS -7 75 WILVAVGQMM1.6 i QSWYMVI&667 7]1 LVAVGQMST 1Z550 8 1 VAVGQMMSTM 0.270 6 ILVAVGQMMS 0.127 9 1 AVGOMMSTMF 0.007 LEJI -WILVAVQM 0.001] 7311 WYMI VAVGQ 0.0001 2 1 SWYWILVAVG 0.000] Table XIV84ILA.A0201-10mers1 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 10 amino adds, and the end position for each peptide is the start position plus nine.

[Sortre Table Xt.V941LAA0201.l1mers 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amIno acids, and the end position for each peptide is the start pnine.

Start H S ii5.89 SALYPLPTQPA 58 4 AMTALYPLPT 5.382 19 YPLPTQPATL j.373 TQPATGYVL 0.888 18 11 LGYVLWASNI I.370 17 TLGYVLWASN 0.127 16 ATLGLWAS 0.066 12 IPTQPATLGWJ0.035 21 WAMTAYPL F031 15 IIPA TLGYVA 0.019 31 W MAMT 1 D 10.005 SLYPLPTQPAT 10002 1o PATLG 0.002 1111 LPTQPATLGY 0001 MTALYPLPT 0.001 6 TALYPLPTQP .001 14 O QPATLGYVLW .001 1 J AYWAMTALYP 0.0 PG'VLWASNIS 9.9000 Table XiI.V1.HLA-A3-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start posio is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Start1Subs ore 772] GYCWEEY6000 [351 MMSTMFYPL 5.400 F470 ASFYWAFHK 14.500 449 GVWTfLNWV 4.500 I FmIL 86 LLYFNIFSC 4.500 j446 ji GLF 3.645 660 GMCVDTLFL 3.600 F633 HLNYYPI 542 KCCLWCLEK 3.600 241 VLSLLFILL 3.600 42 F LLFILGYIV 3.000 II 393 NISSPGCEK 3.000 325:][ LMLIFLRQR 2.700 I ILGYIWGI 2.700 322 I ILLLMIFL 2.700 239 ALVLSLLFI 2.700 641 IMTSILGAY 2.700 598 LLFFGKLLV 2.000 [260 VULGVLGV 1.800 265 LGVLAYGI 1.800 gT3 QIARVILEY 11.800 I 7[ GVGVLSFFF 1.80 Table XII-V14ILA-A-9mers- 24P4C12 Each peptide Isa portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start p lus eight [stat Subsequence [50 SLAFGAUL 1.200 F-662 CVDTLFLCF 1.200 349 GQMMSTMFY 1.080 443 QYGVIGLF 1.012 321 R AILLLMIF 0.900 590 VLDKVTDLL 0.900 326 I MLIFLRQRI 0.900 268 VLAYGIYYC 0.900 107 GLQCPTPQV 0.900 613 LSFFFFSGR 0.900 318 VLEAILLLM 0.900 232 I ILVALGVAL 0.900 518 ILEYiDHKL 0.900 452 WTLNWVLAL 0.810 596 DLLLFFGKL 0.729 645 1 ILGAYVIAS 0.720 258 VLVLILG V 0.608 49 IWGIVAWL 0.608 41 FLLFLGYI 10.608 54 VAWLYGDPR]060 665 TLFLCFLED 0.600 95 ILSSNIISV 0.600 457 VLALGOCVL 0.600 282 VLRDKGASI 0.600 554 FLNRNAYIM 0.600 39 VIFLFILG 0.600 315 IVAWEAIL 10.600 638 WPIMTSiL 0.600 434 LIQRS NL F-0.540 612 VLSFFFFSG 0.540 611 GVLSFFFFS 0.486 647 GAYVIASGF 0.450 580 LLARNIVRV 0.4 364 LLUCLAYW 0.450 564 IYGKNFCV 0.45 237 0.4VALSLL 38 11 VFIFIL ;M0 I 537 IMCCFKCCL 1.800 WGIVAWLY 1.800 686 YMSKSLLKI 1.800 RLVAGPLVL 1.800 593 KVTDLLLFF 1.800 358 PLVFVUL 1.620 544 CLWCLEKFI 1.500 689 KSLLKILGK 1.350 525 KLRGVQNPV 1.350 170 FUPSAPAL 1.350 547 CLFIKFL 1.350 597 ULFFGKLL 1.350 365 11W AYWA 1.3 F ILTLVQI 1.350 148 11 CLPGVPWNM 1.350 Table XIV-HLA..A3.9mers- 24P4C12 Each peptide is a portion of SEQ ID NO. 3; each start position Is specified, the length of peptide is 9 amino acids, and the end I position for each peptide is the start position plus e*ght.

Star Subsece Score 204 GISGIDSL _10.405 VICCVLLL7F0.405 317~ AVLEAILLL 045 240 LVLSLLFIL 0.405 66871 LCIFLEDLER 040 388 11 VLWASNS .0 48971 SAIFIRTILRY 10.400 211 I[SLNARDISV 0.400 -YLLYFNIFS 10.360 Table XiI-V3-HLA-A39mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position Is specified, the length of peptide is 9 amino adds, and the enid position for each peptide is the Start ILS eun cr 9 I ITPPAL 0.030 2I RCFPWTNIT 10.022 -J NITPPALPG 1.00 41 GRC PTN 0.002 7 11 TNTPP 000 3 FCFPWNITP 0.000 Table XI-VSHLA-A3.9mers- 24P4C12 f Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peplide is 9 amino acids, and the end position for each peptida is the start posltow pus eiht [Start susuenca Scre Table XI-VT-HLA-A3-9mners- 24P4C12

I

Start ifSubse uence Score] 8 ifAVGQMMSTM 0.030] 4 WILVAVGQM D.027] I6 IILVAVGQMMS 0.008] 7 VAVGQMMST 0.007] 11SWYWILVAV 0.002 2 WYWILVAVG 10.000 3 wLVAVGO 0.0 Table X1111VS.HLA.A3.9mers- Each peptide is a portion of SEQ ID NO: 17: each start position is specitied, the length of peptide is 9 amino adds, and the end position for each peptie is the start position plus eighL start I Subsequence IScore] 4 1 WLPIMRNPI 10.60D ~1[7 FQTSILGAY 10.0811 1 N W[IM 0.0401 1 i LHVFQTSILG 0.0201 13 UPTGHVFQT 003 I QTSILGAYV 0.010 16 11 GHVFQTSIL 0.003 15-I TGHVFQTSl 10.002 15 1_ LPIMIRNPIT 10.002 F10l NTPGV1.01 I JL2- ITPTHVFQI~I0 114 HI PTGHVFQTS 10.0011 18 J[VFQTSILGA 10.001 I F6 DD~~l0.0 f3 Ii YWLPIMRNP IOR00-0 ITable XiI.V941LA.A3-9mers.

24P4C12 1 Table XII.W.-HLA-A3-9mers- I24P4C12 5 Strt -ubsequence lscore SEach peptide is a portion of SEQ ID N&r 15; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position plus eight 11ILVAVGQMM 10.450 Each peptide is a portion of SEQ ID NO: 19 each.start position Is specified, the length of peptide is 9 amino adds, and the end position for each peptide Is the start position plus eig iartiI Subsequence Morel ATLGYVLWA 0.4051 16 I TLGYVLWAS 0.2701 6 ALYPLPTQP 0.150 H I PTOPATLGY 0.0601 94 PLPTQPATI 1O060 2 WAMTALYPL 0.041 MTALYPLPT 0.030 3 AMTALYPLP 0.020 131 QPATLGYVL 0.018 i18 GYVLWASNI 0.008 12 TQPATLGW 0003 E- 8j YPLPTQPAT 0.002 TALYPLPTQ 0.001 7 LYPLPTOPA 0.000 LPTQPATLG 0.000 1 4] PATLGYVLW 0000 1 1 LGYVLWASN 000 1YWAJLYP r0.000 Table XW-VI.ILAA3-10mers.

24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino adds, end the end position for each peptide is the start position plus nine.

Table XIII-VI-HLA.A3-10mers* 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amnino acids, and the end position for each peptide is the start osition pus ine.

Start Subseuence Scre 354 TMFYPLVrF 3.000 72 GAYCGMGENK 3.000 324 LLMLFLRRQ 2.700 660 GMCVDThFLC 2700 467 GAFASFYWAF 2.700 243 SLLFILLLRL 2700 83 KPYLLYFNIF 2700 42 LLFILGYIW 2.000 518 ILEYIDHKLR r00I 161 SLQQELCPSF 2000 337 AALLEASK 2.000 362 FV FVLICIAY 1.800 650 VIASGFFSVF 1.800 606 WGGVGVLSF 1.800 507 ULTLVQIAR 1.800 329 R.RQRIRIJ 1.800 318 VLEAILLML 1.800 624 GLGKDFKSPH 1.800 309_ WLAAIVLAV 1.800 312 AuvLALEA 1.800 469 FASFYWAFHK 1.800 64 WYPRNSTGA 1.500 364 LLLICIAYWA 1.350 657j SVFGMCVDTL 1.350 YLLYFNIFSC 1.350 220 YJFEDFAQSW 1.350 264 GVLGVIAYGI .215 315 vLAVLEAILL 1.200 237 GVALVLSU.F 1.2001 554 FLNRNAYIMI 1.200 590 3VKVTDL. 1.200 265 VLGVLAYGIY 1.200 VICCVLFLLF 1.200 53 11IVAWLYGOPR 1.

4T1 GLW 1.200 268 VLAYGIYCW 10900 413 1HLVNSSCPGL 0.900 Table XiII.VI-HLA-A3-l0mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each star position is specified, the length of peptide is amino acids, and the end position far each peptide is the start position plus nine Str Subsequnce 2_75i1 YCWEEYR-VtR 1 F232-11 iLvALGVALv 59 1 325 1 LMLIFLRORI jr_9_ II CVLAGAASF 0.900 F5_25_1 KLRGVQNPVA 0.900D F5061I AJLTLVOIA 0.900 I603 JrKLLWGGVGV 0.9001 633 HLHYYWI.PIM 10.900) 510 IITIVQIARVIL 0.900 1365 I LICAY M 0.900I F-5-127 VQIAWVLEY 00.8101 604J LLWGGVGVL 810 251 ifRLVAGPLVLV 0.675 261 VULGVLGVL 0.5608 F-4]1 FILGYIWVGI 10.6081 107 GLQCPTPQVC 0.600 327 LIFLRQRIRI 0.600 r 3261 MLIFLRQRIR 0.600 F597 11LLLFFGKLLV 0.600 F4871 LISAFIRTILR 0.60 1I201 CPEDPwrVGK 10.600 351 MMSTMFYPLV 0.6 00 252 11 LVAGPVL 9 1 F363 I1 VILLICIAYW I 0.45 0 5711 MLLMRNIVRV 045 F95-11 ILSSNIISVA045 STable YJII.V3-HL-A-A3-Omtes. 1 24P4C12 Sart Subs uence 3 ScoreJ Table XIII-VII-HILA-A3A10mers- I24P4Ci2 SStartl Subseuence I Scorel Each peptide is a portion of SEQ ID NO: 7; each start position Is specified, the length of peptide is 10 amino adds, and the end position for each peptide is the start position plus nine.

19 1 NITPPALPGI 10.1351 FpwTNITPPA 060@5] 3 jRCFPWTNITp 010031 I :1 IT-PPALPGIT F 7IU 1 WTNITPPALP 0.0021 171 LGc N I 0.001 2 1 RFWNI .0 4 ICTNITP PG 0.0001 Table XIII4-5ILAA3.10mers- 24412 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10amino adids, and the end position for each peptide Is the "start position plus nine.

Stj eece Scro M6 ILLLVLIFLR j00 F871 LLVLIFLRQR 2.700 i1 11 WIFLRQRIR 0.600 VLE AILLL. 0.! 4 1F EAILLLVUF 0.0541 3 ILULVLI 10.0031 Table XJII-V6-HLA.A3.1 Ormers- 24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the str position plus nine.

Start ifSubsequence rSco0Re] 7[ GUIPRSVFNL .4 2 GYSSKGLIPRM IPRSVFNLOI 0.036 8 IILIPRSVFNLQ FO 009] 5 SKGLIPRSVF M0.03 10 PRVLQ IY0.001 YS11 GIPRS 0.000 4 1SSKQLIPRSV 0.000 I 1[ OGYSSKGUJP FO000 6_71[ KGLIPRSVFN IF 000 Table XJII.V7-HLA.A3.l0mers-1 24P4C12 Strt1 Subsequence sco re] Each peptide is a portion o E 1-I NO: 15; eac- start position i specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start psition plus nine.

9 AVGQ0M MS TM F 0.200 6. ILGQMMS0.1201 5 ][~CIMM 10045 7 I VAVGQMSM .0 2I SWYWLVAV 0.0001 1F VAV M 000 lI IYWILVAVGQ 0.000 Table XIMIV-HLA-A3-lmems 240C2 Each peptide is a portion of SEQ ID NO: 17; each start position is speciflid, the length of peptide Is 10 amino acids, and the end position for each peptide is the start psition pus nine.

rt Subs uence Scorel .18HQT ILGA 0.3070 rable XIV-VI-HLA-AII101 9mers- 24P4CI2 Start] Subsequence Score] 689 KSLLKILGK 0 .180 516 RVJLEYIDH 10.180 609 ][GVGVLSFFF 0.8 4815 ][FPLISAFIR 10.180 446][1 GVLGLFWTL 0.180 267[ GVLAYGIYY 010 27 IYYCWEEYR I.O1601 ILILVOIAR 1.6 668 LCFLEDLER 10.16 698] EA0. K I AYW HK10.1201 701I1 APPDNKKRK F D 595f TDW.FFGK 0_90 38 CVLFLLFIL 11.090 240 IfLVLSLLFIL 11.090 54 VAWLYGDPR 172 IfLPSAPALGR 0.0801 349 IfGQMMSTMFY 10.0721 334 IfIRIAIALLK 0.060 545 fLWCLEKFIK_] 0.0601 5677 GKNFCVSA 676 317 11 AVL.EAJLLL j0060 6997 IfNEAPPDNKK jj0.060 151 1i GVPWNMTV1 1.00 [237J GVALVLSLL 1000 [257 IILVLVLILGV 1000 [75207 DPSFRGPIK 0.0601 [5 1 KNAFMLLMR 100481 [212 LNA.RIISVX 0.O040 [3597[I LVTFVLLUl F 00401 [TI6I PVKYOPSFR 0.O040 704] SVQETWLAA 0.0401 [619 d SGRIPGLGK 0.4 50 I WGIVAWLY 0.0 -68 RYYMSK 0.4 CVDTLFLCF 0.040 DEDDEAYGK IOOB [K831 KPYLLYFNI 0.3! FT 1 GYIWVGIVA 10.036 251] RLVAGPLVL rC03 13311GQPQ)YVLWA 0.036 Table XII-V9.tILAA3l0mers.

24P4C12 U6d peptIde Is a pition of SEQ NO: 19; each start position is specifie, the lenth of peptide is amino adds, and the end position for each petde is the start Position plus nine.

Staf Subsequence More 71 ALYPLPTQPA 12.250 !4 ATALPLPT O.6006 iiII LPATLGY 0.08 13 If U 16 ATGYIfA I 0030 97 TIf YVLASA 0.0203 18 I LGYVILWASNI T0.009 able XIV V1.HLA.A1101-9mers- 24P4C12 Start Subseuence 49 IWG1VA W 314 I AVEA 0 456]1 WVLALGQCV 0.030 589 VVLO)KVTDL 0.030 452 WRNWVLAL F030 141 YTKNRNFCL .030 498 HTGSLAFGA0.03 605 LWGGVG 362 FVCIA 0.030 611 GVLSFFFFS 0.0271 137 I[ YTKNIR 0.02 564 AIYGKNFCV 0.024 272 GYCWEEY 0.024 RQMYPR 0.0241 421 GLMCMQGY D.024 467 GAFASFYWA 0.0241 449 GLFWTWV 0.024 660 -IrMCVD-LFL .2 496 RYHTGSLAF 0.024 [i JLVOIARVIL 0.020 218 SVrJFEDFA .2 [~iB 10.0201 [33 II GVLV0 -22 1 SFRGPIKNR 0.020 175 1I CGMGENKDK 0.2 414 J LVNSSCPGL 0.020 LVAGPLV 00 571 CVAKNAFM 347 AVGQMMrTM 534 ARCIMCCFK 0.020 527 RGVQNPVAR 0.018 34DVICCVLL 10-018 693 KILGKKNEA 0.018 461 GQCVLAGAF JJO-018 4 KQRDEDDEA 1 M1 331 EQRIRIAIA 0.018 DEAYGKPVK j.18 4421 LQIYGVLGL 0.018 255 GPLLIL 0-018 598 LLFFGKLLV 0.016 42 UFILGYIV 0.016 244 UFIOLRL 0.016 327 UFLQR F -016 Table XIV-V3-NLA-AI 101-9mers 24P4C12 Start Subsequence Score Each peptide Is a portion of SE] ID NO: 7; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight 9 ITPPALPGI 0i01 6 WTNITPPAL 0.010 2 :11CFPWTNIT *0.001 8 NITPPAPG 0.0o ,171 GRCFpwrMi 1 S4 11 FPwriTPP 1lOozo 77- 11 CFPWTNITP 0.000 [7 1 TNITPPALP 0.000 =1PWTNITPPA g frable XJV-VS.HLA-AI 10i.9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide Is the start position plus eight StartI Subsen oe 1 LLLVLIFLR 0.360 i-8 I LVUFLRQR 10.060 F-47 AILLLVLIF 10.012 11 ILLLVLIFL 0.012 I VLEAILLLV 0.008 9 FLRQRI .006 I77 LLVLIFL 0.001 112111 LEAILLLVL 0.001 Iu EAI LLLV ][0-.001 Table XV.V6-HLA-AlIIO09mers.

24P4C12 Start Subsjuence Scon Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the Stan sei h Table XIV-V4I-LA-AI 101 9mers- 24PC12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amIno adds, and the end position for each peptide is the Start 11 S core[ 8 1AVGQMMSTM j 0.020 5 I ILVAVGQMM 0.006 4 WILVAVGQM rL;0 i 6 LVAVGQMMS0.

2 1 F -WLVAVG 0.0011 7 VAVGQMMST 0.001 I S LVAV 0.0-] able XI-V4LA.A11101 Smers 24P4C12 Each peptide is a portion of SEQ ID NO- 17: each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptida is the.

start position plus eight.

Start Subsequence IjScore I j N'YWLPIMR 110.320 20 QTSILGAYV 0.010 17 HVFQTSLG MJR-4 19 F FTSILGAY~ oo 18 VFQTSILGA 10.004 4 WLiPI 1.00 10 NPITPTGIV 10.003 Table XIV.V8.HLA-AI 101 .9mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start Subsequence Nisre 2 YYWLPIMRN IFOO02 9 RNPITPTGH N0.001 i 12 [rPTGRQ 0.001 16h 1 GHVFQTSIL 10.001 F-i731 TPTGHVFQT100a F11 PITPTGHVF 0.00 7 [MRNPITPT VT" PIMRNPT 0.000 TGH QTSI 0.

1 6 II IMRNPITP 10.000 1 14 1 PTGIVFQTS 0.000 8 MRNP*1! TG 0.000 YWLPIMRNP P0.9 able XIV.VBHLAAIIO1-9mers 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Start i Subsuence Scorel 14 PATLGYV.W 10 17 I LGYVLWASN 0.000 II YWAMTALYP Table XV-VI-A1101-10mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start sition us nine.

Erill Subsequence ~score RVILEYIDHK 9.000 594 VTOLLLFFGK 3.000 134 1 QTVGEVrfTK 3.000 F3-3-3-1 2.400 5441 CLWCLEKFIK 2400 621 RIPGLGKDFK 1.200 5579 AYIMIAIYGK 1.200 7= I[GAYCGMGENK 1.200 NIVRWVLDK 1.200 I680 SDRPYYMSK 0.800 F469 FA"FYAFHK 10.6001 2721 GIYYCWEEYR 0.480 428 1GYSSKGLIQR 10.48qj 337 0.400 1 AWiKEASK .0 53 IVAWLYGDPR O.

211 SLNARDISVK FO-07 322 IU MLIF 0.

423 MCQGYSSK1 1578 FMUMRNIV 0.4 120] CPEDPWrVGK 0.200 533 VARCIMCCFK 0.2200 264 GM GI 0.180 609 GVGSFFFF 10.180 Table XV4V1-A101O-mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specfied, the length of peptide is 10 amino acds, and the end position for each peptide is the start position plus nine.

Startj Subs uence Score 684 11 PYYMSKSLK 0.160 667 7F 7ELER I 0.160 171 LLPSAPALGR 0.160 611 RDEDDEAYGK 0.120 SKNEAPPDNKK 0.120 484 TFPLISAFIR 1 0.120 72371 GVALVLSLLF 0.120 I74 YCGMGENKDK 0.100 6891 KSLLKILGKK 0.090 S-I yviASGFFSV 0.090 281 RVLRDKGASI 0.090 VLSFFFFSGR 0.080 48711 LISAFIRTLR I10.0801 17] YCWEEYRVLR .080 4]38[ SVFNLQIYGV I08 I697][ KKNEAPPDNK ]10..0]0 I 392][ SNISSPGCEK .060 577ljCVSAKNAFML 12591 LVLILGVAGV 0.060 49 IWGIVAWLY 0.060 240 LLSUFILL 0.060 317 AVLEAILLLM 0., 362 FVLLICIAY 0.060 433 GLIQRSVFNL 0.054 449 GLFWTLNWVL 0.048 493 RThRYHTGSL 0.045 518 ILEYIDHKLR 0.040 252 LVAGPLVLVL 0.040 618 FSGRIPGLGK 0.040 688 SKSW(ILGK 0.040 606 WGGVGVLSF 0.040 541 FKCCLWCLEK 0.040 657 SVFGMCVDTL 0.040 360 VTFVLLLICI 0.040 233 LVALGVALVL ]0.040 331 RORIRIAIAL 0.036 589 WWDKVTh 0.030 Table XV-VI.A111014j0iers- 24P4C12 Each peptide Is a portion of SEQ ID NO, 3: each start position is specified, the length of peptide is amino adids, and the end position for each peptide is the start position plus nine.

F Start I -Sbence re [i 15 1 TVITSLQQEL 1.3 [4-63- CVLAGAFASF 10.030 [5-8-i I VVLDKVTDL 0.030 EAPPONKKRJ( 0.030 7317 I IVLAVLEAL H0.030 74-67 wvLALGQCVL 003 [T[LVLVILGVL 11,3 34i DVICCVFL 007 1'611 GVLSFFFFSG0.2 59~. GDPRQVLYPR0.2 2201 KIFEOPQW .2 654[ GFFSVMC 0.2 548 LEKFIKFLNR 004 467 GAFASFYWAF 004 674 1j LERNNGSWDR0.2 3477I AVGQMMSTMF j2O 566 11YGKNFCVSAK I0.020 353 IISTMFYPLvTF 0.020 IVRVVUKV 0.020 7011II APPDNKI(RKK 002 II SVQETWVLML 10.020 380 I1 ATSGOPQYVL 10.020 II ~0.020 414 I LVNSSCPGLM 0.020 19 jjYDPSFRGPIK 0.020 116 jICVSSCPEOPW 0.020 1 NVTPPALPGI 0.020 642[ MTSILGAYVI 0.020 ~512 J~VQIARVILEY 0.018 478 I KPQDIPTFPL 0.018 471 GYIVGIVAW 10.018 41] GQCVLAGAFA 10.018.

23 ALVISILFIL 10.018 :41 KQRDEDDEAY 10.018 603 KLLWGGVGV 10.018 553 f K~lRAYIM U0.018 10.018 Table XV.VI-Al I01 -lOm-ers- 24P4C12 Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptie is the start rScore 267 GVLAYGIYY C f Table XV-V3.I4LAAliO1.

l0mers-2434C12 Each peptide Is a portion of SEQ ID NO: 7; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

Str [Subsequence fS-core 9 fNITPPALPGI IFO0 5 FPWThITPPA FO 00 3IJ RCP TP 0.002 7] WThITPPALP 0.001 4 IfCFPWTNITPP 0.00 I LGRCPWTGI0.000 -8 2 If PAFP G 0 000 A-l GR[ F IT .000 Table 110mers-2414412 Each peptide is a portion of SEQ ID NO: 11; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start poition pus nine.

6 ILLLVLIFLR 0.360 1 ]I VLEAILLLV 0.00 4 I EAILLLVU 10.002 Table XV-HLA-A11 101.

l0mers-24P4C12 Each peplide Is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide Is 10 amino adds, and the end position for each pepfide is the s postionplusnine.

WO E~q~s ju csore Table XV-V6-HLAAI 101.

10mers-24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the startsition pus nine.

Start Subsquence Ilcore 2 GYSSKGLIPR 0O.480 7 GUPRSVFNL 0.054 9 IPiRSVFNLQIl 0.004 8 LUPRSVFNLQ 0.00 5 -SKGUPRSVF 10.0001 6 KGLIPRSVFN 0.000 1 j QGY KLP 1.000 110oj PRSVFNLQJY 110.] 4 71 SSKGLIPRSV0.0 3 jfYSSKGLIPRS0.0 Table XV.V7-HLA-A11101.

l0mers-24P4C12 Each pelpfide is a porton of SEQ ID N~r 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the 6 postionplusnine.

Start Suseuence Sor 9 AVGOMMSTMF0.

5 jWILVAVGQMM 0.06 7 1LVAVGQMMST o.ODj =8i WYVAVGQ T 0.0 6 S1WILV AVMM 0.001j 0.00 Table XV-V7-HLA-A1101iOners-24P4C12 Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is amino adds, and the end position for each peptide is the start position plus nine.

1 YWI R 0.VAVG 032 1 TableXVILHLA-AI 1012 Each peptide is a portion of SEQ ID NO: 17; each start position is specifed, the length of pepticle is amino adds, and the end position for each peptide Is the start psiion plus nin Start c i-r 8 1I HVFQTSILGA 0.080a 1 ILNYWLPIMR [q032 2 QTSILGAYV I 02 2 FQTSILGAW 1.006 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start iton pus nine.

Start Subsequence Score] 13 IITQPATLGYVL 0.012 [T7 ALYPLPTQPA 0.008 711 1LPTPATIGY 0.004 7-9] YPLPTQPATL 0.003 16 ATLGYVLWAS 0.003 14 ]I QPATLGYVLW 0.002 19 I GYVIWASNIS 0.002 1 AYWAMTALYP 0.002 12 PTQPATLGW 10.01 5 MTALYPLPTQ 0.001 4 AMTALYPIPT 0.001 8 LYPLPTOPAT 0.000 U1 LGLWASNI 5.000 15 1 PATLGYLWA 0.000 F-7- TLGYVLWASN OM-000 3 WAMTALYPL 0.000 2 YWAMTALYPL H-.000 H T 1 TALYPIPTOP 0.000 10 I[ PLPTQPATLG 0.000 Table XV-VI-HLA.AZ4-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specifted, the length of peptide is 9 amino acids, and the end position for each peptide Is the start psto lsegt Start][ S ene 881 YFNIFSCIL 0 P -F 3.00 666

LFLCFLEDL

150 450 LTLWVL '0 503 AFGALILTL 0 84 PYLLYFNIF 1 D 0 T 00 658 jVFGMCVDTL -0 L553 KFLNRNAYI NO50 II P0 513

RNIVAGPVL

0 484 PMKL 417 FGYIGIV 1 1 30I1 AY0SVOETW 1 468 AFASFYWAF II0 139 VFYTKNRNF Jl NPITPTGHVF 13 [IPTGHVFQT 19 VFQTSILGAY

NYWLPIMRN

I RNPITPTGHV 115 PTGHVFQTSI

LPIMRNPITP

8 f IMRNPITPTG

WLPIMRNPIT

4 JYWLPIMRNPI 9 MRNPITPTGH S TPTGH QTS IT II TGHVFQTSIL

JGHVFQTSILG

7 PIMRNPITPT [T ]WPIMRNP 121 PITPTGHVFO 0.003 0.003 0.002 0.002 0.001 0.001 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.000 000 0000 0.000 Sable XV-V9HLA.AIO1.

0murs.24P4C12 Table XV1.V.lILAA24-9mers* 24P4C12 Each peptide Is a portion of SEQ ID Na. 3; each start position Is specified. the length of peptide is 9 amino acids, and the end I position for each peptide is te start Position Plus eighL Start Subsequence Score 154I 1 WNMTVITSL 800 311 II AIVLAVl- 8.40D 261 ii ULGVLGVL 18.400 -440 II FNLQIYGVL I8.4001 72-34I VALGVALVIL J 7683 II RPYYMSKSL .0 333 11 RIRIAIALL J8-70 1[ LYPRNSTGA J7.500 32 IFLRQRIRI 7.500 317] FLPSAPL 7.200 YPLVTFVLL 7.200 [236 I LVALVLSL 7.200 621 l RIPGLGKDF 7.200 158~!. VITSLQQEL 6.336 13051 VQETWLMAL 6.000 115 ~[KPVKYDPSF 6.000 f -5T47( CLEKFIKFL 16.000 597 1[LLLFFGKU. 6.000 565I IYGKNFCVS 6. 000 34 11 DVICC 1 6.000 308I TWLAALIVL 6.000 IWTNVTPAL 6.000 316 ]ILAWLEALL 16.000 2 200 TIQQISGL 6.000 [635 II NYYWLPIMT 6. 000 140 FYTKNRNFC r6.O0 0 673I DIERN'NGSL 6.000 442 ILQIYGVLGL 6.000 4114 1[LVNSSCPGL J6.000 4441 IYGVLGLFW 16.000 Table XVIVI-HLA-A24-9mers- 24C2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acis, and the end position for each peptide is the Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino adids, and the end position for each peptide Is the 7-971 ITPPALPGI 1i1.800' 727 CFPWTNIT .7 =3 NITPPALIP 0.075 8 WNITPPA 0.014 4L~. PWTNITPP 00.010 '[Table XVI.V3.HLA.A24-9niers- 24P4C12I Table XVI.V6HLA-A249mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight SStartScare 6 IIGLIPRSVIFN Boie 3 If SSKGLIPRS 10.120 8 IPRSVFNLQ 0.020 4 f SKGLIPRSV 10.014 2 I YSSKGLIPR 911 PRSVFNLQI Table XVI.VI-A-A24-9mers.

24P4C12 Each peptide is a portion of SEQ ID N0r 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight [i Subsequence Scorel 11ILVAVGQMM 11.2601 IY4I[ WLVAVGQM 0.750 F-2 WYWILVAVG 110.6001 :Tr AVGQMMSTM ]1ug r71 VAVGQMMST 0.150 Fil SWYWILVAV 0.140 6 LVAVGQMMS T.10 3 ]I YWILVAVGQ 0.0211 Table XIV8-HLA-A24-Smers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is thi start ton lus el Start I SseueneIscore NYYWPIMR 0.600 [iL PITPTGHV 0.240 10 NPiTPTGHV 0.150 LPIMRNPIT 0.150 19 FOTSLGAY 0.140 20 QTSILGAYV 0.120 13 TPTGHVFOT 0.100 7 IMRNPT 0100 F RNPITPTGH 0.001 1 YWLPIMRNP 0.025 14 PTGHVFQTS 0.020 12 ITPTGHVFQ 0.0151 17 HLG o.oio ]1 jRN FG 0.003 PtMRNPI1P 0.002 Table 'XVI VSLAA29mers* 24P4C12 Each peptide is a portIon of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino adds, and the end position for ead peptide is the rosition Plus eight.

Start Subsequence Score 18

GYWWASNI

7 LYPIPTOPA 9.000 2 J WAMTALYPL 6.000 13 OPATLGWL 4.800 9 P.PTQPAT 0 SI YPLPTOPAT 0.180 151 ATLGYVLWA 12 i TQPATLGYV 16 TLGYVLWAS 0.140 171 LGYVLWASN 0.120 4 MTALYPLPT 0.100 11 PTQPrTLAT 0.018 [Table XVIVI.-HLA-A24-l0mers- I24P4C12 Each peptide isa portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start ionusnine.

Start Subsequence Score 356 FYPLV F00 301 AYQSVQETW 00 .0 87 LYFNIFSCIL 0 140 KNRNFCL 0 274 YCWEEYRVL 1 F1 00 370 AYWAMTALYL 00 00 2D.0 18 11 KYDPSFRGPI 00 00 636 YYLPIMTSI 4391 VFNLQIYGL -0 355 MFYPLVTFL 6 00 169 SFLUPSAPAL 0 2I 6 01 4251[ FQGSSKGL 0.00] 00 00 Table XVII-VI-HLA.A24-lOmers 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the s position plus nine.

Starti Subsequence Score 616 FFFSGRIPGL 224 DFAQSWYWIL .40 478 01 PTPL U4 0 [i1 EFSQTVGEVF 14001 14.00 658 VFGMCVDTLF 69 NFCVSAKNAF Z KSPHLNWL 493 RTLRYHTGSL 3312 M3 RQRIRIAIAL_ 517 VILEYIDHKL

D'

i LFLLFILGY I 'V5 1.08 589 WLDKVTDLL

F

157 TVlTSLQEL 9.9141 620 EYID1KLRGV .0 386 QYVLWASNIS (900 445 YGVGL T16 240 LVISLFIL 8.640 248 W.RVAGPL 1.7 400 257 LVLVULGVL 8.400 48 I YIWGIVAWL 8.400 260 j VUILGVLGV 8.400 236 LGVALVLSLL 8.400 F-34 7 DVICCVLFLL 8.400 683 RPYYMSKSLL 8.000 648 AYVASGFFS 7.500 41 7 GYIWGIVAW 7.500 Table XVIIV1-HLA.A24-Omers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3, each start position is specitied, the length of peptide is 10 amino adds, and the end position for each peptide is the s ostin lu nine.

FSlart I I[ LYPRNSTGAY 7. 0f [53 KIFTIAYIM 7.500 [254 AGPLVLIL 7.200 [304 SVQETWAA 7.200 [23171 W1LVALGVAL .200 637 1 YWLPIMTSIL 7.200 162 LQQELCPSFL 7.200 17 f ALSLFIL 7.200 1318 VLEAILLLML 7.200 314 IVLAVLEAIL 7.00 37 CC F IL 7.0 D 15461 WCLE IKFL 7.200 F350 QMMMfMFYPL 7.2001 99-] NIISVAENGL 7.200 203 QGISGUDSL 7.200 243 SLLFILLLRL 7.200 229] WYW[LVALGV 7.000 31 MSCDVICCVL 6.720 441 NLQIYGVGL 6.000 357 YPLVTFVW. 6.000 604 LLWGGVGV1 6.000 1510 TVARVL 6.0001 5961 DWLFFGKLL 6- 536 1 CIMCCFKCCL 6.000 588 WADKVTDL 6.000 4331 GLIQRSVFNL 659 FGMCVDTLFL q.000 456 WVLALGQCW 6.000 413 HLVNSSCPGL 6.000 290 ISQLGF1TNL 6.000 321 WIALLMUFL 6.000 316 LAVLEAILLL 6.000 57 1 LYGDPRQVLY 6.000 [91 IFSCILSSNI 6.000 1 [77 MGENKDKPYL 6.000 EK I] QQELCPSFU 6.000 [A i TTIQQGISGL][.000 Table XVII.Vi-HLA-A24-lOmer.

24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start sition us nine.

[start Subsequence Score [00 OSLAF L 6.000 [83 KPYLLYFNIF 5.760 3 10 LAALIVLAVL 5.600 233 LVALGVALVL 5.600 227 OSWYWILVAL 5.600 661 MCVDTLFLCF 5.184 279 EYRVLRDKGA 5.000 635 NYYWLPIMTS 5.000 273t IYYCWEEYRV 5.000 444 IYGVLGLFWT 5.000 L86 YMSKSLKIL 4.800 56 1WLYGDPRQVL 4.8001 ffi ALGVALSL498j 252 JLVAGPLW 4.800 449 1 GLWTLNWVL 4.800 ]502ILAFGALILTL 4.800 [-25I LGKDFKSPHL 4.800 F ]49 HTGSIAFGAL 4800 I4 VSAKNAFM .800 542 KCCLWCLEKF 4.40 442 LOWGLF 420 3681 CIAYWAMTAL 4.000 [241 11 VSLLFLL I 4.00 STable WlV1I.3iLA-A24-10merso I24P4C12J Each peptide is a portion of SEQ1 ID NO: 7; each start positi isj specified, the length of peptide isi posaitnfor acpide nd the achtids, and the] start position plus nine.

FPwTrNrr 04 4l CFPWTNITPP 0.7 3 ]IRCFPwflIITP 10.0241 f 1 TNITPPALPG 10.015, r-7]I WTNITPPALP 0.015 GRCFPWThIT 10.0121 [Table XV11I-V54ILA.A24-l10meirs.: 2413402 Each peptide is a portion of SEQ ID NO: 11: each start position is specified, the length of peptide is amino acids, and the end position for each peptide Is the str position plus nine.

strtSubsequence Score 2 11 AILLLVL M8.200 4 EAILIIVLIF 3.600 9 LVLIFLRQRI i2.160 M 2 VLA- Y .20 i LALLLVLI 0.1201 7 VUFLRQ 10.0251 6 ILlVFLR 0.018 VLIFLRQRIR 0.015 8ii LLVUFLRQR 0 .015 Table XVIV6.HLA-A24-l10mers-I 24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is amino acids, and the end position for each peptide Is the start position plus nime.

7 GLIPRSVFNL 720 IPRSVFNLQI 1.000] 2 IIGYSSKGUPR 050 Table XVII-V-HLAA24-10mors-1 24P4C12 Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amnino acids, and the end position for each peptide is the start poii lus nine.

Start][ Subs. ueneIsoe AVGQM I270- 3 YWLVAVGQ .0 "I [AVGMMS0.50 171 QWY~lLAV 10.140 -77 LVAVGQMMST 10.100 2= SWYW1LVAVG 10.012 Table XVII-V8-I4LA-A24-0mers- 244C12 Each peptide is a portion of SEQ IDNO, 17; each start positionlIs specified, the length of peplide is 10 amino adids, and the end position for each peptide is the sub us nine.

Start quence S1core 72- NYYWLPIMRN [F50-0 [16 1TGiVFQTSIL 4.000 11 1NP ITPTGH VFF 00 Table XVD.V944LAA24-1Omers-j S24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position pl us nine.

Strt Subseuence [iZcr-e 19 GYVLWASNIS j9.00I 8 LYPPTQPAT 71.500 13T PAGY 9 YPLPTQPATI. 17.2001 2 YWAMTALYPL I4.000I 18 LGYVLWASNI 100 1 AYWAMTALYP 050 16 ATLGYVLWAS 020 7 ALYPLPTQPA 0.1441 7IT77- TLGYVLWASN 0.120 B 4 AMTALYPILPT 010 U~ QPATLGYVLW 11.1O 11 LPTPA-TLGY 010 iTPTQPATLGYV 1.1 %6 TALYPLPTQP V 3T WAMTALYPLP 1008] 15 TPGYVIWA 101 M5_ MAYLTQ 10.01 0 00 00 C0 00 00 Table XVI.Vg9-HLA-A24-lOmers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is amino acids, and the end position for each peptide is the start position Plus nine.

Startll Subsequence Score PIPTOPATLG 0.002 Table XVIII-VI-HLA.B7.9mors 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino ads and the end position for each peptide is the start sition lus eiht Start Subseuence Score 240 L IL 605 LWGGVGVLL' 34 1DVICCVLL 0 00 589 VWLDKVTDL 347 AVGQMMSTM I[Is TO 573 SAKNAFMLL 12.00

VAGPLVLVL

IAYWANTAL L 120O 22V5 FAQSWWIL 1 2.00] 213 NARDISVKI 514 tARVILEYI [jj WNM-0SL 316 LAMAUI7 1200 00 234

VALGVALLMO

396 SPGCEKVPI 83 KPYLLYFNI 406 TSCNPA 01 381 TSGQPOVL .00 571 CVSAKNAF M 15.000 r261 LILVGG 4000 315 VLAVEAIL 114.O 91q SMGMFTNL 1.000 WLPIMTSIL

=O

ZB V VLILGVLJ.0 F-42 wn~wvLAL 14000 128 KRCTDVI Table XVIIIVI-HLA4B7-9es1 24P34C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the Start'Subsenc GPKNCT ]3-.000! 482 IPF IS] 13.000! 344 ASKAVGQMM J3.000! 343 1 EASKAVGOM J3.7000 14911 LPGVPWNMT 3.000!0 .s8i f LMRNIVRVV J2.0001 531 0 PVRMC J2.0000 188 I TPPALPGrr 2.000 1121 TPQVCVSSC 2.0 IfDPRQVLYPR 2.000 525 KLRGVQNPV 2.0 314 I ViAVLEAI 167 CPF LLS 151 11 GVWMV _1200 192 11LPGIThD1T 2.0000 359 If LvTFvLLLi- 2.0006 252 IfLVAGPLVLV 1.50 491 1 FIRTLRYHT J1.500 530i QNPVARCIM 1.I500 23 0ALVLSLLFI 1.0 Table XVIII.V-HLAB7.9mers- 24P4C12 rE 8 C peptide is a portion of SEQ ID N(Yr 7; each start position is specified, the length of peptide is 9 amino acids. and the end position for each peptide Is the start position plus eighL 8 NJTPPALPG 1Oijo01 3 1CFPWTNITP 11o01 Table XVIII-V5HLA.B7.Omers- 24P4C12 rEach peptidelis a portion of SEQ ID NO:. 11; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start poAiio plus eight I tr Subseauerice liScore fable XVIII-V6HLA-B7-gmers- 24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 9 amio acids, and the end position for each peptide is the Sat Subsequence Iscorel rable XVIII-V6.HLA-B79mors.

?4P4C12 Each peptide is a portion of SEQ~ ID NO. 13; each start position Is specified, the length of peptide is 9 amino acids, wa the end position for each peptide Is the start Dosition olus elaht Table XVII.V84LAB7-mes 24P34r.12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is t start poition pus eig ht tart I!Subsequence Scre, 107! NPITPTGHV 6.000 51 I ILPIMRNPIT l2.000 ::3=If VQ JF 2000 7 11 WIMRNPIP 01.500 I TGHVFQTSI I040 71-67 GHVFQTSIL 10.400 M IQTSILGAYV_0.0 1-7- HvFQTS!LG 0.050 19 FITSILGAY 0.020 118 11 VFQTSILGA 0.010 F 7 1 ITPTGHVFQ D.0101 9 RNPITPTGH 0.010' 6~ PIMRNPITP 0.003 2 YYWPIMRN 0.003 114 PlTPTGHVF 0.002 14 PTGHVFQTS; 0.0 -3 YWLPMRNP 1001 1 1 NYYWLPIMR 11 I~' Table XVII-H-A7.9mers-1 24P4CI2 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amrino ads, and the end position for each peptide is the Start Subseuence cre 8 YPLPTQPAT ]F2-0-0 9 LPTQPATL 0.4001 101 LPTQPATLG 10.3001 il- ATLGYVLWVA 0.300! ~12 11 TQPATLGYV 0.O 2001 4 I( MTALYPLPT .1000 -1TALYPLPTO .05 F-1 _8 IGoVLo40N A -MTALYPLP 000 1 6 ALYPLPTQP 0.3 171 LGYVLWASN 0.020 16 Tj[ VWA 10.00 7 1 I4KjI40i5 141 PATLGYVLW 10.006 1i! PTQPATLGY 0.002 1 YWAMTALYP ]M00 Table XI-V-HLA.BT10mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptid is 10 amino acids, and the end position for each peptide is the srt pstion Dlus nine.

Start 1Subs uence Sore] 478 KOITP 2.

63 RPTYMSKSLLso'0 ~0 _PLVTFVLLL r02 31 RORIRIAJAL j400] 571 ICVSAKNAFML 27 LVWVLILGVL r0 58E1 66TD GAC00 =4 LVALLLL 00 oo 3_[14 CVLLL_ j200 221LVAGLVLVL]100 101.00 380 ATSGQPOYVL 18.0 37 AVLEAILLLM 150 q A ILLNUFL ~502 LAFGALIT 00 00 C0 00 Table XIX-V1 -HLA-67-1 Orners* 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end I position for each peptide is the start position plus nine.

tart FSubse uence score 243 SLLFIW.LRL ]4-.000- 371 CCVLFU.FIL 14.000 4491 GLFWTLNWVL 14.0001 182-1 LOOELCPSFL 4.000 605 LGKDFKSPHL ]E00 22 QSWYWILVAL 4.000 Z48 HGSLAFGAL 4.000 ___YIWGIVAWL 4.000O 604 LLWGGVGVL 4.000 149 [LPVWMV 4.0001 500 GSLAFGAUIL 4.000 546 1 WCLEKFIFL. 400 241 VI.SLLFILLL 14.000 539 CCFKCCLWCL 400 445 YGVLGLFwTL J500 ETWLAAUVL I4.D 435 R lQRSVFNLQI 14.000] [51-711 VILEYIOHKL [Th-9]9 TriQQGISGL 4J 73- SCTDVCCVL 4.000 1 78 1LGRCFPWTNV 3.000 F33 EASKAVGQMM 3.000 346 -KAVGQMMSTM 3.000 58 LMRIVRVVN 3.000 [7 [SAKNAFMLLM 3.000 F-62- If AS*SG .000 F 4-02-1 TCNP 2.000 FI182 I1 FPWTNvTPPAI2.0 1 528 If GVQNPVARCI W r-2E8I7 RVLRDKGASI 2.000 1 16 NVTPPALPGI .0 Table XIX-Vl.HLAB-l0mers- 24P4C12 Each peptide is a porton of SEQ ID NO: 3; each star position is specified, the length of peptide is 10 amnino acids, and the end position for each peptide is the ITable XIX-V3-HLA-B7-l0mers- I24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specifid, the length of peptide Is 1aino acids, and the end position for each peptide is the Start I Msuence Scot] ij LGRCFPWNI 6.000 F- FPWTNITPPA 2.0MOO0 9l NITPPALPGI 10.4001 1 1 ITPALPGIT 0.1001 F 6711 PWTNIPPAL 0.0401 8] TNITPPALPG 0.0151 7 WTITPPALP 001 RCPWTNITP 0.010 1 2FNTP 0.010 SGCPWTNITFPTTP 0.01 jTable XIXV5.HLA87.l0mors I24P4C12 Table XIX.V7-HLAB71 Omers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 10 amino adds, and the end position for each peptide is the start position pusn e Tabe IX-94LA-B7-l0mers- 24PC12

I

00 00 Table XX-Vi .HLA-B35-9meres.

24P4C12 Eac;pptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight 7KF iS ubeuence !SCor78 731-1 AALIVLAVL 300 253I VAGPLVLVL 113000 651~ IASGFFSVF IF3.00 F-9-1 GAY iASGF 3000 [225 1 FAQSWYWL J3.0001 SAPALGRCF 3.000 F2-34 VALGVALVL 3.000~ F-369 IA~AMTAL 3.000 [1417II YTKNRNFCL 3.000 F-4]J TLRYHTGSL 3.000 F-67781 NGSLDRPYY 3.000 249J LLRLVAGPL 3.000 117T VSSCPEDPW 2.500 [28 ]jKNRSCTDVI 2.400 W1 AVAJLLL 12-000 266[ LGVLAYGIY 2.000 363 VLLLICIAY r2.000 267C GVLAYGIYYY 2.000 GPIKNRSCT [2 0j0 045 VNSSCPGULi 12.000 WVGIVAWLY 12.000 I589 VVLDKVTDL 2.000 I272 GIYYCWEEY 2.00)0 1188 TPPALPGIT 2.00 1432 KGUQRSVF .2000 152 VPWNMTVIT 2.000 1192 LPGITNDTTI 2000- 531 NPVARCIMC 12.000 583 RNIVRVVVL 2.000 366 UICIAYWAM 12.000 546 WCLEKFIKF 12 000 554 FLNRNAY1M 2000 53 QIARVILEY 2.0-00 92 FSCILSSNI 2.000 530 CtPVARCIM 2000 Table XX-VI-HLA-835.9meres- ARMC1 FEach peptide is a portion of SEQ 10 NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start posto pls eht.

Start 1 Suseuenc Icr Table XX(-V3-HLA-B35.gmers- 24C2 Each peptide Is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

=Start Subse uence- Score 21 RCFPWTNIT 0.0 ILI GRCpwl 10.040 7J TNITPPALP j0.-010 8 NITPPALPG 0.010 3[ CFPWTNITP j 0.00 _AJL PWVTNITPPA 10.001o Table XX-V6-MILA-B3354h.&m1 24P4C12 Each peptide is a portion of SEQ ID NO: '13; each start position Is specife, the length of peptide is 9 amino adldsx and the end position for each peptide is the sait psition plus ei-- t.

Start Subs uec Score 7 LIPRSVFNL r 000 8 IPRSVFNL 0.600 6 GLIPRSVFN 0.100 Ai YSSKGLIPR0.050 4 SKGLIPRSV 10.020 9 I PRSVFNLQI r.004 1 I GYSSKGLIP 0.001 Table XX-V8-HLA-B35-gmers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specfied, the length of peptide is 9 amino acids, and the end position for each peptide Is the startpoiinpu gL SartISusqec 18I VFQTSILGA 0.010 14 PTGHVFOTS 0.010 17 0 010 12 IIITPTGHQJ1 6 PIMRNPTP 0 3 1YWLWMRNP 10.00i Te" mRNPiTp G 'rO 001 SI NWPI =0.001 Table XXV9.HLAB359mers- 2i.P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start pion lus ei Startore 13 Il QPATLGYVL p00 F-2 ir WAMTALYPL F03.000 Table XX-V8-HLAB35-9mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start sition us eight Start Subsuence More NPITPTGHV 4.000 13 IF2H000 19 FQTSIGAY I LPIMRNPIT 1200 j( ITGHMTSI 10-00j j WLPIMRNPI 0.400 7 IMRNPITPT 110.3001 QTSILGAYV 0.200 11 PITPTGHVF 0.100 716 11 GHVFQTSIL 0.100 7-9-11 RNP cjTPTl o0.2 LI2 l YYWLPIMRN 10.010 Table )OU.VI4ILA.535-10mers- 24P4C12 Each peptide is a portion of SEQ ID Na. 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the s'awt Psition Plus nine. 1 itirt Subsequence ]Scorel [57211 KAFL 1 5.000 [22T I! YLVAL 5.000 fGSLAFGALIL 5.0001 [417I SSCPGLNICVF 5.0001 F2 20-1 ISGFTNL 5.00 761 GMGENKDKPY ]4.000 68 ][RNSTGAYCGM 14FO7 149 LPGVPWNM1V. 14.000 676 IRNNGSLDRPY 4.000 310J LMLIVLAVL 3.000 31 LAVLEAIU.L 13.000 f320 IfEAIU.LMLIF .0 467LI GAFAFWAF 13.0001 395~. SSPGCEKVPI 3. 000D 647 1GAYVIASGFF 3.0001 I67 NNGSI.DRPYY 3.000 502 ILAFGALILTL 3.000 430] SSKGUQRSV 3.00 381 ][TSGQPQYVLW 2.500 39§J VLFLLFILGY 2.000 188J TPPPLPGITN 2.000 1152 11VPWNMTVITS 2.000 348 IIVGQMMSTMFY Z000 31i SCTDVICCVL 2.000 3114 IfQPQYVLWASN 2.000 409 IINPTAHLVNSS 2000 613[ LSFFFFSGRI 2 0 220 1F KFEDFAOSW 2.000 110 ]j CPTPQVCVSS 2.000 54 1[ WCLEKFIKFL 2.000 271 J YGIYYCWEEY 2.0 RSCTDVICCV 2.00 172 E- LPSAPALGRC 2.0001 162 1f LQQELCPSFt. 2.0001 Table XXI-VI.HILA.B935-10mers- 244C1 2 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 10 amino adids, and the end position for each peptide is the start Position Plus nine.

[Start Subsequence Sore 73- 71 SPGCEKVPIN 2.000 F LGVLAYGIYY 2.000 F-4027 VPINTSCNPT 2.000 1 378 If YLATSGQPQY 12.000 F3851 If CIAYWAM 12.000 fLGF1TLSAY 2.000 262T ]ILGVLGVLAY 12.000 I286 11KGASISQLGF 2.000 529 ][VQNPVARCIM 2.000 678 JrNGSLDRPYYM 2.000 49 IWVGIVAWLY 2.000 147 JrFCLPGVPWNM 2.000 265f VLGVL.AYGIY 2000 304r SVQETWLAAL 2.0001 464 VIAGAFASFY 2000 20 ~JDPSFRGPIKN 2.000 661 IfMCVDTLFLCF 2000 92 FSCILSSNII 2.-000 512 IfVOIARVILEY 2000 182 ILFPWTNVTPPA 2.000 639 J[LPIMTSILGA 2.000 [570 FCVSAI(NAFM 2.000 FRTLRYHTGSL 2.000 6~33 HLNYYV&PIM 2.000 53 PCIMCC 2.000 6221 JIPGL FK] 2.000 4851 FPLIART 12.000 542 If CggCLE] I2.000 589 IfVLDKvTDLL 2.0 517 IfVILEYIDHKL 2.000] 344~ ASKVGMMS 1.500] 465 JjLGAFAEW J 1 .500J 300 SAYQSVQETW H 1 iS5O0 659 JFGMCVDTLFL 11500 31 AF AVEA-Il s]n 18 SSCPEDPWTV U1~ Table 24P4C02 Each peptide is a portion of SEQI ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptlde Is the [statson lusnin [Sat Subse uence Score F576 NAFMVLLMRNI 1.200 435 IQRSVFNLQI 1.200 ITable MU.V3ILA..B35-1OCmers- S24134C12 Each peptide is a portion of SEQ ID NO:?7; each staut position is specified, the length of poptide is 10 amino adds, and the end posItIon for each peptide is the startsition plus nine tat[ ubseuno soe 5FPWTNITPPA 2.000 i urLGRCFPWTNI 1.200 V 11NITPPALPGI 0.4001 a CPITP 002 '1 [TITPPALPG 0.1 PTITPL 000 7 CWT NITPL 10.0201 2 GRCFPWTNITG 0.010] CFPWTNITPP 0.010] Table XU-VS HL-A-B35-10mers- f 24P4C12 Each peptide is a portion of SEQI ID NO. 11; each start position Is specified, the length of peptle is 10 amino acids, and the end position for each peptide Is the start position Plus nine.

rt Subsequence Score.0 4 EAL LV I 3lF J 0I 5 AILLLVLIFL 1.0001 9~J LVUFLRQRJ 10.4001 1l AW.EAILLLV 0.400 Table XCI-HLA.35-l0mers.1 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is amino adids, and the end position for each peptide is the start *ostion pus nine.

Start Susuene FS-eor- II LLVIFi i.10 VUFLRQRIR 0.010 .fiI LLLVLIFLRQ J .010 8 LLVLIFLRQR I .010 Table XXi.V7-HLA.535-10mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start sion lus snine.

startf Susqec Score YWILVAVGQM 0.200 6 LVA MS 0.100 7 fLVAVGMT0.00 3 WYWIGQ0.001 Table XX-VO-HLA-B35-10Omers- 24P4C12 Table XXI-Vg-HLAB35.l0mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the str *ton pus nine. I Start Subsequence IScorel 2 YWAMTALYPL 1.000 13 j TOPATIGYVI 1.000 9 YPLPTQPATL 11.000 18 LGYV1.WASNI 11.000 14 QPATLGYVLW 0.01 11 LPTQPATLGY 0 .200 16 1 ATLGYVLWAS 10.150 19. GYVI WASNIS 10.100 4 AMTALYPLPIT 10.100 8I LYPLPTQPAT 10.10 17 1 TLGYVLWASN 10.100 1 ALPTQPA 0.100 12 1 PTOPAThGYV 0O.020 16 1 TALPPT 10.010 If L~vw 0.1 iII AYWAMTALYP 101 107ri PLPTOPATLG 11OW Table XXI.V741LA-1B35-1Omers.- 24P34C12 Each pepUde Is a portion of SEQ ID NO: 15; each start position Is specified, the length of peplide is amtno acids, and the end position for each peptide Is the start position plus nine.

Start j Subseuence HScoe 8 11VAVGOMMSTM I1I IfWILVAVGQMM I~ 9I AVGQMMSTMF ]1.000 1 II SWYWILVAV 1.I 000 00 Tables XXll.XLX, Tab UWI-I.I-HA-01mers- 24P4C12 Each peptide is a portion of 00 SEQ ID NO: 3: each start 00 position is specifed, the length of peptide is 9 amino adids, and the end position for each peptide Is the start position 00 plus eight.

N Pea 123456789 scoe N!KDKPYI4.Y 34 58 YGDPRQ A-Y 33 222 FEDFAQS-WY 26 QRDEDDEAY 00 77 MGENKDKRPY 263 L&VLGVAY 24 489 SAFIRTLRY 23 513 OIARVIIEY 23 628 DEK(SPHINY 22 LEILFILGY 21 267 G_ LAY6IYY 21 383 VLLLICIAY 21 421 GLMCVFQGY 211 VYGIVA~t.Y 318 VLEAILLILM 629 FliSPHL.iYY 1133 SgTVGEY]FY 19 437 RSVFNLglY 19 662 C:VDTLFLCF 19 11 EAYGKPVKY 18 370 AYWAMTALY 18 18 KDPSFJBGP 17 32 C I VC VI 17 66 YERNSTGAY 17 277 WkEYRLRD 17 379 LATSGQEQY 17 594 VTULLFG 17 165 ELCPSFLLP 16 353 SIMFYPLVT 16 398 G CEKVPiNT 16 552 IKFLNRNAY 16 590 VLDKVTgLL 16 678 NGSLDRPYY 16 7able)OUIV3HLAAI.Smers- 2442 Each peptide is a portion of SEO ID NO: 7; each start positin Is specified, the length of papflde is 9 amino acids, and the end position (or each peptide is the start position plus aighi Pos '123456789 score TableXllI.VU4LA-AI-9meli- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified. the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight.

Pos 1 23456789 score 8 NITPPALPG 11 9 ITPPALPGI 6 WTNOIEAL 6 3 CfFPWNITP TableXXiI-V54ILA-AI-9mers- NNWC1 Each peptide Is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 1 23456789 score 1 VLEAILLLV 20 7 LLVLIFLRO 10 TableX~lt-V64LA-All-Omers- 24134C2 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 2 YSSKGL!PR 12 1 G(_SSKdIIP 7 3 SgKGUERS 7 8 IPRSVFNLQ 7 9 PE3SVFNjQI 7 6 GI4PRSVFN 5 TableXMIl-VT-HLA.AI-9mers- 244C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eighL Pos 123456789 score IL VA VGQMM 5 3 YW1LVAVGQ 4 7 VVGQEMST 4 6 L.VAVGQMMS 3 1 SWYW1LVAV 2 2 WXYWILVAVG 2 TableXXOl.V8.HL-A.All9mers- 24P4C12 Each pepfide is a portion of SEQI 10 NOr 17; each start position is specified, the length of pepfide is 9 amino acids, and the end position for each pepbde is the start position plus eight.

Pos 123456789 scor e 19 FQTSILGAY 16 14 PIGHVFgTS 11 12 IfiPTGHMFQ 8 18 VEQTSILGA 7 20 QTIlLGAYV 7 TableXXIlV4MILA-A1.-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 11 PIQPATLGY 31 15 AILGYVLWA 16 TabWeXX$iV1-HLA.A0201- Bniers.24C2 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 12345%789 score TableX~ill-VI-HLA-AD2011- Omers-24*4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 260 VLILGYLGV 31 244 UYFILILIRL 29 580 LLMRNIVRV 29 95 ILSSNIISV 28 204 GISGLIDSL 28 261 LILGVLGVL 28 322 ILLLMLIFL 28 506 AULTINQl 28 170 FLLPSAPAL 27 252 LVAGPLVLV 27 449 GLFWTLNW 27 487 LISAFjRTL 27 604 LL.WGGVGV 27 45 lLGYIWVGI 26 232 ILVALGVAI. 26 233 LVALGVALV 26 315 VLAVLEAIL 26 501 SLAFGALIL 26 521 Y1DHKLRGV 26 42 LLFILGYIV 107 GLQCPIPQV 200 T1QQGISGL 211 SLNARDISV 239 ALVLSLLFI 257 LVLVL!LGV 258 VLYLIIGVL 282 VLRDKGASI 317 AVLEAILLL 457 VLALGQCVL 598 LLFFGKLLV 650 VLASGEFSV 686 YMSKSLLIKI 41 FLLFLGYl 24 Tabt.XXll.VII.HLA.A020I1 gmer-241114C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and t end position for each peptide is the start position plus eight Pos 123456789 score 49 IWGIYAWL 24 310 LAALIYLLAV 24 311 MLIVLAVL 24 333 RIRIAIALL 24 434 LIQRSjVFNL 24 509 LTLVQ.IARV 24 525 KLRGV-QNPV 24 564 AIYGKN~FCV 24 581 LMRNI'jRW 24 596 DILLLFEFGKL 24 605 LWGGVGVL 24 V1CCVWFLL 23 56 WLYGDERQV 23 240 LVLSLLFIL 23 251 RLVAGPLVL 23 253 VAGPLVLVL 23 309 WLAL!IVLA 23 340 LIJ(EASKAV 23 358 PLvrFyLLLL 23 494 TLRYHIGSL 23 518 ILEYDHKL 23 547 CLEI(FIKFL 23 589 WLDK~/JDL 23 590 VLDKVI-DLL 23 597 LLLFFdKI1 23 100 IISVAENGL 22 241 VILSILL[ILL 22 248 LLLRLVAGP 22 249 LLRLVAGPL 22 265 VLGVLAYGI 22 446 GVLGLFWTL 22 452 WTLNWVLAL 22 578 FMLLMBNIV 22 638 WLPIMISII. 22 660 GMCVDTLFL 22 158 VITSLQQEL 21 187 VTPPALPGI 21 191 ALPGIIWDT 21 237 GVAILVISILL 21 247 ILLLRLVAG 21 313 LM..AyLEA 21 314 IVLAVLEAI 21 442 LQIYGYIGL 21 507 ULTLVQIA 21 537 IMCCFIKCCL 21 599 LFFGKLLWV 21 693 KILGKKNEA 21 TabeXXIl.V1.IILA-A0201- Slmers.24124C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus Pus 34 38 44 207 228 234 238 242 319 326 339 364 417 503 633 644 673 690 48 245 255 262 268 291 318 323 329 351 365 414 464 544 617 666 86 231 235 243 336 355 369 380 394 439 459 510 511 eight 123456789 score DVICCVLFL 20 CVLFLLFIL 20 FILGYIWVG 20 GLIDSLNAR 20 SWYVLVAL 20 VALGVALVI. 20 LGVALVLSL 20 LSLFILL.L 20 LEAILLLML 20 MLirLBORi 20 AUKEASKA 20 WJCIAYW 20 SSCIPGLMCV 20 AFGALILTL 20 HLNYYWLLPI 20 SILGAYViA 20 IDLERN1NGSL 20 SW<KILGKK 20 YIWVG!VAW 19 LFILLLRLV 19 GPILVL LIL 19 ILGVLGVLA 19 VLAYdrYYC 19 SQLGF1TNL 19 VLEAILLLM 19 LLLMLIFLR 19 FLIRQRIRIA 19 MMSTME'fPL 19 WLCI.AYWA 19 LVNSSCPGL 19 VLAGAEASF 19 CLWaggKi 19 FFSGRIPGL 19 LFLCIFLEDL 19 LLYFNIFSC 18 WILVALGVA 18 ALGVALVLS 18 SLLFILLLR 18 LAIALLKEA 18 MFYPLVTFV 18 IAYWAMTAI. 18 ATSGQQYV 18 ISSPGCEKV 18 VIFISLQIYGV 18 ALGQCVLAG 18 TLVQIARVI 18 LVQtABVIL 18 TabloX)hII-VI -HLA-A0201 9mers-2434IC1 2 U6~ peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 ami~no acids, and the end position for each peptIde is the start position plus eight Pos 123456789 score 514 IARVILEY1 18 517 VILEYjDHK 18 583 RNIVRVVVL 18 602 GKUNVYGGV 18 645 ILGAYVIAS 18 46 LGYIWYGIV 17 128 GKNEFSQTV 17 154 WNMTV!ITSL 17 '177 ALGRCEPWT 17 184 WTNVTEPAL 17 213 NARDISVKI 17 246 FILLI±RLVA 17 289 SISQLGFT 17 300 SAYQSQET '17 305 VQETWLAAL 17 3 12 ALIVLAVLE 17 325 LMLIFLRQR 17 335 IRIAIALLIKE 17 354 TMFYPLVTF 17 359 LVTFVLLLI 17 453 TLNWVLALG 17 456 WVLALGQCV 17 502 LAFGALILT 17 5034 FGAULTWV 17 513 QIARVILEY 17 554 FLNRNAYIM 17 560 YIMIA!YGK 17 586 VRVVDKV 17 642 MTSILGAYV 17 658 VFGMCVDTL 17 31 SCTDVICCV 16 43 LFILGYIWV 16 64 VLYPRNSTG 16 90 NIFSCjLSS 16 119 SCPEDEV 16 144 NRNFCLPGV 16 148 CLPGVEWNM 16 161 SLQQELCPS 16 230 YWILVLGV 16 254 AGPLVLVLI 16 308 rWLAALIVIL 16 316 LAVLEAILL 16 320 EAILLLMLI 16 357 YPLVTEVLL 16 362 FVLLLICiA 16 373 AMIALXLAT 16 376 ALYLATSGQ 16 00 00 TabIeXXII.V14iLA-AO2OF- 9mers.24 4Cl2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peplide is the start position plus eight Pos 123456789 score 407 SCNPTAHLV 16 458 LALGQGVLA 16 637 YWLPIMTSI *16 640 PIMTSJLGA 16 52 GIVAWLYGO 15 141 YTKNR!AFCL 15 225 FAQSWXjWIl 15 250 LRLVAGPLV 15 264 GVLGVLAYG 15 275 YCWEEXRVL 15 366 LICIAYWAM 15 368 CIAYWANTA 15 371 ?VVAMTALYL 15 374 MTALYLATS 15 406 TSCNPIAHL 15 433 GLIQRSVFN 15 443 QIYGVLGLF 15 491 FIRTLRkYHT 15 573 SAKNAEMLL 15 657 SVFGMCVDT 15 663 VDTLFLCFL 15 TabteXMlI.V34itLA-A0201- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Pos 123456789 score 9 ITPPAI-PGI 22 6 WTNITPPAL 17 8 NITPPALPG 1i 2 RCFPWINIT 10 TableXXIII.V5-HLA-A020i- 9mers-24P4132 Each peplide is a portion of SEQ ID NQ 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for eact) peptide is the start position plus eight Pos 123456789 score ILLLVILIFL 28 TabteXXII..-AOA20I- 9mes-24P4CI2 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 1 VLEAJLLLV 25 9 VI-FLERI 21 2 LEAILLLVL 20 6 LLLVL!FLR 19 3 EAILIIVU 18 4 AILLLVLUF 18 7 LLVLIFLRQ 13 8 LVUIFLRQR 13 TableX~aII-VB-HLA-A0201gmers-24P4C12 Each peptide Is a portion of SEQ 1t) NO: 13; each start position is specified, the length of peplide is 9 amnino acids, and the end position for each peptide Is the start position plus eight Pos 123456789 score 2 YSSKGLIPR 12 1 GYSSKGLIP 7 3 SSKGLIPRS 7 8 IRSVF7HLQ 7 9 PERSVFNLOI 7 6 GjUPRSVFN 5 Tabte)OII-V7ILA-A0201- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peplide Is 9 amino acids, and the end position for each peptide is the start position ptus eight.

Pos 123456789 score 1 SWYWILVAV 4 VILVAjGQM 18 5 ILVAV GQMM 16 7 VAVGWJMST 13 8 AVGQMMSTM 12 6 LVAVG~QMMS TabteX)llV8-HLA.A0201- 9mers-24P34C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 4 WLPIMENPI 19 7 IMRNPITPT 19 20 QTSILGAYV 17 10 NPITPIGHV 16 GHVFQJSIL 12 15 TGHVF-QTSI 11 18 VFQTSILGA 11 12 ITPTGtIVFQ 5 LPIMRN~PIT 9 13 TPTGII FQT 9 TableX~tll-V9HLA-A0201- 9mers.24P34M1 Each peptide Is a portion of SEQI ID NO: 19; each start position is specified, the length of peptide Is 9 amino acis and the end position for each peptide is the start position plus eight.

Pos 123456789 score 9 PLPTQEATL 21 2 WAMTALYPL 15 ATLGYVLWA 6 ALYPLPTQP 16 12 TQPATLGYV 14 13 QPATLGYVL 14 16 TLGYVLWAS 14 5 TALYPLPTQ 13 4 MTALYELPT 12 8 YPLPTQPAT 12 3 AMTALYPLP 1i TabIeXXIV-VI.-HLA.A0203- 9mers-24P34C12 Pos 1234567890 soore NoResultsFound.

TableXXI[V.V3-HLA.A0203gmers-24P4C12 Pos 1234587890 score NoResutFound.

TabteXXI.-HLA-A0203- 9mers-2434C12 Pos 1234567890 Score NoResultsFound.

TableXXIV.V64.HLA-A0203- 9mers-24P4C1 2 Pos 1234567890 Score NoResuttsFound.

TableXXI[V.V7-HLA.A0203- 9mers-24P4C12 Pos 1234567890 score No~tesutFound.

TableXXIV.VB.HLA.A0203gmers.24P4C12 Pos 1234567890 score lNoResuttsFound.

Table)OUJV-V9-HLA-A0203gmers-24P4C112 Pos 1234567890 score NoResuttsFound.

TableX(XV.Vl-HLA-A3-9mers- 24P Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 585 IVERVVMDK 29 424 CVEOGXLSSK 27 64 VLYPRNSTG 26 135 WTgEVFYTK 26 251 11G1.VLd- 26 506 ALILTLVQI 24 513 016RVILEY 24 603 KLLWGGfVG 24 690 SLILKILGKK 24 267 G\LAYi YY 2 282 WLRDKGASI 23 312 AL!IVIAg[E 23 334 IR1AtIlLK 23 102 SVAENa1,QC 22 232 ILVALGVAL 22 247 ILLLR-LVAG 22 443 QIYGVLGLF 22 464 VjGAEASF 22 516 RVILEYIDH 22 579 MLfMRFFNVR 22 W61VAWIY 21 212 LNARDISVK 21 281 RVLRD!LGAS 21 321 AtLILLtMjIIF 21 338 IAl)(EASK 21 339 AWL(EAKA 21 376 AL.YLATSGQ 21 Table)0(V-VI-HLA-A3-9mers- 24P Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 393 NISSPGCEK 21 517 VILEYIDHK 21 593 KVIOLLFF 21 619 SGRIPGLGK 21 621 RIFGL -DF 21 44 FLGYJ&VG 20 56 WLYGDPaOV 20 243 SLLFILILLR 20 259 LVIlLffiLG 20 347 AV GQMLTM 20 363 VILLICIAY 20 463 CV1.AGAEAS 20 501 SLAFG6IL 20 606 WV\GMVLS 20 689 KSLLKILGK 20 16 PVkYDPSFR 19 170 FW'PS6PAL 19 186 NVTPPALPG 19 207 GLjDS4LAAR 19 24<6 FILLLSLVA 19 249 LLLELVffPL 19 260 VLILGVLGV 19 262 llI.L IA 19 298 NL.SAYOSVQ 19 317 AVLEAILLL 19 333 RIELAIXlL 19 433 GL1QR~LffN 19 508 IILVQIAR 19 525 KLRGVCLNPV 19 560 YIMLAXGK 19 588 VVVLDKVTD 19 604 LL.WVGraLGV 19 605 LWVGGM9VL- 19 681 LEPYYMSK 19 11 EAYGKPVKY 18 49 IVWGlFV& 18 73 AYCGMGENK 18 220 KIEE0EAQS 18 248 LLLRLVAGP 18 261 LILGVLGVL 18 264 GLGVIAG 1 272 GIYYCVCEY 18 278 EEX[RVLRL)K 18 314 IVLAVLAJ 18 432 KGLIOUVF 18 441 NLQIYLG 18 448 GVLGLEWT 18 TableXXV-VI-HLA-A3-9mars- 24P Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amnino acids, and the end position for each peptide is the start position plus eight Pos 123456789 scoe 457 VLLGgW 18 564 AlYGKNFCV 18 587 RA wVLK 18 649 YVIASGFFS 18 10 DEAYGI- VK 17 63 QV1-YP§-HST 17 121 PEfPWVGK 17 177 ALaRCfPWT 17 211 SLYARDISV 17 233 LVALGVALV 17 235 ALGVALVLS 17 239 ALVLSLLFI 17 252 LVAGPLVLV 17 309 WLAALVLA 17 335 PJRIAA E 17 365i LL!CtAYWA 17 368 CI6YWAMTA 17 401 KVEINLSCN 17 421 GLMCVE9QGY 17 456 IWLALgQCV 17 459 ALGQCVLAG 17 510 TLVQtAV1 17 542 KCCLWCLEK 17 562 MtAIYGKNF 17 580 LLMRNVRV 17 583 RN1VR.LVL 17 644 SILGAYVIA 17 657 SVEGMCVDT 17 662 CVQTLELCF 17 26 PIKNRSCTD 16 34 DV1CCVLFL 16 45 ILGYIYNGI 16 86 LLYFNIFSC 16 15 TV1TSL.QQE 16 165 ELCPSE.LL 16 237 GVt.LVL.SLL 16 258 V'WILGVL 16 289 SISQLGFTT 16 304 SVQETWILA 16 323 LLLMLIELR 16 364 LILLJCIAYW 16 470 ASE1'WAFHK 16 494 TLRYHIGSL 16 511 LV!QIARL 16 554 FLtARNAYIM 16 571 CVSAKN61FFM 16 584 NLVRWVLD 16 TabIeXXV.Vi-HLA-A3-9mers- 24P Each peptide is a portion of SEQ ID Na. 3; each start position is specified,. the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 673 DILERNYGSL 16 693 KILGKKNEA' 16 698 KN1EAIPONK 16 DPSFRGPIK 15 48 YPIWGIVAW 15 58 YGQPRVLY 1 99 NIISV~gNG 15 151 GVPWNMTV1 15 191 AI.EIRDT 15 231 WILVALGVA 15 234 VALGV-6LVL 15 257 LVLV1ALGV 15 318 VLEAILLLM 15 322 ILILMLIFIL 15 327 UIELRQBIR 15 329 FLRCRIRIA 15 532 PVARC MCC 15 589 WIKVTIDIL 15 597 LU.FFGKLL 15 598 LLEFGgLLV 15 622 IP GLGKDFK 15 645 ILAYD!AS 15 651 IA§GFLSVF 15 680 SLDRPYYMS 15 691 LLKILGKKN 15 7 OIERDEAYGK 14 42 LLEILQXIV 14 53 IVAWLY.GDP 14 81 KDKPYLLYF 14 lLSSN1!SV 14 148 CLEGVEPWNM 14 171 LLPSAPALG 14 244 LLEIL!JLRL 14 3111 AAIIVL 14 315 VLVMlIl- 14 324 LLMLIELRQ 14 326 MILIFLEQgRI 14 337 AIALIKEAS 14 359 LVIFVLLI 14 370 AYWAMTAILY 14 378 YLATS-(jPQ 14 388 VLWASNISS 14 453 TLh!WVLALG 14 465 LkGAFAEFY 14 487 LIBAFIRTL 14 496 RY~iTGSLAF 14 523 DHLRIYQN 14 TableXX-V1 .HLA-A3-9meFS- 24P Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide is the start position plus eight Pos 123458789 score 527 RGYQNEMAR 14 528 GVQNPYIRC 14 534 ARCIMCCFK 14 558 NAYIM]~IY 14 567 GKNFCVSAI( 14 596 DLLLFFGKL 14 609 GVGVLSFF 14 638 WLPIMTSIL 14 647 GAjVVESGF 14 665 TLFLCFLED 14 685 YYMSKSLLK 14 694 ILGKKNEaP 14 699 NEAPPDNKK 14 701 APPDNKKRK 14 TableXXV-V3-HLAA3.9mers- 24P4C12 Each peplie is a portion of SEQ) ID NO: 7; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide Is the start position plus eight Pos 123456789 score 8 NIIPPALPG 17 Tab~eXXV-V5-HLA-A3-9mners- 24P4C12 Each peptide is a portion of SEQ ID NO: 11, each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123458789 score 4 AILLLALIF 21 8 ILVLIFEQIR 20 5 ILUMVIFL. 16 6 LLLVLIELR 16 1 VLEAJLLLV 15 7 LL&UELQ 14 9 VLIFLRIP 14 TabI9XXVV6-HLA.A3-Smem.- 2442 Each peptide is a portion of SEQ 10 NO: 13; each start position is specified, the length of peptide is 9 amino adids, and the end position for each peptide is the start position plus eight Pos 123458789 score 6 GULPRSVFN 22 5 KGLJPESVF 18 7 LIl5RSVFNL 11 TabIeXX(V-VT-HLA-A3-§rmers- 24P4C12 Each peptide is a portion-of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 8 AVGQMMSTM 5 IL'IAVGQMM 19 6 LVAVGQMMS 4 WILVAVGQM 14 3 YWJ1LVAVGQ 12 1 SWIWLVAV TableXXV.V8-HLA-A3..9mers* 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 9 amino adds, and the end position for 'each peptide Is the start position plus eight Pos 123456789 score 11 PITPTGHVF 22 6 PIMRNE!ITP 16 4 WLEIMMPl 12 9 RNPITPTGH 11 1 NVWLEIMR 17 4VFQT SILG TabIaXXV.V9.HLA-A39mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptIde is the start position plus eight Pos 123456789 score 6 ALYPLETOP 9 PLTQf!TL 18 11 PTQPAILGY 12 16 TLGYVLWAS 12 Table)OMV-V1.4iLA-A26- 9mers.24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 34 DVICCVLFL 35 49 IWGIVAWL. 28 483 PTFPLISAF 28 805 LWGGVGVL 27 593 KVTDLLLFF 26 317 AVILEAILLL 25 592 DKVTDLLLF 25 138 EVFYTKNRN 24 240 LVLSLLFIL 24 589 WLDKVTDL 24 38 CVLFLLFIL 23 237 GVALVLSLL 23 11 EAYGKPVKY 22 267 GVLAYGIYY 22 285 DKGASISQL 22 452 WTLNWVLAL 22 WVGIVAWLY 20 79 ENKDKPYLL 20 157 1VITSLQQE 20 263 LGVLGVLAY 20 446 GVLGLFWiTL 20 628 DFKSPHLNY 20 641 IMTSILGAY 20 662 CVDTLFLCF 20 236 LGVALVLSL 19 258 VI.VLILGVL 19 307 EThYIAALIV 19 320 EAILLLNLI 19 414 LVNSSCPGL 19 437 RSVFNLQIY 19 513 QIARVILEY 19 609 GVGVLSFFF 19 673 DLERtINGSL 19 32 CTDVICCVL 18 198 DTThQQGIS 18 200 TIQQGISGL 18 204 GISGLIDSL 18 244 LLFILLLRL 18 294 GFTTNLSAY 18 354 TMFYPLVTF 18 360 .VTFVWJLC 18 400 EKVPINTSC 18 511 LVQiARVIL 18 596 DUILFFGKL 18 102 SVAENGLQC 17 TableXXVI.Vi-HLA-A26- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 184 WTNVTPPAL 17 216 DISVKIFED 17 261 ULGVLGVL 17 358 PLVTFVLLL 17 438 SVFNLQIYG 17 442 LQIYGVLGL 17 443 QIYGVLGLF 17 487 LISAFIRTI 17 608 GGVGVLSFF 17 664 DII FLCFLE 17 TabIeXXVI-V3-HLAA26-9mers- 24P4C12 Each peptide Is a portio of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Pos 123456789 score 6 WTNITPPAL 17 9 ITPPALPGI 13 TaboXX)l-V5-HLA-A26-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the lengt of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight Pos 123456789 em 3 EAILLLVLI 19 4 AILILVUF 18 8 IVUFIROR 15 2 LEAILLLVL 14 5 ILLLVUFL 13 TableVAV6-ILA-A26-mes.

24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight 123456789 score LIPRSVFNL 16 KGLIPRSVF 9 TabIeXXVI.V?-HLA-A26-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 8 AVGQMMSTM 12 6 LVAVGIQIMS 11 4 W1LVAVGQM 1 SWYWLVAV 8 5 ILVAVGQMM 6 2 WY'WILVAVG 7 VAVGQMMST TabIeXXVI-V8-HLA-A26-9mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino adids, and the end position for each peptide is the start position plus eight Pos 123456789 score 19 FQTSILGAY 11 PITPTGHVF 17 HVFQTSILG 16 GHVFQTSIL 13 20 QTSILGAYV 14 PTGHVFQTS 9 TableXXVI.V9-HLA-A26-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 9 amino adids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 11I PTQPATLGY 15 ATLGYVLWA 13 2 WAMTALYPL 12 13 QPATLGYVL 4 MTALYPt.PT 9 9 PLPTQPA1L 9 TabloeXXVII.V1IHLA.B0702gmers.24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight Pos 123458789 score 255 GPLVLVLIL 23 357 YPLVTFVU. 23 683 RPYYMSKSL 21 149 LPGVPWNMT 20 396 SPGCEKVPI 20 482 IPTFPLISA 20 631 SPHLNYYWL 20 KPVKYDPSF 19 152 VPWNMIVIT '19 167 CPSFLLPSA 19 GPIKNRSCT 18 172 LPSAPALGR 18 83 KPYLLYFNI 17 188 TPPALPGIT 17 192 LPGITND1T 17 57 LYGDPRQVL 16 232 ILVALGVAL 16 253 VAGPLVLV1 16 479 PODIPTFPL 16 503 AFGALILTL 16 49 IVVGIVAWL 15 120 CPEDPWTVG 15 175 APALGRCFP 15 189 PPALPGITN 15 234 VALGVALVL 15 251 RLVAGPLVI. 15 381 TSGQPQYVL 15 406 TSCNPTAHL 15 583 RNIVRVWL 15 617 FFSGRIPGL 15 DPSFRGPIK 14 34 DVICCVLFL 14 66 YPRNSTGAY 14 204 GISGLIDSL 14 236 LGVALVLSL 14 252 LVAGPLVLV 14 291 SQLGFUNL 14 311 AALIVLAVL 14 317 AVLEAILLL 14 333 RIRIALALL 14 351 MMSTMFYPL 14 419 CPGLMCVFQ 14 452 WTLNWVLAI. 14 499 TGSt.AGAL 14 605 LWGGVGVL 14 660 GMCVDTLFL 14 DPRQVLYPR 13 100 IISVAENGL 13 110 CPTPQVCVS 13 164 QELCPSFU. 13 Tab~eXXVI.V1.HLA-B0702- Ilmers-24P4C12 Each peptide is a portion of SEQ I D NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123458789 score 170 FLIPSAPAL 13 182 FPWrNVrPP 13 228 SWYWILVAL. 13 241 VLSLLFILL 13 249 LLRLVAGPL 13 261 LILGWLGVI. 13 302 YQSVQE1WL 13 319 LEAILLLtvL 13 358 PLVTFVLU. 13 369 IAYWAMTAL 13 371 YWAMTALYL 13 409 NPTAHLVNS 13 442 LQIYGVLGL 13 446 GVLGL.EWTL 13 478 KPODIPTFP 13 487 LISAFIRTL 13 494 TLRYHTGSL 13 501 SLAFGALIL 13 511 LVQIARVIL 13 590 WDKVTDLL 13 622 IPGLGKDFK '13 651 IASGFFSVF 13 32 CTDVICCVL 12 78 GENKDKPYL 12 154 WNM1VITSL 12 184 WTNVTPPAL 12 242 LSLLFILLL 12 244 LLFILLILRL 12 285 DKGASISQL 12 305 VQETWI.AAL 12 308 TWLMLIVL 12 315 VLAVLEAIL 12 322 ILLLMUFL 12 358 FYPLVTFVL 12 373 AMTALYLAT 12 380 ATSGQPQYV 12 457 VLALGOCVL 12 525 KLRGVQNPV 12 547 CLEKFIKFL 12 572 VSAKNAFML 12 589 WLDKVIDL 12 591 LDKVTOLLL 12 626 GKDFKSPHL 12 658 VFGMCVDTL 12 701 APPDNKKRK 12 28 KNRSCTDVI '11 45 ILGYIVVGI 11 79 ENKDKPYLL '11 TableXXVII-VI.HLA-80702- 9mers-24P4C12.

Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 104 AENGLOCPT 11 107 GLQCPTPQV 11 109 QCPTPQVCV 11 112 TPQVCVSSC 11 123 DPWTVGKNE 11 163 QQELCPSFL 11 169 SFLLPSAPA 11 177 ALGRCFPWT 11 191 ALPGITNDT 11 237 GVALVI.SLL 11 239 ALVLSLLI 11 258 VLVLILGVL 11 262 ILGVLGVLA III 275 YCWEEYRVL 11 310 LAALIVLAV 11 332 QRRItAdAL it 343 EASKAVGQM 11 354 TMFYPLVTF 11i 384 QPQYVL WAS 1t 414 LVNSSCPGL 11 426 FQGYSSKGL 11 434 LIQRSVFNL I11 440 FNLQIYGVL 11 450 LFWTLNWVL 11 464 VLAGAFASF 11 518 ILEYIDHKL 11 531 NPVARCIMC 11 537 IMCCFKCCL 11 571 CVSAKNAFM 11 573 SAKNAFMLL 11 574 AJ<NAFMLLM 11i 595 DIILFFGKL 11 597 LILFFGKLL 11 599 LFFGKLLW I i 638 WLPIMTSIL I11 663 VDTLFLCFL 11 686 YMSKSLLKG I 11 702 PPDNKKRKK I1I TableXXVil-V3-HLA-60702.

9mers-24P34CIZ Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peplide is the start position plus eight Pos 123456789 score I 00 00 C0 00 TabIeXXCVI-V3-HLA-B0702- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Pos 123456789 score 4 FPWTNITPP 12 6 WTNITPPAL 12 1 GRCFPWTNI 10 2 RCFPWTNIT 9.

5 PWTNITPPA 9 9 ITPPALPGI 9 8 NITPPALPG 7 TabIeXXVII'.V"41A-B0702gmers-24P14IC12 Each peptidle is a portion of SEQ ID NO: 11; each start position Is specified, the length of peptidle is 9 amino acids, and t end position, for each peptide Is the start position plus eight.

Pos 123456789 score 2 LEAIU±LVL 14 ILLLVUIFL 12 4 AILLLVLIF 11 1 VLEAILLLV 9 3 EAILLLVL-I 9 9 VUFIRQRI 7 TableXXVII-V6-HLA-B0702.

9mers-24P4C1 2 Each peplide Is a portion of SEQ ID NO: '13; each start position is specified, the length of peptide Is 9 amino acids, and the end posifion for each peptidle Is the start position plus eighL Pos 123456789 score 8 IPRSVFNLQ 14 KGIJPRSVF 12 7 LIPRSVFNL 11 9 PRSVFNLQI 10 4 SKGLIPRSV 7 Tableo(VlIV7-HLA-BO702.

9mners-24P4C12 Each peptide Is a portion of SEQ U) NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

POS 123455789 scor SWYWILVAV 9 ILVAVGfQIM 9 AVGQMMSTM 9 VAVGQMMST 8 WILVAVGQm 7 TableX"l.VII-HILA-B0702-Smers- 24P4C12 Each peptide is a portion of SEQ I D NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptidle is the start position plus eight.

Pos 123456789 score '19 FQTSILGAY 20 11 PITPTGHVF 15 17 HVFQTSILG 15 16 GHVFQTSIL 13 20 OTSILGAYV 10 14 PTGHVFQTS 9 TableXXV11IV-HLA-0702.9mers- 24P4C12 Each peptidelIs a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 13 OPATLGYV1. 23 8 YPLPTQPAT 19 10 LPTQPATLG 14 15 ATLGYVLWA 13 2 WAMTALYPL 12 7 LYPLPTQPA 11 9 PLPTQPATL 11I YableXXVIll.VI-HLA-BOB08-mers Each peptide Is a portion of SEQ ID NO.- 3; each start position Is specified, the length of peplidae is 9 amino adds, and the end position for each poptide is the start position plus eight.

Pos 123456789 score 79 ENKDKPYU.. 32 14 YTKNRNFCL 29 282 VLRDKGASI 29 573 SAKNAFMLL 26 249 LLRLVAGPIL 23 494 TLRYHTGSL 23 26 PIKNRSCTD 22 329 FLRORIRiA 22 TabIeXXV1III.VI .tLA-BO849mers Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle is 9 amnino acids, and the end postion for each peptidle is the start position plus eight Pos 123456789 score 589 WLDKVTDL 22 333 RIRIAIALL 21 583 RNIVRVVVL 21 591 WDKVTDLU. 21 626 GI<DFKSPHL 21 687 MSKSLLUKIL 21 340 W<KEASKAV 474 WAFHKPQDI 523 DHKLRG VON 540 CFKCCLWCL 617 FFSGRIPGL 2 GGKQRDEDD '19 232 ILVALGVAL 19 255 GPLVLVLIL 19 631 SPHLNYYWL 19 694 ILGIKKNEAP 19 139 VFYTKNRNF 18 170 FLLPSAPAL 1B 241 VLSLLFILL 18 247 ILILLRLVAG 18 258 VLVLJLGVL 18 315 VILAVLEAIL 18 322 IW.LMLIFL 18 357 YPLVTFVLL 18 457 VLALGQCVL 18 501 SLAFGALIL 18 514 LARVILEYI 18 518 ILEYIDHKL '18 546 WCLEKFN(F 18 547 CLEKFIKR. 18 683 RPYYMSKSL 18 11 EAYGKPVKY 17 213 NARDISVKI 17 216 DISVI(IFED 17 358 PLVTFVLLL 17 533 VARCIMCCF 17 590 VU)KVTDLL 17 596 DLLLFFGKL 17 597 LLLFFGKLL 17 673 DLERNNGSL 17 691 W(ILGKKN 17 45 ILGYIWGI 16 64 VLYPRNSTG 16 81 KDKPYLLYF 16 100 IISVAENGL 16 158 VITSLQQEL 16 204 GISGLIDSL 16 211 SLNARDISV 16 244 LLFILLLRL 16 00 00 00 00 TableXXVIII.Vi-HLA-808-9meI'S Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 251 RLVAGPLVL '16 253 VAGPLVLVL 16 338 IAI.LKEASK 16 369 LAYWAMTAL 16 433 GLIQRSVFN 16 551 FIKFLNRNA 16 638 WLPIMTSIL 16 702 PPDNKKRKK 16 35 VICCVLFLL '15 200 TIQQGISGL 15 225 FAQSWYWIL 15 234 VALGVALVL 15 316 LAVLEAILI. 15 331 RQRIRIAIA 15 396 SPGCEKVPI 15 434 LIQRSVFNL 15 487 LISAFIRTL 15 553 KFLNRNAYI 15 564 AJYGKNFCV 15 579 MLLMRNIVR 15 693 KILGKKNEA 15 TableXXV1I.3-IILA-B08-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position Plus eight.

Pos 123456789 score 6 WTNITPPAL 11 4 FPWTNITPP 8 1 GRCFPWTNI 7 9 ITPPALPGI 7 TableXXVI-5.BO-9msrs- 24PAIC12 Each peptide is a portion of SEQ I) NO: 11; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight Pos 123458789 score IU±LVLIFL 18 3 EAILLLVLI 14 9 VLIFLRQRI 13 4 AILLLVLIF 12 TableXXVIII.V5-BD8-9n10rs 24P4C12 Each pepfide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids. and the end position for each peptide Is the start position plus eight.

Pos 123456789 score 2 LEAILLLVL 10 6 LLLVLIFLR 8 TableXXVIII.V6-.HLA-BOB-9mers- 244C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 6 GLIPRSVFN 16 7 LIPRSVFNL 15 3 SSKGLIPRS 13 8 IPRSVFNLQ 13 1 GYSSKGUP 11 9 PRSVFNLQI 8 TableXXVII.V74LA.B08.9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of poptide is 9 amino adids, and the end position for each peptide is the star position plus eight.

Pos 123456789 score 5 ILVAVGQMM 7 4 WILVAVGQM 6 7 VAVGOMMST 5 1 SWYWILVAV 4 TabIeXXVIII.V&HLA.BO8-9iirs- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptlde Is39 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 5 LPIMRNPIT 15 4 WLPIMRNPI 12 16 GHVFQTSIL 11I I1I PITPTGHVF 10 7 IMRNPiTPT 8 13 TPTGHVFQT 7 TableXXVII.VM4LA-B889mers- 24P4C12 Each peptide is a portion of SEO ID NO: 17; each start position is specified, the length of peptide Is 9 amino ecids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 15 TGHVFQTSI 7 TableXXOlII.V9-H-11A-O& 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eiglht Pos 123456789 score 9 PLPTQPATL 16 13 OPAILGYVI 16 2 WAMTALYPL 14 16 TLG'rVLWAS 8 18 GYVLWASNI 8 8 YPLPTQPAT 7 TabIeXXX-V1 -HLA-81 510-9mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of pepticle Is 9 amino adds, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 275 YCWEEYRVL 16 583 RNIVRVL 16 57 LYGDPRQVL 232 ILVALGVAL 253 VAGPLVLVL 381 TSGQPQYVL 487 USAFIRTL 605 LWGGVG VI 49 IWGIVAWL 14 78 GENKDKPYL 14 100 IISVAENGL 14 170 FLLPSAPAL 14 184 WTNVrPPAL 14 200 TIQQGISGL 14 204 GISGUDSL 14 251 RLVAGPLVL 14 357 YPLVTFVLL 14 369 tAYWAMIAL 14 457 VLALGQCVL 14 617 FFSGRIPGL 14 32 CTDV1CCVL 13 TableXXIX-VI.HLA-B1 510.gmers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eighL Pos 123456789 sore 79 ENKDKPYU. 13 228 SW'rWILVAL 13 234 VALGVALVL 13 255 GPLVLVLIL 13 261 LILGVLGVL 13 302 YQSVQETWL 13 308 TWLAALIVL 13 440 FNLQIYGVL 13 446 GVLGLFWTL 13 499 TGSLAGAL 13 511 IVOIARVIL 13 518 ILEYIDHKL 13 537 IMCCFKCCL 13 547 CLEKFIKFL 13 572 VSAKNAFML 13 163 QQELCPSFL 12 237 GVALVLSLL 12 244 LLFIlLLRL 12 258 VLVUILGVL 12 305 VQE1'AIAAL 12 311 AALIVL.AVL 12 315 VLAVLEAIL 12 317 AVLEAILLL 12 322 ILLLMLIFL 12 356 FYPLVTPVL 12 371 YWAMTALYL 12 406 TSCNPTAHL 12 412 AI4LVNSSCP 12 442 LQIYGVLGL 12 450 LFWTLNWVI. 12 452 WTLNWVLAL 12 476 FHKPQDIPT 12 497 YHTGSLAFG 12 501 SLAFGAUL 12 503 AFGAJJLTL 12 523 DIIKLRG VON 12 589 WLDKVTDL 12 626 GKDFKSPHL 12 651 1ASGFFSVF 12 658 VFGMCVDTL 12 660 GMCVDTLFL 12 673 DLERNNGSL 12 34 DVICCVLFL 11 88 YFNIFSCI1L 11 141 YTKRNFCL 11 154 WNM1VITSL 11 158 VITSLQQEL 11 1164 QELCPSFLL 11 TableXXIX-V1.HLA.B1510-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptida is the start position Pos 236 241 242 285 291 319 332 333 351 354 358 414 434 479 494 590 591 631 684 35 38 124 225 240 249 316 343 418 426 477 483 540 573 596 597 632 638 663 666 683 687 33 36 217 347 432 461 607 plus eight.

123456789 score LGVALVLSL 11 VLSU.FILL 11 LSLLFILLL I11 DKGASISQL 11 SQLGFTTNL I11 LEAILLLNIL 11 QRIRIAIAL 11 RIRtAIALL I11 MMSTMFYPL I11 TMFYPLVTF I11 PLVTFVLLL 11 LVNSSCPGL 11 LIQRSVFNL I11 PQOIPTFPL 11 TLRYHTGSL I11 VLDKVTDLL I11 LDKVTDLLL 11I SPlILNYYWL 11 PYYMSKSLL 11 V1CCVI.FLL 10 CVLFLLFIL 10 PWrVGKNEF 10 FAQSWYW1L 10 LVLSUFIL 10 LLRLVAGPL 10 LAVLEAILL 10 EASKAVGQM 10 SCPGLMCVF 10 FQGYSSKGL 10 HKPQDIPTF 10 PTFPLISAF 10 CFKCCLWCL 10 SM(NAFMLL 10 DLLLFFGKL 10 LLLFFGKLL 10 PHLNY'YWLP 10 WLPIMTSBL VDTLFLCFL 10 LFLCFLEDL 10 RPYYMSKSL 10 MSKSLLKIL 10 TDVICCVLF 9 ICCVLFLLF 9 ISVKIFEDF 9 AVGQMMSTM 9 KGLIQRSVF 9 GQCVLAGAF 9 VGGVGVLSF 9 TableXXIX-Vi-HLA-Bi 510-Smers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 679 GSLDRPYYM 9 15 KPVKVDPSF 8 81 KDKPYLLYF 8 132 FSQTVGEVF S.

139 VFY11(NRNF 8 148 CLPGVPWNM 8 162 LQQELCPSF 8 174 SAPALGRCF 8 287 GASISQLGF 8 415 VNSSCPGLM 8 464 VLAGAFASF 8 468 AFASFYWAF 8 496 RYHTGSIAF 8 53D QNPVARCIM 8 570 FCVSAKNAF 8 608 GGVGVLSFF 8 609 GVGVLSFFF 8 647 GAY VIASGF 8 48 Y'V WGVAW 7 69 NSTGAYCGM 7 214 AROISVKIF 7 238 VALVLSLLF 7 318 VLEAILLLM 7 321 AILILLILIF 7 366 -UCIAYWAM 7 443 QIYGVLGLF 7 533 VARCIMCCF 7 546 WCLEIFIKF 7 554 FLNRNAYIM 7 562 MIAYGKNF 7 571 CVSAJ(NAFM 7 574 AI(NAFMLLM 7 593 1(VDLLLFF 7 621 RIPGLGKDF 7 634 LNYYWLPIM 7 653 SGFFSVFGM 7 TableX0(iX-V3.HLA-BI 510-9merr- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 6 WTNITPPAL 13 00 00 C0 00 TableXXI-V-B1510-9mert- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specife, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 2 LEAILLLVL 13 5 ILLLVLIFL 12 TableXJXV6B61510.9mers.

24P4C12 Each peplide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 ,:amino acids, and the end position for each pepfide is the start position plus eight Pos 123456789 score 7 LIPRSVFNL 11 KGLIPRSVF 10 3 SSKGUPRS 5 6 GLIPRSVFN 5 TableXXIX.WO51-9rers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15. each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Ps 123458789 score 8 AVGQMMSTM 9 4 WILVAVGQM 8 ILVAVGQMM 8 I SWYVIILVAV 3 *2 WYWILVAVG 3 3 YWILVAVGQ 3 6 LVAVGQMMS 3 TableXXIX-8411510.9mers- 24PAC12 Eahpeptide is a portion of SEQ ID NO: 17; each start position is specified, Mhe length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123458789 score 16 GHVFQTSIL 21 11 PITPTGHVF 10 13 QPATLGYVL 13 9 PLPTOPATL 12 2 WAMTALYPL 10 TableXYX-XVg-81510-9mers-24P4C12 Each peplide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 13 QPATLGYWL 13 9 PLPTQPATL '12.

2 WAMTALYPL 10 TableXXX.-Vl.HLA-B2705.9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peplide is 9 arnino acids and the end position for each peptide is the start position plus eight Pos 123456789 score 334 IAIALLK 26 332 ORIRIAIAL 25 675 ERNNGSLDR 24 214 ARDISVKIF 23 534 ARCIMCCFK 21 620 GRIPGLGKD 211 5 QRDEDDEAY 20 204 GISGLIOSL 20 446 GVLGLFWrL 20 689 KSU..KILGK 20 251 RLVAGPLVL 19 424 CVFQGYSSK 19 436 QRSVFNLQI 19 483 PTFPLISAF 19 583 RNIVRVWL 19 608 GGVGWLSFF 19 15 KPVKYDPSF 18 22 SFRGPIKNR 18 179 GRCFPWTNV 18 200 TIQQGISGL 18 207 GLIDSLNAR 18 234 VALGVALVL 18 244 LLFILLLRL 18 255 GPLVLVUIL 18 291 SQLGFTTNL 18 317 AVLEAILLL 18 330 LRQRRAI 18 333. RIRIAIALL 18 496 RYHTGSLAF 18 527 RGVQNPVAR 18 847 GAYVIASGF 18 688 LCFLEDLER 18 683 RPYYMSKSL 18 690 SLLKILGKK 18 49 IWOiVAWI 17 78 GENKDKPYL 17 154 WNMTVITSL 17 TabISXXX-VI-HLA-B270"-mers- 24P4Ci2 Each peptide is a portion of SEQ ID NO:, 3; each start position is specified, the length of peptide is 9 amino acids, and t end position for each peptide is the start position plus eight Pos 123458789 score 237 GVALVLSLL 17 242 LSLLFILLL 17 261 LILGVLGVL 17 287 GASISQLGF 17 311 MLVLAVL 17 338 IALU(EASK 17 354 TMFYPLVTF. 17 381 TSGQOYVL 17 429 YSSKGLUQR 17 477 HKPQOIPTF 17 503 AFGALILTL 17 516 RVILEYIDH 17 546 WCLEKFIKF '17 549 EKFIKFLNR 17 605 LWGGVGVL 17 621 RIPGLGKDF 17 111 EAYGKPVKY 16 23 FRGPIKNRS 16 137 GEVFYTKNR 16 139 VFYTNRNF 16 170 FLLPSAPAL 16 283 LRDKGASIS 16 285 DKGASISQL 16 321 AIWNLUF 16 322 ILLLMLIFL 16 323 LLLMLIFLR 16 327 LIFLRQRIR 16 432 KGUJQRSVF 16 440 FNLQIYGVL 16 442 LQrYGVLGL 16 443 QIYGVLGLF 16 457 VLALGQCVL 16 508 ILTIVOIAR 16 517 VILEYIDHK 16 589 WLDKVTDL 16 617 FFSGRIPGL 16 626 GKDFKSPHL 16 699 NEAPPDNKI( 16 10 DEAYGKPVK 40 LFLLFILGY 60 DPRQVLYPR 73 AYCGMGENK 81 KDKPYLLYF 124 PWITVGKNEF 212 LNARD1SVK 217 ISVKIFEDF 228 SWYMLVAL 236 LGVALVLSL TableXXX.Vi-HLA-B27054flers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide is the start position TableXXX.V1.HLA-B327O5-9mers- 24P4C1 2 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is 9 amino adids, and the end position for each peptide Is the start position plus eigIht.

Pos 123456789 238 VALVLSLLF 243 SU.FILLLR 253 VAGPLVLVL 258 VLVLILGVt 308 TWLAALIVIL 316 LAWAEAILL 369 IAYWAMTAL 461 GQCVLAGAF 470 ASF'TWAFHK 518 ILEYIDHKL 542 KCCLWCLEK 543 CCLWCLEKF 547 CLEKFIKFL 567 GKNFCVSAK 579 MLLMRNIVR 586 VRVVVLDKV 593 KVrDLLLFF 596 DLLLFFGKL 607 VGGVGVL.SF 609 GVGVI.SFFF 622 IPGLGKDFK 651 IASGFFSVF 684 PYYMSKSU.

.698 KNEAPPONK 34 DVICCVLFL 38 CVLFLLFIL 61 PRQVLYPRN

CGMGENKDK

83 KPYLLYFNI 84 PYLLYFNIF 135 1VGEVFYTK 148 CLPGVPWNM 158 VITSLQQEL 162 LQQELCPSF 164 QELCPSFLL 232 ILVALGVAL 240 LVLSLLFIL 263 LGVLGVLAY 267 GVLAYGIYY 272 GIYYCWEEY 278 EEYRVLRDK 325 LMLIFLRQR 379 LATSGQPQY -418 SCPGLMCVF 434 UQRSVFNL 437 RSVFNLOIY 450 LFWTLNWV1.

452 WTLN 1

WLAI.

score 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 '15 15 15 115 15 14 14 14 14 14 14 114 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 plus eight Pos 123456789 464 VLAGAFASF 485 FPILISAFIR 487 LISAFIRTL 488 ISAFIRTIR 489 SAFIRTILRY 501 SLAFGAL 513. QIARVILEY 515 ARVILEYID 552 IKFLNRNAY 556 NRNAYIMIA 558 NAYIMIAIY 560 YIMLIYGK 575 KNAFMLIIR 585. IVRVW1J)K 595 TDLLLFFGK 613 LSFFFFSGR 643 TSILGAYVI 659 FGMCVDTLF 660 GMCVDTLFL 679 GSLDRPYYM 700 EAPPDNKKR 701 APPONKKRK 702 PPDNKKRKK 7 DEDDEAYGK 36 ICC VLFLLF 172 LPSAPALGR 241 VISLIFILL 249 LIRLVAGPL 250 LRLVAGPLV 273 IYYCWEEYR 275 YCWEEYR VI 280 YRVLRDKGA 294 GFTNLSAY 319 LEAILLLML 347 AVGQMMSTM 348 VGQMMSTMF 349 GQMMSTMFY 356 FYPLVTFVL 357 YPLVTFVLL 358 PLVTFVLLL 363 VLLUCIAY 492 IRTLRYHTG 495 LRYHTGSLA 506 AIJLTILVQI 526 LRGVQNPVA 545 LWCLEKFIK 570 FCVSAKNAF 572 VSAKNAFML score 114 14 14 14 14 14 '14 14 14 114 14 14 14 114 14 14 14 14 14 14 14 14 14 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 Table)0(X-Vi.I4LA-B2705-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide is the start position Plus eight Pos 123456789 Score 582 MRNIVRV 13 590 VLDKVTDLL 13 592 DKVTDLLLF 13 610 VGVLSFFFF 13 637 YWLPIMTSI 13 648 AYVIASGFF 13 653 SGFFSVFGM 13 666 LFLCFLEDL 13 681 LDRP'rYMSI< 13 682 DRPYYMSKS 13 685 YYMSKSLLK 13 686 YMSKSLLKI 13 29 NRSCTDVIC 12 32 CTDV1CCVL 12 33 TDVICCVLF 12 35 VICCVLFLL 12 57 LYGDPRQVL 12 58 YGDPRQVLY 12 79 ENKDKPYLL 12 80 NKDKPYLLY 12 93 SCILSSNII 12 100 IISVAENGL 12 121 PEDPWIVGK 12 132 FSQTVGEVF 12 144 NRNFCLPGV 12 151 GVPWNMTVI 12 163 QQELCPSFL 12 190 PALPGITND 12 193 POITNOTTI 12 239 ALVLSLLFI 12 276 CWEEYRVLR 12 302 YQSVQETWL 12 305 VQETWIAAL 12 315 VLAVI.EAIL 12 320 EAILLLMvLI 12 328 IFIRORIRI 12 343 EASKAVGQM 12 371 YWANTALYL 12 386 QYVLWASNI 12 393 NISSPGCEK 12 406 TSCNPTAIIL 12 414 LVNSSCPGL 12 421 GLMCVTQGY 12 426 FQGYSSKGL 12 468 AFASFY'WAF 12 490 AFIRThRYH 12 500 GSLAFGALI 12 510 TLVQLARVI 12 00 00 C0 00 TableXXXVi.HAk2705-9mers- 24P4C1 2 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptde is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 519 LEYIDHKLR 12 537 IMCCFKCCL 12 540 CFKCCLWCL 12 553 KFLNRNAYI 12 557 RNAYIMIAI 12 562 MIAIYGKNF 12 591 WDKVTDLLL 12 597 LLLFFGKLL 12 614 SFFFFSGRl 12 6 19 SGRIPGLGK 12 628 DFKSPHLNY 12 631 SPHLNYYWL 12 634 LN'rYWLPIM 12 658 VFGMCVDTL 12 662 CVDTL.FLCF 12 683 VDThFLCFL 12 673 DLERNNGSL 12 687 MSKSLLKIL 12 7ableXXX-V3.HLA-B27OS-9mers- 24P4C12 Each peptide Is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is59 amino acids, and t end position for each peptide is the start position plus eight Poa 123455789 scoe 1 GRCFPWTNI 24 6 WTNITPPAI. 11 TableXX0(-VS-HLAB2705-9mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Pea 123458789 score 4 AIU..LVLIF 17 ILLLVUFL 17 6 WYLLIFLR .16 2 LEALLLV. 14 8 LVUFLRQR 14 3 EAILLLVLI 12 9 VLIFLRQRI I1I TableXXX.V6-HLA-B2705-9m01's- 24P4C12 Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 9 PRSVFNLQI 19 5 KGLIPRSVF 17 2 YSSKGLIPR 16 7 LIPRSVFNL 14 3 SSKGLIPRS 9 TableXXX.V7.HLA-B2705flmers-24P4C112 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptlde Is 9 amino acids, and the end position for each peptide Is the start position plus eight Pos 123456789 score 8 AVGQMMSTM 13 4 WILVAVGQM 12 5 ILVAVGQMM 11 3 YW1LVAVGQ 6 TableXXX.V-HLAB270549mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 16 GHVFQTSIL 15 I NYYWLPIMR 14 8 MRNPITPTG '14 9 RNPrTPTGH 14 111 PITPTGHVF 12 15 TGHVFQTSI 11 19 FQTSILGAY 10 2 YYWLPIMRN 8 4 WLPIMRNPI 7 7 IMRNPITPT 7 17 HVFOTSILG 7 TableXXX.V9-HLA-B2705-9mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123458789 score 18 GYVILWASNI 13 QPATLGYVL 13 2 WAMTALYPL 12 9 PLPTOPAT1. 12 11 PTQPATLGY 6 ALYPLPTQP 8 15 ATLGYVLWA 7 TableX00(J.V1.HLA.B2709.

gmerse-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123458789 score 332 ORIRIAIAL 23 179 GRCFPWTNV 22 250 LRLVAGPIN 21 214 ARDISVKIF 436 QRSVFNLQI 144 NRNFCLPGV 19 330 IRORIRIAI 19 582 MRNIVRVVV 19 586 VRWVLDKV 19 255 GPLVLVLIL 17 583 RNIVRVVVL 17 251 RLVAGPLVL 16 683 RPYYMSKSL 16 78 GENKDKPYL 170 FLLPSAPAL 334 IRIAIALLK 446 GVLGLFWTL 620 GRIPGLGKD 647 GAYVIASGF 660 GMCVDTLFL 49 IWGIVAWL 14 228 SWYWILVAL. 14 234 VALGVALVL 14 244 LLFILLLRL 14 317 AVLEAILLL 14 333 RIRIAAILL 14 452 WLNWVLAL 14 602 GKLLWGGV 14 626 GKDFKSPHL 14 679 GSWDRPYYM 14 23 FRGPIKNRS 13 34 DV1ICCVLFL 13 83 KPYLLYFNI 13 TableMIM.V-HLA-B2709- 9merse-24P34Cl2 Each peptide is a portion of SEQ ID N(Y. 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 107 GLQCPTPQV 13 204 GISGLIDSL 13 232 ILVALGVAL 13 236 LGVALVLSL 13 237 GVALVLSLL 13 240 LVILSLLIFIL 13 242 LSLLFILLIL 13 253 VAGPLVLVL 13 291 SQLGFTTNL 13 311 AALIVLAVL 13 322 ILLLMLIFL 13 357 YPLVTFVU. 13 358 PLVTFVLLL 13 369 [AYWAMTAL 13 440 FNLQIYGVL 13 442 LQIYGVLGL 13 449 GLFINWV 13 496 RYHTGSLAF 13 500 GSLAFGALI 13 .515 ARVILEYID 13 557 RNAYIMIAI 13 589 WLDKY'TDL 13 KPVKYDPSF 12 38 CVLFLLFIL 12 ILGYIWVGI 12 56 WLYGDPRQV 12 61 PRQVLYPRN 12 81 KDKPYLLYF 12 158 VITSLQQEL 12 164 QELCPSFLL 12 258 \VVLLGVL 12 261 LILGVLGVL 12 287 GASISQLGF 12 308 1'WLAALIVL 12 316 -LAVLEAILL 12 321 AILLLMLIF 12 328 IFLRQRIRI 12 355 MFYPLVTFV 12 371 YWAMTALYL 12 414 LVNSSCPGL 12 432 KGLIQRSVF 12 434 UQRSVFNL 12 461 GQCVLAGAF 12 492 IRTLRYKTG 12 495 LRYHTGSLA 12 501 SLAFGAUL 12 503 AFGALILTIL 12 506 ALILTLVOI 12 TableXXXI-V1 .HLA-B2709gmerse-24PAIC12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide Is the start position plus eight.

Pos 123456789 score 518 ILEYIDHKL 12 553 KFLNRNAYI 12 593 KVTDIILFF 12 596 DUILFFGKL 12 597 LLI.FFGKLL 12 605 LWGGVGVL 12 608 GGVGVLSFF 12 621 RIPGLGKDF 12 637 YWLPIMTSI 12 666 LFLCFLEOL 12 6814 PYYMSKSLL 12 5 QIRDEDDEAY 11 28 KNRSCTDVI 11 29 NRSCTDVIC 11 32 CTDVICCVL 111 41 FLIFILGYI 11 42 LLFILGYIV 11 46 LGYIWGIV 11 67 PRNSTGAYC 111 79 ENKDKPYLL 11 87 LYFNIFSCI 111 100 IISVAENGL 11 128 GKNEFSQTV 11 139 VFYTKNRNF 11 151 GVPWNMTVI 11 184 WTNVTPPAL 11 217 ISVKIFEDF 111 225 FAQSWYWIL 11 230 YWILVALGV 11 238 VALVLSULF 11 239 ALVLSLLFI 11 249 LLRILVAGPIL 11 257 LVLVLILGV 11 260 VULGV1.GV 11 280 YRVLRDKGA 11 283 LRDKGASIS 11 285 DKGASISQL 11 297 TNLSAYQSV 11 310 LAALIVLAV 11 314 IVLAVLEAI 11 319 LEAILLLML 11 351 MNSTMFYPL 111 354 TMFYPLVTF 11 381 TSGQPQWVL 111 386 QYVIWASNI 11 427. QGYSSKGU 11 480 QDIPTFPLI 11 483 PTFPLISAF I11 TableXXX-VI-HLA-B2709gmerse-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each stairl position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Pos 123455789 score 509 LTLVQIARV I11 510 TLVOIARVI I11 5111 LVQIARVIL 11 526 LRGVQNPVA 11 534 ARCIMCCFK 11 537 IMCCFKCCL 11 564 AIYGKNFCV 11 572 VSAKNAFML 11 591 LDKVTDLLL 11 592 DKVIDLLLF 11 598 LLFFGKLLV 111 599 LFFGKLLW 11 609 GVGVLSFFF 11 614 SFFFFSGRI 11 617 FFSGRIPGL 11 631 SPHU4YYWL 11 634 LNYYWLPIM 11 643 TSILGAYVI 11 653 SGFFSVFGM 11 658 VFGMCVDTL 11 663 VDTLFLCFL 11 675 ERNNGSLDR 11 687 MSKSLLKIL 11 Table)WO-V3-IILA-2709- 9nmers-24P34C12 Each peptide is a portion of SEQ ID NO: 7; each stat position is specified, the length of peptide Is 9 amino acids, and the end position for each peptidle is the start position plus eight Pos 123456789 score 1 GRCFPWTNI 22 6 WThITPPAL 11 9 ITPPALPGI 11 TableXXi-5-82709.9mers- 2434C12 Each peptide Is a portion of SEQ ID NO: 11; each start position Is specified, the length of peptide is 9 amino adids, and the end position for each peptide Is the start position plus eight Pos 123456789 score 4 AILLIWIF 13 ILLLVLIFL 13- 2 LEAILLLVL 11 1 VLEAILLLV 10 3 EAILLLVLI 10 9 VLIFLRQRI 10 TableXXXIl.V6-HLA-B2709- 9mers-24P4C12 Each peptide Is a portion of SEQ ID NO: 13; each start posiion Is speciffied, the length ot peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 9 PRSVFNLQI 20 KGLIPRSVF 12 7 LIPRSVFNL 12 4 SKGLIPRSV 9 TabtXXXI-V74ILA-B2709.

9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score I SWYWILVAV 12 4 WILVAVGQM 12 ILVAVGQMM 10 8 AVGQM.MSTM 9 TableX)00-V8-HLA-B2709- 9mers-24P4C12 Each peptide is a partion of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 16 GHVFQTSIL 14 8 MRNPITPTG 13 11 PITPTGHVF 10 NPITPTGIIV 9 4 WLPIMRNPI 8 TGHVFQTSI 8 QTSILGAYV 8 TabIeXXXI-V9-ILA-82709.

9mers-24P4C1 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123458789 score 18 GYVLWASNI 14 2 WAMTALYPL 11 13 QPATLGYVL 11 9 PLPTQPATL 10 12 TQPATLGYV 8 TablI-i-HLA.B4402.

9mers-24P4C12 Each peptide Is a portion of SEQ ID N0r 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start poiEon plus eight.

Pos 123455789 score 164 QELCPSFLL 22 319 LEAILIIML 22 222 FEDFAQSWY 21 78 GENI<DKPYL 20 306 CI1WLAALI 20 483 PTFPLISAF 20 3 17 AVLEALLL 19 332 QRIRWAAL 19 503 AFGALILTL 18 506 ALILTLVQI 18 552 IKFLNRNAY 18 58 YGDPRQVLY 17 170 FLLPSAPAL 17 214 ARDISVKIF 17 242 LSLLFILLL 17 583 RNIVRVWVL 17 11 EAYGKPWKY 16 40 LFLLFILGY 16 48 YIWGIVAW 16 81 KDKPYU.YF 16 121 PEDPW1VGK 16 228 SWYWILVAL 16 253 VAGPLVLVL 16 254 AGPLVILU 16 311 MALIVLAV1. 16 320 EAILLLMLI 16 321 AILLLMLIF 16 363 VILUCIAY 16 382 SGQPQYVLW 16 452 WTLNWVI.AL 16 480. QDIPTFPLI 16 487 LISAFIRTL 16 489 SAFIRTIRY 16 617 FFSGRIPGL 16 TableXKXII-VILA-B4402.

9mers-24P4C12 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 629 FKSPHLNYY 16 699 NEAPPONKK 16 34 DVICCVLFL 79 ENKDKPYLL 130 NEFSQWVGE 154 WNMTVITSL 204 GISGUDSL 234 VALGVALVL 241 V1.SUFILL 263 LGVLGVLAY 278 EEYRVLRDI( 294 GFTTNLSAY 354 TMFYPLVTF 370 AYWAMTALY 399 CEKVPINTS 442 LQIYGVLGL 488 AFASFYWAF 477 HKPQDIPTF 499 TGSLAFGAL 513 QIARVILEY 547 CLEKFIKFL 66 YPRNSTGAY 14 80 NKDKPYLLY 14 84 PYLLYFNIF 14 93 SCILSSNII 14 104 AENGLQCPT 14 193 PGITNDTI 14 223 EDFAQSWYW 14 239 ALVLSLLFI 14 244 LLFILLLRL 14 258 VUVLGWL 14 261 LILGWLGVL 14 285 DKGASISOL 14 291 SQLGFTTNL 14 301 AYQSVQETW 14 305 VQETWLMAL 14 308 TWLAALIVL 14 316 LAVLEAILI. 14 322 ILLLMLIFL 14 330 LRORIRIAl 14 333 RIRIAIALL 14 356 FYPLVTFVL 14 357 YPLVTFVLL 14 358 PLVTFVLLL 14 384 LWLCIAYW 14 418 SCPGLMCVF 14 432 KGLIQRSVF 14 446 GVLGI.EWTL 14 00 00 TabloXX)GI-V1.lILA-84402- 9mrs-24P4C!2 Each peptide is a portion of SEQ NO: 3; each start position is Specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123458789 score 496 RYHTGSLAF 14 546 WCLEKFIKF 14 558 NAYIMIAIY 14 573 SAKNAFMLIL 14 577 AFMLLMRNI 14 592 DKVTDLLLF 14 593 KVrDLLLFF 14 596 DLLLFFGKL 14 597 LLLFFGKLL 14 821 RIPGLGKDF 14 641 IMISILGAY 14 643 TSILGAWVI 14 651 IASGFFSVF 14 662 CVDTLFLCF 14 671 LEDLERNNG 14 678 NGSLDRPYY 14 QRDEDDEAY 13 7 DEDOEAYGIK 13 32 CTDVICOVI 13 36 ICC VLFLLF 13 49 IWGIVAWL 13 57 LYGOPRQVL 13 77 MGENKD1KPY 13 87 LYFNIFSCI 13 137 GEVFYTKNR 13 146 NFCLPGVPW 13 174 SAPALGRCIF 13 176 PALGRCFPW 13 184 WINVTPPAL 13 187 VTPPALPGI 13 200 TIOQGISGL 13 209 IDSLNARDI 13 213 NARDISVKI 13 232 ILVALGVAL 13 237 GVALVLSLL 13 238 VALVLSLLF 13 251 RLVAGPLVL 13 255 GPLVLVLIL 13 277 WEEYRVLR1) 13 342 KEAS)VGO 13 351 MMSTMFYPL U3 440 FNLQIYGVL 13 443 QIYGVLGLF 13 448 LGLFWTU'JW 13 481 GQCVLAGAF 13 486 AGAFASFYW 13 501 SLAFGAUIL 13 518 ILEYIDHKL 13 TableXXXII-VI-HLA-64402- 9mers-24P4C12 Eachi peptie Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 519 LEYIDHKLR 13 529 VQNPVARCI 13 543 CCLWCLEKF 13 570 FCVSAKNAF 13 589 WLDKVrOL 13 590 VLDKVrfJLL 13 605 LWGGVGVL 13 831 SPHLNYYWL 13 631 YWLPIMTSI 13 648 AYVIASGFF 13 674 ILERNNGSILD 13 687 MSKSLLKIL 13 33 TDVICCVLF 12 35 VICCVLFLL 12 38 CVLFLLFIL 12 50 WGIVAWVLY 12 100 IISVAENGL 12 132 FSQTVGEVF 12 133 SQ1VGEVFY 12 139 VFYTKNRNF 12 1141 YrKNRNFCL 12 163 QQELCPSFL 12 217 ISVIKIFEDF 12 221 IFEDFAQSW 12 236 LGVALVLSL 12 240 LVLSLLFIL 12 249 LLRLVAGPL 12 267 GVLAYGIYY 12 269 LAYGIYYCW 12 275 YCWEEYRVL 12 287 GASISQLGF 12 314 IVLAVI.EAI 12 326 MLIFLRQRI 12 328 IFLRQRIRI 12 349 GQMMSTMFY 12 369 lAYWAMTAL 12 371 YWAMTALYL 12 406 TSCNPTAHL 12 421 GLNCVFQGY 12 426 FQGYSSKGL 12 434 UQRSVFNL 12 437 RSVFNLQIY 12 450 LFWTLNWVL 12 457 VLALGQCVL 12 464 VLAGAFASF 12 479 PQDIPTFPL 12 510 TLVQIARV1 12 511 IVOIARVIL 12 TableX=OI-V1-HLA-B4402.

9mers-24P24C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified. the length of pepfide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 548 LEKFIKFLN 12 553 KFLNRNAYI 12 557 RNAYIMIAI 12 562 MIAJYGKNF 12 572 VSAKNAFML 12 591 LDKVTDLLL 12 607 VGGVGVLSF 12 608 GGVGVLSFF 12 610 VGVLSFFFF 12 630 KSPHLNYYW 12 638 WLPIMTSIL 12 647 GAYMASGF 12 658 VFGMCVDTL 12 659 FGMCVDTLF 12 660 GMCVDTLFL 12 663 VDTLIFLCFL 12 666 LFLCFLEOL 12 673 DLERNNGSL 12 677 NNGSLDRPY 12 683 RPYYMSKSL 12 686 YMSKSLLKI 12 10 DEAYGKPVK 11 15 KPVXYDPSF 11 28 KNRSCTDVI 11 37 CCVLFLLFI 11 41 FLLFILGYI 11 45 ILGYIWVGI 11 117 VSSCPEOPW 11 124 PWTVGKNEF 11 151 GVPWNMTV] 11 197 NDTIIQQGI 11 201 IQQGISGLI 11 266 LGVLAYGlY 11 302 YQSVQE1WL 11 359 LVTFVLLLI 11 361 TFVLLLICI 11 379 LATSGQPQY 11 381 TSGQPQYVL 11 436 QRSVFNLQI 11 444 IYGVLGLFW 11 465 LAGAFASFY 11 474 WAFHKPQDI 11 484 TFPLISAFI 11 494 TLRYHTGSL 11 533 VARCIMCCF I11 538 MCCFKCCLW 11 540 CFKCCLWCL 11 614 SFIFFFSGRI 11I TabIOXXXUII-HLA.B4402.

9mers-24134C12 Each peptide is a portio of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position Plus eight.

Pos 123456789 score 626 GKDFKSPHL 11 628 DFKSPHLNY 11 684 PYYMSKSLL 11 19 YDPSFRGPI 10 83 KPYLLYFNI 10 88 YFNIFSCIL 10 158 VISLQQEL 10 162 LQQELCPSF 10 225 FAQS WY WMt 10 272 GIYYCWEEY 10 315 VLAVLEAIL 10 348 VGQMMSTMF 10 386 QWLWASNI 10 396 SPGCEKVPI 10 414 LVNSSCPGL 10 500 GSLAFGALI 10 514 IARVILEYI 10 537 IMCCFKCCL 10 544 CLWCLEKFI 10 555 LN'RNAYIMI 10 609 GVGVLSFFF 10 TableX)OUI.V3HLAB4402.

9mers-24C2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position Plus eight Pos 123456789 score 6 WThITPAL 13 9 ITPPALPGI 13 1 GRCFPWTNI 8 2 RCFPWrNIT 7 7 TNITPPALP 6 8 N[TPPALPG 6 TabIeXXXG-V5HLA.4M02- 9mers.24P4C12 Each peptide Is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight.

POS 123456789 score LEAILLLVL 23 EAILLLVLI 17 AILLLVLIF 17 ILLLVLIFL 14 VLIFLRQRI 12 TableXXXII.V8.HLA-B4402.

9mers-24P4C1 2 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 scoe 5 KGLIPRSVF 14 7 LIPRSVFNL 13 9 PRSVFNLQI 11 6 GIJPRSVFN 8 TableXXOUI-V7-HLA-B4402.

gmers-24P14C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 1 SWYWILVAV 6 3 WV1LVAVGQ 6 8 AVGQiMSTM 4 4 WILVAVGQM 3 2 WYWLVAVG 2 TableXXXJI*8HLA442- 9mers-24P4C12 Each peptide Is a portio of SEQ ID NO: 17; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 11 PITPTGHVF 15 19' FOTSILGAY 14 4 V&PIMRNPI 11 16 GHVFQTSlL 11I 115 TGHVFQTSI 8 TabteXXOfll.V9-HLAB4402.9mers- 24P4C12 Each peptidle is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peplide is the start position plus eight.

Pos 123456789 score 11 PTQPATLGY 9 PLPrTQPATL 14 2 WAMTALYPL 13 14 PATLGYVLW 13 13 QPATLGYVL 12 18 GYVLWASNI 6 ALYPLPTOP 8 15 ATLGYVLWA 7 TableXXXIII-VI.H-LA-351101 Smers-24P34C12 Each peptide is a portion of SEQ ID NO, 3; each start position is specified, the length of peptide is 9 amino acids, andl the end position for each peptide is the start position plus eight.

Pos 123456789 score 234 VALGVALVL 27 213 NARDISVKI 46 LGYIVVGIV 24 83 KPYULYFNI 24 311 AALIVLAVL 24 253 VAGPLVLVL 23 310 LAALIVLAV 23 357 YPLVTFVLI. 23 369 LAYWAMIAL 23 474 WAFHKPQDI 23 514 IARVILEYI 23 683 RPYYMSKSL 22 254 AGPLVLVLI 21 255 GPLVLVLIL 21 320 EAILLLMLI 211 396 SPGCEKVPI 21 427 QGYSSKGLI 21 i i EAYGKPVKY 193 PGITNDTTI 316 LAVLEAILL 123 DPWTVGKNE 19 236 LGVALVLSL 18 314 IVLAVLEAI 18 599 LFFGKLLVV 18 686 YMSKSULKI 18 60 DPRQVLYPR 17 150 PGVPWNMTV 17 225 FAQSWYWIL 17 261 LILGVLGVL 17 269 LAYGIYYCW 17 300 SAYOSVQET 17 504 FGALILTLV 17 00 00 TableXXXIII.VII.HILA.35101- 9mers-24P4C12 558 NAYIMIAIY 17 573 SAKNAFMLL 17 651 IASGFFSVF 17 182 FPWTNVTPP 16 192 LPGITNQTT 16 328 IFIRORIRI 16 355 MFYPLVTFV 16 359 LVTFVLLLI 16 458 LALGQCVLA 16 502 LAFGALILT 16 505 GALILTLVQ 16 510 Tl.VQIARVI 16 581 LMRNIVRWV 16 631 SPHLNYYWiL 16 9 DDEAYGKPV 15 ILGYIWVGI 15 56 WLYGDPRQV 15 110 CPTPQVCVS 15 120 CPEDPW1TVG 15 1151 GVPWNMTVI 15 172 LPSAPALGR 15 224 DFAQSWYWI 15 275 YCWEEYRVL 15 308 TWLAALIVI. 15 336 IAIALLKEA 15 338 WALKEASK 15 375 TALYLATSG 15 485 FPRLISAFIR 15 529 VQNPVARCI 15 564 AIYGKNFCV 15 582 MRNIVRV 15 596 DLLLFFGKL 15 637 YWIPIMTSI 15 643 TSILGAYVI 15 647 GAYVIASGF 15 700 EAPPDNKKR 15 DPSFRGPIK 14 41 FLLFILGYI 14 43 LFILGYIW 14 72 GAYCGMGEN 14 87 LYFNIFSCI 14 119 SCPEDPWTV 14 152 VPWNM1VIT 14 188 TPPALPGIT 14 190 PALPGITND 14 209 IDSLNARDI 14 230 YWLVALGV 14 238 VALVLSLLF 14 257 LVLVLILGV 14 409 NPTAHLVNS 14 411 TAHLVNSSC 14 450 LFWTLNWVL 14 465 LAGAFASFY 14 467 GAFASFYWA 14 482 IPTFPLISA 14 TableXXXJI11.HL11A-B51101.

9mers-24134Cl2 499 TGSLAFGAL 14 509 LTLVQIARV 14 576 NAFMLLMRN 14 586 VRVVVLDKV 14 589 WI.DKVTOL 14 602 GKLLWGGV 14 605 LWGGVGVI. 14 639 LPIMTSILG 14 701 APPDNKKRK 14 702 PPDNKKRKK 14 19 YDPSFRGPI 13 28 KNRSCTDVI 13 34 DVICCVLFL 13 54 VAWLYGDPR 13 66 YPRNSTGAY 13 112 TPQVCVSSC 13 149 LPGVPWNMT 13 174 SAPALGRCF 13 176 PALGRCFPW 13 187 VTPPALPGI 13 189 PPALPGITN 13 201 lQQGISGLI 13 239 ALVLSLLFI 13 252 LVAGPLVLV 13 282 VLRDKGASI 13 285 DKGASISQL 13 293 LGFrrNLSA 13 322 ILLLMLIFL 13 330 LRQRIRAI 13 340 LLKEASKAV 13 343 EASKAVGCQI 13 356 FYPLVTFVI. 13 361 TFVLUICI 13 384 QPQYVLWAS 13 478 KPQOIPTFP 13 487 USAFIRTL 13 489 SAFIRTLRY 13 500 GSLAGALI 13 506 ALILTLVQI 13 521 YIDHKLRGV 13 531 NPVARCIMC 13 553 KFLNRNAYl 13 555 LNRNAYIMI 13 563 LAIYGKNFC 13 578 FMLLMvRNIV 13 580 LLMRNIVRV 13 TableXXXII-V3.HLA45IOI1- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peplide Is the start position plus eight 123456789 score FPWTNITPP ITPPALPGI 14 GRCFPWTNI 11 WTNITPPAL 8 TableX)OU11.V.l4LAB5101-9mers- 24P4C12 Each peptide is a portion of SEQ I D NOr 11: each start postion Is specified, the length of peptide is 9 amino acids, and the end position for each pepflde Is the start position plus eight.

Pos 123458789 score 3 EAILLLVLI 22 5 IU±VLIFL 14 2 LEAILLLVL 13 1 VLEAILLLV 12 9 VLIFLRQRI 12 TableXXXIIIV6-HLA-B5101- 9mers-24P4CI2 Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino adds, and the end positi on for each peptide is the start position Plus eight.

Pos 123456789 score 8 IPRSVFNLQ 16 7 LIPRSVFNL 12 9 PRSVFNLQI 12 5 KGUPRSVF 11 4 SKGUPRSV TableXX(YtIIl.WHLA-65101- 9mers-24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide Is the start position Plus alghL Pos 123458789 score 1 SWYWILVAV 14 7 VAVGQMMST 12 2 WYWVILVAVG 6 3 YW1LVAVGQ 6 TabIeX)O~tI.V84It.A.851O1-9mers- 24P4C12 I Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the ength of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 1D NPITPTGHV 21 Is TGHVFQTSI 18 13 TPTGHVFQT 14 4 WLPIMRNPI 13 LPIMRNPIT 13 rableXXXUlI-V9-HLA.BSIOI- 9mers-24P4C12 Each peptide is a portion of SEQ I D NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 13 OPATIGYVI 20 2 WAMTALYPL 18 TALYPIPTO 16 8 YPLPTQPAT 15 IPTOPATIG 14 12 TQPATLGYV 13 17 LGYVLWASN 12 9 PLPTQPATL 11 14 PATLGYVI.W 11 18 GYVLWASNI 11 TableXXXIV.V1.lILAA10lmars.

24P4C12 Each peplide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is amino adds, and the end position for each peplide Is the start position plus nine.

Pos 1234567890 score 221 IFEDFA§S WY 25 488 I_$AFIRILRY 25 39 VLFLLFILGY 23 58 YdGDPRQ LLYP 23 79 EHIKDKPYLLY 23 262 ILGVWGVLAY 23 512 V-QLARajEY 22 627 Ik~FSPjLNY 21 132 FaQTVGEVFY 20 266 LGVLAY GIYY 20 362 FVLLICIAY 20 590 V'LDKVTDLLL 20 594 VIDLLLFGK 20 318 VLEAILLNL 19 32 CTDVICCVLF 18 TablgXA0UV.V1-HLA.A11mers- 24P4C1 2 Each peptide Is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pus 1234567890 score 49 IWVGIV WLY 18 378 YfATSGQPQY 18 420 PGLMCVEQGY 18 464 V1AGAF6SFY 18 10 DEAYGIFEVIO' 17 57 LYGDPR.QVLY 17 121 PEDPWTGKN 17 265 VLGVLAYGIY 17 271 YGrYYCWEEY 17 276 CWEEYRVLLRD 17 369 IAYWAMIM..Y 17 551 FIKFLNfiNAY 17 80 NKDKPYLLYF 16 348 V90MMSIMFY 16 676 RNNGSLDRPY 16 677 NEGSLDRPYY 16 4 KgRDEDDEAY 15 18 KYDPSFEGPI 15 65 LYPRNSIGAY 156 76 GMGENKDKPY 214 ARDISVKIFE 15 293 LG&rrLSAY 15 436 QSVFNLQIY 15 479 FPQDIPTEfPLI 15 557 RtjAYIMI-AIY 15 628 DEKSPH!:NYY 15 640 PIMTSIILGAY 15 664 DILFLCELED 15 283 LRDKGASISQ 14 521 YIjDHKLR;GVQ 14 673 D)LERN4GSLD 14 141 YIKNRNiFCLP 13 305 VFQETWLAALI 13 382 SGQPQYM]LWA 13 407 SCNPTAHLVN 13 518 fIkY 1R 13 547 CLEKFIKFLN 13 670 FIEDLERNNG 13 680 SLDRPYYMS< 13 7 DEDDEAYGKP 12 35 VICCGWLLF 12 159 ITSLQQECLCP 12 163 QQELCP§FLL 12 242 LULLFI.I1R 12 618 FSGRIPGLGK 12 626 CLMFKSE-HLN 12 698 KbAEAPPflNKK 12 rableXXXIV.V3-HLA-AI1 Omers- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 score 10 ITPPALEGIT 3 ROFPWTNITP 9 7 WINITPfALP 8 8 Tt!ITPPALPG 6 9 NITPPALGI 4 10mars-24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide Is 10 amino acids, and thle end position for each peptide is the start position plus nine.

Pos 1234567890 score 2 VLEAIL1LVL 19 7 LLLLIFLRQ i AVLEATLLV 9 TabIXJOUV.VB.HLAAI-l0mel'S 24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specilied, the lengt of peptide is 10 amino adids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 10 PRSVFNLQIY I Q YSSCGUP 7 4 SSKGURRSV 7 9 IpRSVFNLOI 7 Tab~eXXiV-V7.HLA-A11-lCmen;- 24P4C12 Each peptide Is a portion of SEQ, ID NO: 15; each start position is specified, the length of paptide is 10 amino acids, and tihe end position for each peptide is the start position plus nine.

Pos 1234567890 score 1 02WfYVMLVAV 4 2 SWYWILVAVG 4 4 YWILVAVGQM 3 5 W!LVAVaQMM 2 6 iCVAVGQMMS 2 00 00 C0 00 8 VAVGQMMSTM 2 9 AVGfMi-TMF 2 TabIXXXVV8-HLA-A1- I Omers-24012 2 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 10 arruno acids, and the end position for each peptide is the start position pIus nine.

Pos 1234567890 score 19 VFQTSILGAY 16 4 YAWLP1iMNPl 7 13 IIPTGHVFQT 7 21 QITSILG2AWVI 7 TableXXXJV.V944LAAI-l0mers- 24P4C12 Each peptide is a portion of SEQ I D NO: 19; each start position is specified, the length of peptide is 10 amio acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 11 LPTQPATLGY 21 12 PIQPATLGY 10 TableXXXV-VI-HLA-A020i.

10mers-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle is amino adds, and the end position for each peptide is the start position plus nine.

Pos 1234587890 score 235 ALGVALVLSL 29 44 FILGYJWGI 28 232 ILVALVALV 28 243 SLLFILLLRL 28 309 WLAALIVLAV 28 579 MILLMRNIVIRV 28 244 LLFIlLLRL 27 260 VJLILGVLGVL 27 433 GIQR0FSVFNIL 27 508 ILTLV.QIARV 27 580 LLNRNIVRW 27 598 LLFFGIELLW 27 48 YIWGIVAWL 26 94 CILSShWISV 26 239 ALVLSJLFIL 26 241 VLSLLILLL 26 251 RLVAGRLVILV 26 321 AIUI.MUFL 26 TableXXXV.VI-HLA-AO201 l0mers-24P4Cl2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 score 441 NLQIYgVLGL 26 502 LAFGALILTL 26 517 VILEYIDHKL 26 603 KLLWGGVGV 26 604 LLWGCGVGVL 26 45 ILGYIWVGIV 25 252 LVAGPLVLVL 25 304 SVQETWLAAL 25 312 ALIVLAV1LEA 25 318 VLEAILLLML 25 486 PUSAEIRTL 25 657 SVFGMCVDTL 25 665 TLFLCELEDL 25 248 LLLRLYAGPL 24 259 LVLILGVLGV 24 310 LAALIVLAVL 24 339 ALLKEASKAV 24 597 LLLFFGKLLV 24 41 FLLFILGYIV 23 42 LLFIL YIWV 23 56 WL.YGDERQVL 23 231 WILVALGVAL 23 249 LLRLVAGPLV 23 .256 PLVLVLILGV 23 313 LIVILAVLEAI 23 315 VLAVLEAILIL 23 438 SVFNLgIYGV 23 459 ALGQW~LGA 23 686 YMSKSLLKIL 23 99 NIIS VAENGL 22 257 LV'LVL!LGVL 22 354 TMFYPLVTFV 22 413 HLVNSSCPGL 22 449 GLFWrINWVL 22 506 ALILTILVQIA 22 510 TLVQARVIL 22 513 QIARVILEYI 22 581 LMRNIVRVW 22 585 IVRVVYLDKV 22 590 VLDKVIDLLL 22 199 TTIcqQ~isG 21 247 ILLLRLVAGP 21 253 VAGPl~-LVLI 21 316 LAVLEAILUL 21 501 SLAFG ALILT 21 505 GALILTLVQI 21 641 IMTSIIGAWV 21.

86 LLYFNIFSCI 20 TableXXXV.VI.HLA-A0201 l10mers-24P141C1 2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 95 ILSSNIISVA 191 ALPGITNDTr 238 VALVWLFI 261 LILGVjLGVLA 314 IVLAVJEAIL 325 LMLIFLRQRI 329 FLRQR!RIAI 350 QMMSTMFYPL 358 PLVTVFAL 368 ClAY WAMTAL 393 NISSPGCEKV 554 FLNRiAYIMl 596 DLLLFFGKLL 645 ILGAYVIASG 649 YVIASGFFSV 34 DV1CCM-LFLL 19 64 VLYPRNSTGA 19 85 YLLYFNIFSC 19 186 NVTPPALPGI 19 233 LVALGVALVIL 19 264 GVLGVIAYGI 19 317 AVLEA:ILLLM 19 327 LIFLRQRIRI 19 335 RIAIAJ.EA 19 351 MMSTMEFYPLV 19 357 YPLVTEVLLL 19 363 VILLICIAYW 19 364 LLLICIAYWA 19 365 LLICIAYWAM 19 380 ATSGQEQYVL 19 457 VLALGQCVLA 19 536 CIMCCEKCCL 19 588 VVVLDKVTDL 19 633 HLNYYWLPIM 19 644 SILGAyYIAS 19 39 VLFLLFILGY 18 157 TV1TSLQQEL 18 203 QGISGILIDSIL 18 208 UDSLN!ARDI 18 240 LVILSILLFILL 18 246 RIELLVAG 18 262 ILGVL-QVLAY 18 281 RV'LRDKGASI 18 322 ILLLMIFLR 18 332 OR1IRIAL 18 360 VTFVLLLICI 18 388 VLWASHISSP 18 448 LGLFWTLNWV 18 TablXXXV.VI-HLA.A0201l10mers-24P4C12 Each peptide is a portion ot SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 493 RTLRYHjTGSL 18 525 KL.RGVQPVA 18 589 WLDK'jFDLL 18 616 FIFFS32GIPL 18 662 CVDTLFLCFL 18 685 YYMSKSLLKI 18 130 NEFSQJJVGEV 17 143 KNRNFCLPGV 17 148 CLPGVPWNMT 17 170 FLLPS6PALG 17 211 SLNARDISVK 17 227 QSWYW!VAL 17 254 AGPLVLUL 17 296 17NLSAYOSV 17 324 UJLlLIFLROR 17 373 AMTAIZIYLATS 17 481 DIPTFPLISA 17 546 WCL.El F11fL 17 563 IAIYGKNFCV 17 582 MRNIVRVVVL 17 IFILFI1LGYI 16 108 LQCPTEOVCV 16 118 SSCPEDPWTV 16 169 SFLULP APAL 16 200 TIQQGISGLI 16 207 GLIDSU4ARD 16 212 LNARDISVKI 16 236 LGVALVLSLL 16 292 OLGFUNLUSA 16 307 E1WLAAJVL 16 319 LEAILLLMLI 16 .337 AIALLK EASK 16 366 LICIAYWAMT 16 405 NTSCNPTAHL 16 451 FWTIMWVLA 16 458 WVLALGOCVL 16 458 LALGQCVLAG 16 503 AFGALILTLV 16 509 LTLVQiARVI 16 637 YWLPIMTSIL 16 33 TDVICCWFL 15 36 ICC V'LLLFl 15 NIFSC!LSSN 15 161 SLQQEICPSIF 15 225 FAQSWLYWILV 15 234 VALGV&VLS 15 250 LRLVAGPLVL 15 284 RDKGASISQL 15 TableXXXV.VI.KLA-AO2O1- 10mers-24P4Cl2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the legth of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234587890 score 323 LLLNIFLRQ 15 340 LLKWAKAVG 15 378 YLATSGQPOY 15 379 LATSGQPQYV 15 430 SSKGL!QRSV 15 464 VLAGAFASFY 15 498 HTGSLAFGAL 15 520 EYIDHISRGV 15 539 CCFKCCLWCL 15 601 FGKLL VGGV 15 690 SLLKIIGKKN 15 26 PIKNRSCTDV 14 30 RSCTD%1CCV 14 37 CCVLFLLFIL 14 102 SVAENgLQCP 14 149 LPGVPWNMTV 14 153 PWNMTVITSL 14 162 LQQELCPSFL 14 165 ELCPSELLPS 14 171 LLPSAPALGR 14 1177 ALGRCFPWTN 14 220 KIFEDEAQSW 14 273 IYYCWEEYRV 14 338 IALLKEASKA 14 353 sThIFYLvri, 14 370 AYWAMIALYI. 14 395 SSPGCEKVPI 14 416 NSSCPGLMCV 14 445 YGVLGLFWTL 14 483 P1FPL!SAFI 14 500 GSLAFGAILIL 14 571 CVSAKt-AFML 14 577 AFMLLMRNIV 14 595 TDLLLFFGKL 14 606 WGGVGVLSF 14 639 LPIMTfilLGA 14 680 SLDRPfl'MSK 14 693 KILGKKNEAP 14 694 ILGKXIEAPP 14 Tabl*XO(V.V34ILA.A0201.

l10mers-24PAIC12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide Is 10 amino acids, and the end positon for each peptide Is the start position plus nine.

Pos 1234567890 score 9 NITPPALPGI 23 10 ITPPALPGIT 12 TableXXXCV.V5-HLA-A0201- 110mers.244C12 Each peptide Is a portion of SEQ ID NO: 11; each start positionr is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 5 AILLLV'UFL 26 1 AVLEA!LLLV 2 VLEAIILLVI. 3 LEAILLLVLI 18 6 ILLLVLIFLR 18 8 LLVLIFLRQR 16 9 LVUIFRQRI 16 7 WL!LFLRQ 10 VUFLBQRIR 12 TableXXXV-V6-HLA.A0201lfiners-24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 GLIPRSVFNL 29 4 SSKGUIPRSV Tab~eXXXV-VT.HLA.A0201.

I10mers-24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peplie is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score I 0, WYWILVAV 4 2 SV(YWILYAVG 4 4 Y;MLVAVGQM 3 5 WILVA\VGQMM 2 6 ILVAVGQMMS 2 8 VAVGQMMSTM 2 9 AVGQMMSTMF 2 Table)0V-HLA-A0201l1rners.24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peplide is the start position plus nine.

Pos 1234567890 score 4 YWLPIMRNPI 15 V&PIMRNPIT 115 18 HVFOII ILGA 15 7 PIMRNEITPT 14 13 ITPTGHVFQT 14 8 IMRNPITPTG 13 21 QTSILGAYVI 13 FQTSILGAYV 12 PRTGHVEQITSI 11 RNPITPTGHV 10 16 TGHVFQTSIL 10 12 PITPT.QHVFQ 8 TableXX(V-V9.HLA-A0201l0mens-24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start posit is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 9 YPLPTQPATL 20 2 YWAMTALYPL 19 7 ALYPL;TQPA 19 12 PTQPAILGYV 17 16 ATLGYVLWAS 15 4 AIMALYPLPT 14 MTALYELPTQ 13 17 TLGYVLWASN 13 13 TQPATLGYVL 11 18 LGWVLWASNI 11 PATLGVVLWA 9 TabeX)O(V-14LA.A0203l0mors-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 303 QSVQETWLAA 19 168 PZFLILP APA 18 330 LRQRRIAIA 18 459 A4,GOC\ AGA 18 481 GgCVLAGAFA 18 304 S.YQETWLAAL 17 3 1G!QRDE5DEA 10 TabIeXXXV -HLA.A0203.

l0mers-24P4C12 Each peptide is a portion of SEQ ID NO. 3; each start position is specified. the length of peptide Is 10 amino adds, and the enid position for each peptide is the start position Pos 46 64 95 166 182 205 217 226 230 245 261 279 292 302 308 312 328 335 338 361 364 367 371 382 403 450 457 466 481 494 497 506 525 550 555 565 568 639 643 692 4 47 65 96 167 169 183 206 plus nine.

1234567890 score L.GYIVVGIVA 10 VjYPRNBTGA 10 ILSSNIISVA 10 LCPSFLLPSA 10 FEWTNVIPPA 10 ISGLIDSLNA 10 ISVKIFEDFA 10 AQSWWILVA 10 YWILVALGVA 10 I.EILLLRLVA 10 LILGVLGVLA 10 EXRVLRDKGA 10 &GF11IALSA 10 YQSVOErWLA 10 TWLAAL!VLA 10 AIVLAYLEA 10 IELRORIRIA 10 RIAIALLKEA 10 IALIKEASKA 10 TFVLLLICIA 10 ILLLICIAYWA 10 iYWAv A 10 YWAMTALYLA 10 SGQPQWYLWA 10 PINTSCNPTA 10 LFWTLWV 10 V~kGQgjVLA 10 AGAFASEYWA 10 D!PTIFIPLISA 10 TILRYHT GSLA 10 YHTGSL.FGA 10 ALILTL.VQIA 10 KLRGVQNPVA 10 IKIFQtRNA 10 ItANAYIMIA 10 IYGKNFCVSA 10 KNFCVSAKNA LEIMISILGA TILGAYVLA 10 LIILGKKNEA 10 KRDEDPEAY 9 GYIVVGIVAW 9 LYPRSIGAY 9 ILSSNiIIVAE 9 CESFLLPSAP 9 SELIPSAPAL 9 PWfTNVrEPAL 9 SGLIIDSNAR 9 TableX)"VV-tLA.A0203l0nmers-24P4C12 Each peptide is a portion of SEQ I D NO: 3; each start position Is specified, the length of peptide is amIno acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 218 SVKIFEDFAQ 9 227 QL$WYWILVAL 9 231 W!LVALGVAL 9 246 FILLIRI-VAG 9 262 lLGVLGMIAY 9 280 YRVLRDKGAS 9 293 LGFUFNLSAY 9 309 WLAALIYLAV 9 313 LIVILAVLEAI 9 329 FLRQRIRIAI 9 331 RQIRIAAL 9 336 IkIALLSEAS 9 339 ALLKEASKAV 9 362 FVLLLICIAY 9 365 ILCtAYWAM 9 368 CkAWAMTAL 9 372 WANTALYLAT 9 383 G~qPYVL WAS 9 404 IltTSCNETAH 9 451 FWTLNWVLLAL 9 458 LALGOCVLAG 9 460 LGQCVLAGAF 9 462 QCVLAGAFAS 9 467 GAJASIFYWAF 9 482 ETFPLISAF 9 495 LBYHTGaLAF 9 498 HTGSLAFGAL 9 507 LTLLVdIAR 9 526 LRGVQNPVAR 9 551 FIKFLNRNAY 9 556 NRN'AYIMIAI 9 566 YGKNFCMSAK 9 569 NfCVSAKNAF 9 640 PIMTSILGAY 9 644 SILGAYVIAS 9 693 K1LGKIKAEAP 9 TabteXXX(VI-V3HLA.A0203-10merrs 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position Is specified, t length of peplide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 5 FEWTIIPPA 6 PATNITEPAL 9 TableXXXV-HLA-A0203l-fllers- 24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amnino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 WINITPE-ALP 8 TableXXXVI.V5.HLA.A0203l0mers-24P4C12 Pos 1234567890 score NoResuttsFoufld.

TableXXXV1.V6-HLA-A0203.

10mers-24P4C12 Pos 1234567890 Score NoResultsFound.

TableXXXVl.V7-HLA.A0203-10mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 15: each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 1 QLSWYWLVAV 9 2 SWYWILVAVG 8 TableXXXVI-V8.HL1A.A0203-10mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234587890 score 18 HVFQTSILGA 10 19 VFQTSILGAY 9 F.QTSILGAYV 8 TableXXXVM-V9.HLAA0203-10mars- 24P4C12 Each peptide is a portion of SEQ ID -NO: 19; each start position is specified. the length of peptide Is 10 amino adds, and the end position for each peptide Is the staut position plus nine.

Pos 1234567890 score 7 ALYPLPTQPA 10 PATLGYYLWA 10 8 LYPLPTQPAT 9 16 ATLGYVLWAS '9 YPLPTQEATL 17 T.LGWVLWASN TableX)O(Vi-IHLA-A3-l0mers 24P4 C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino adids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 333 RIPLAWJJ(K 32 211 SLfjAR2I-SVK 30 .337 AIALLKEASK 28 516 Rq[LET!DHK 28 281 RVL:RDKQASI 27 680 SLDRPYYMSK 27 464 VIAGAFASFY 25 584 NIYRVALDK 24 621 RIPGLGKDFK 24 49 IWGIVAWLY 23 463 CVLAGAFASF 23 233 LVALGVALVL 22 262 ILGVLGVLAY 22 376 AL71LLSGQP 22 443 QIYGWLGLFW 22 525 KLEZGVQU~PVA 22 587 RVyVLaKTD 22 603 K(LLWVGGVGV 22.

56 WLyGofflQVL 21 63 QVLYPRNSTG 21 177 ALGRCL-YN 2 584 AIfIGK1ECVS 21 606 VGVLSF 21 39 VtlFLLFILGY 20 53 IVAWLYGDPR 20 171 LLPSAPALGR 20 251 RLYAGPLVLV 20 252 I.V GP1LVi 20 282 VL.KSIS 20 362 FVLLLIQIAY 20 378 YLATSGQPQY 20 544 CLWCLEKFIK 20 650 VIASGEESVF 20 95 ILS.SNIISVA 19 170 FLEPSARALG 19 191 AlP GITNDTT 19 237 GV ALVLSLLF 19 248 LLLRLYAGPL 19 260 VL1LGALG VI 19 261 LILGVLGVLA 19 298 NL_ AY0SVQE 19 312 ALIVLAVLEA 19 314 lVLAVLEAIL 19 317 AVLEALM 19 322 ILLLMLIFLR 19 24P4C12 Each peptide is a portion of SEQ ID MrY. 3; each start position Is specified, the length of peptide is 10 amino adds, and the end position for each peptide Is the start positlor) plus nine.

Pos 1234567890 score 340 LLIEASKAVG 19 347 AV GQMMSTMF 19 494 TLRYHTGSLA 19 605 LVjGGVGVLS 19 618 FSGRIPGLGK 19 645 ILGAYVASG 19 673 DLERNNGSLD 19 6 RDFDDEAYGK 18 64 VLY.PROITGA 18 231 WILVAJLGVAL 18 235 ALGVA LVLSL 18 247 IL[LRLVAGP 18 258 VLVU!GVLG 18 324 LI.MUFLRQR 18 456 WVL~aLQC VI 18 532 P\VARCIMCCF 18 72 GAYCGhffENK 17 86 LLXFNIFSCI 17 161 SLQQELCPSF 17 207 GLIDSUIARD 17 220 KIFEDEAQSW 17 232 ILVLALGVALV 17 249 LLBL\FA-PLV 17 257 LVLVLILGVL 17 264 GVLGVLAYGI 17 265 VLGAYGY 17 292 QLGF {LSA 17 309 WL6&VLAV 17 326 MLjFLfflRIR 17 364 LLLICiAYWA '17 388 VLAS1N1SSP 17 392 SNISSPCEK 17 486 PL!SAFIRTL 17 506 AL1LTLYQ[A 17 551 FINFLAENAY 17 580 LLMRNIVRW 17 598 LLFFGjE1W 17 612 VLSFFFFSGR 17 624 GL-QKDEKSPH 17 649 YV1AS~EFSV 17 657 SVFGMCVDTL 17 667 FLCFLEDLER 17 684 PYVMSKSW(K 17 689 KSILUGK 17 9 DDE-AYGKPVK 16 44 FLLGYLVVGI 16 126 T\VGKNff SQT 16 165 ELCPSFLLPS 16 TableXXXVII.V-HLAA3I1 Omers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 243 SLLFILLLRL 16 246 FILLLVAG 16 259 LVLILG.VLGV 16 272 GIfl'CWEEYR 16 304 SVQE1ffAAL 16 318 VLFAIjJLML 16 339 ALLKEASKAV 16 363 VILLIC-IAYW 16 453 TLI1WVLGQ 16 457 VLAJ.GXVLA 16 459 ALGQCVLAGA 16 487 LlIAFIRTLR 16 508 IILVQARV 16 518 ILEYIDHKLR 16 559 AYIMvIAIYGK 16 566 YGdNFLSAK 16 571 CVSAIK4AFML 16 579 MLLNIRNIVRV 16 596 DLLFE-GKLL 16 640 PIMTSILGAY 16 690 SUJ(ILGKKN 16 693 KICGK-{AP 1 VlrCVLELLF 15 41 FLLFILGYIV 15 42 LLEILEIW 15 107 GLQCPIEQVC 15 120 CPEDPWIfVGK 15 180 RCEPWIW/P 15 323 LLLMLLELRQ 15 329 FILRORIRIAI 15 367 ICIAYWKAMTA 15 369 IAYWVAMTALY 15 423 MCFCSSK 15 446 GVLGLE.W 15 491 FlRTLRYHTG 15 507 LILTILDQtAR 15 510 TLYQIAEVIL 15 585 IVBVVV1DKV 15 597 U4,FFGKLLV 15 604 LLWVGGVG Vi 15 688 SK SLLKILGK 15 694 ILjKKL1EAPP 15 697 KKNEAPPDNK 15 698 I0IEAPDNKK 15 Table0O(Vll-3-HLA-A3I0mers.

24P4C12 Each peptide Is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 3 RCEPWIN-TP 11 9 NIIPPA1,PGl 11 8 TN!TPPALPG 9 '10 ITPPALPGIT 7 7 W~tAfeEfALP 5 TableXX(VlI.V5.HLA-A3-1Omers- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10D amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 score 1 AVLEAILLLV 19 2 VLEAILLLVL 19 6 ILIVIJFLR 19 8 LLYVLIELRQR 18 10 VL!FLEQRIR 17 7 LLLVL IFLIRQ 15 5 AILLLYLIFL 14 9 LVLIFLRQRI 14 4 EAILILYLIF 11 7abIeXXX(ViI-V6-HLA.A3-10mers- 2434C12 Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 GLIPRYffNL 16 5 SKGLIPRSVF 14 I QGYSKGLIP 12 8 LIRRSYENLQ 11 9 IPRSVFNLOI 11 6 KGLIPLSVFN 10 4 SSKGI4ERSV 7 TableXXXVU.-V7HLA-A3-0mers- 2442 Each peptide is a portion of SEQ ID NO: 15; each 3tar position Is specified, the length of pepfide Is 10 amino adds, and the end position for each paptide is the start position plus nine.

Pos 1234567890 score 9 AVPQMNLSTMF 19 6 IL.VAVggMMS 16 5 WILVAVGQMM 14 7 LVAVG2!4MST 14 2 SWILVAVG 12 8 VAVGQMMSTM 9 24P4C12 Each peptide is a portion of SEQ ID NO, 17; each start position is specified, the length of peptide Is amno acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 12 PIIPTCiIVFQ 11 NPITPTGHVF 14 18 HVEQTSILGA 13 7 PIMRNEITPT 12 5 WLPIMRNPIT 11 I L-NjYWLPIMR 8 IMRNPITPTG 21 QTSIlkGAYVI 9 MRIAPIIETGH 9 6 LPIMRNPITP 8 19 VF-QTSILGAY 8 TableXKXVMl.V9-HLA-A3-10mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 ALYPLPIQPA 17 TLGYMWASN 10 PLPTQPATLG 14 9 Y;LPTCPATL 13 1 AYWAMIALYP 11 18 LG)VLWMSNI 4 AMTALXELPT 9 11 LPIQOP6ILGY 9 13 IQPATGYVI. 9 TableXXXVIII-VI-iILA-A2B-1 Omers- 244C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score TableXXXVIII.VI.HLA.A26.l0mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3: each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide, Is the start position plus nine.

Pos 1234567890 sore 34 DVICCVLFLL 34 138 EVFYTKNRNF 32 307 E1YVLA.LIVL 31 657 SVFGMCVDTL 28 199 1TIQQGISGL 26 304 SVQETWLAAL- 26 588 WVLDKVTDL 26 592 DKVTOLLLFF 25 49 IWVGIVAWLY 24 606 WGGVGVLSF 24 157 TVITSLQQEL 23 252 LVAGPLVLVL 23 257 LV VLIIG VI 23 320 EAILvLIF 23 628 DFKSPHLNYY 23 79 ENKDKPYLLY 22 353 STMFYPLVTF 22 362 FVLLLICIAY 22 662 CVDTLFLCFL 22 672 EDLERNNGSL 22 48 YIWGIVAWL 20 198 DTTIQQGISG 20 216 DISVKIFEDF 20 240 IVISILFILL 20 293 LGFTTNLSAY 20 640 PIMTSILGAY 20 DEAYGKPVKY 19 39 VIFLIFILGY 19 131 EFSQTVGEVF 19 233 LVALGVALVL 19 237 GVALVLSLLF 19 347 AVG(QfMSThIF 19 438 SVFNLQIYGV 19 463 CVLAGAFASF 19 498 HTGSLAFGAL 19 512 VQIARVILEY 19 520 EY1DHKLRGV 19 571 CVSAI(NAFML 19 589 WU)KVTDLL 19 33 TOVICOVIFI 18 203 QGISGLIDSL 18 314 IVLAVLEAL 18 456 WVI.ALGQCVL 18 481 DIPTFPLISA 18, 486 PUSAFIRTL 18 493 RTLRYHTGSL 18 502 LAFGAUJLTL 18 516 RVILEYIDHK 18 7ableXXX(ViII.VI-HLA.A26-10mers- 24C2 Each peptide is a portion of SEQ ID NQ 3: each start position is specified, the length of peptide is 10 amino adds, and the end position for each peptide is the start position pus nine.

Pos 1234567890 score 532 PVARCIMCCF 18 549 EKFIKFLNRN 18 609 GVGVLSFFFF 18 99 NIISVAENGL 17 102 SVAENGLQCP 17 156 MTVITSLQQE 17 236 LGVALVLSLL 17 260 VLILGVLGVL 17 316 LAVLEAILLL 17 317 AVLEAILLLM 17 321 AILLILMLIFL 17 360 VTFVLLLUCI 17 442 LQIYGVLGLF 17 596 DLLLFFGI(LL 17 604 LLWGGVGVL 17 616 FFFSGRIPGL 17 664 DTLFLCFLED 17 665 TLFLCFLEDL 17 682 DRPYYMSKSL 17 32 CTDVICCWIF 16 37 CCVLFLLFIL 16 123 DPWTVGKNEF 16 165 ELCPSFLLPS 16 186 NVTPPALPGI 16 224 DFAQSWvYWIL 16 239 ALVISILIL 16 262 ILGVLGVLAY 16 266 LGVLAYGI'YY 16 332 ORIRIAIALL 16 359 LVTFVLULIC 16 380 ATSGQPQYVL 16 400 EKVPINTSCN 16 405 NTSCNPTAHL 16 424 CVFQGYSSKG 16 433 GLIQRSVFNL 16 539 CCFKCCLWCL 16 593 KVTrDLLLFFG 16 TableX)O(\Vll-V3-HLA.A26. 0mers- Each 24P4C12 Eahpeptide is a portion of SEQ ID NO: 7; each start position Is specified, the length of peptide Is 10 amlno adds, and the end position for each peptide is the start position plus nine.

Pos 1234567890 Score 6 PWrNITPPAL '10 9 NITPPALPGI 10 10 ITPPALPGIT 7 WTNITPPALP 3 RCFPWTNITP 8 TNITPPALPG 4 CFPWTNiTPP TabeX)OMll.V5.HLA-A26l0mers-24C2 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 sore 4 EAlLLLVU-F 27 1 AVLEAILLLV 17 5 AILU.VUFL 17 2 VLEAILLLVL 13 TabIeXXX(V1II-V84ILA-A26- 10mers-2442 Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 123458890 score 7 GLIPRSVFNL 17 10 PRSVFNLQIY 14 5 SKGLIPRSVF TabeOXXVIIW.V7HLA.A26- 10mers-2434C12 Each peptide is a portion of SEQ ID NO: 15: each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus ine.

Pos 1234567890 score 9 AVGQMMSTMF 19 7 LVAVGQMMST 11 4 YWILVAVGQM TabIeXXXVIIIV8-HLA-A26- 10mers-24P4C12 Each peptide is a portion of SEQ ID NO: '17; each start position Is specified, the length of peptide Is amino adds, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score

HVFQTSILGA

VFQTSILGAY

NPITPTGHVF

ITPTGHVFQT

TGHVFQTSIL

PTGHVFQTSl TableXXXVII.-V9-HLA.A26-10oflrs- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 12 PTQPATLGYV 14 MtvALYPLPTQ 13 16 ATLGYVLWAS 13 2 YWAMTALYPL 12 11 LPTOPATLGY 12 9 YPLPTQPATL 10 13 TQPATLGYVL 10 PATLGYVLWA 6 TableXXXIX.V1.HLA-B0702l10mers-2424C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 score 357 YPLVTFVLLL 23 478 KPQDIPTFPL 23 883 RPYYMSKSt.L 21 182 FPWTNVrPPA 19 83 KPYLLYFNIF 18 192 LPGrTNDTTI 18 482 IPTFPLISAF 18 639 LPIMTSILGA 18 149 LPGVPWNM1V 17 252 LVAGPLVLVL 17 380 ATSGQPOWVL 17 402 VPINTSCNPT 17 485 FPLISAFIRT 17 123 OPWVTVGKNEF 16 235 ALGVALVLSL 16 254 AGPLVLVUIL 15 370 AYWAMTALYL 15 659 FGMCVDTLFI. 15 33 TDV1CCVLFL 14 56 WLYGDPRQVL 14 175 APALGRCFPW 14 233 LVALGVALVL 14 241 VLSLLFILLL 14 TabIOXXXUX.VI.HLA-BO7O2l10mers-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 331 RQRIRLPJAL 14 405 NTSCNPTAHL 14 451 FWTLNWVLAL 14 502 LAFGALILTL 14 582 MRNIVRVVVL 14 590 VLDKVTDLLL 14 15 KPVKYDPSFR 13 60 DPRQVLYPRN 13 66 YPRNSTGAYC 13 110 CPTPQVCVSS 13 120 CPEDPWrVGK 13 167 CPSFLLPSAP 13 172 LPSAPALGRC 13 226 AQSWVYWILVA 13 227 OSWYWILVAL 13 231 WILVALGVAL 13 250 LRLVAGPLVL 13 284 RDKGASISQL 13 290 ISQLGMTNL 13 301 AYQSVQETWL 13 310 LAALIVLAVL 13 314 IVLAVLEAIL 13 318 VLEAIU.LML 13 321 AILLLMLIFL 13 350 QMMSTMFYPL 13 355 MFYPLVTFVL 13 356 FYPLVTFVU. 13 368 CLAYWAMTAL 13 396 SPGCEKVPIN 13 441 NLQIYGVLGL 13 498 HTGSLAFGAL 13 500 GSLAFGALIL 13 510 TIVOIARVIL 13 525 KLRGVONPVA 13 571 CVSAKNAFML 13 572 VSAKNAFMU. 13 657 SVFGMCVDTL 13 686 YMSKSLLKIL 13 20 DPSFRGPIKN 12 48 YIWGIVAWL 12 169 SFLLPSAPAL 12 183 PWTNVTPPAI. 12 189 PPALPGITND 12 239 ALVLSLLFIL 12 243 SLLFILLILIRL 12 304 SVQETWLAAL 12 307 ETWLAALIVL 12 309 WLAALIVLAV 12 TableX)OWX.V-IILAB0702l0mars-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptlde is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 316 LAVLEAILLL 12 409 NPTAHLVNSS 12 419 CPGL.MCVFQG 12 425 VFQGYSSKGL 12 456 WVLALGQCVL 12 493 RTLRYHTGSL 12 581 LMRNIVRVVV 12 588 VVVLDKVTDL 12 604 LLWGGVGVL 12 606 WGGVGVLSF 12 622 IPGLGKDFKS 12 637 YWI.PIMTSIL 12 662 CVDTLFLCFL '12 701 APPDNKKRKK 12 18 KYOPSFRGPI 11 25 GPIKNRSCTD 11 31 SCTDVICCVL 11 44 FILGYIWGI 111 77 MGENKDKPYL 11 78 GENKOKPYLL 111 140 YKINRNIFCL 11 152 VPWNMTVITS 11 153 PWNMTVITSL 11 162 LQQELCPSFL 11 188 TPPALPGITN 11 224 DFAQSWYWIL 11 236 LGVALVLSI± 11 240 LVLSLLFILL 11 248 LLLRLVAGPL 11 257 LVLVULGVL 11 260 VLILGVLGVL 11 274 YYCWEEYRVL 11 312 AUVLAVLEA 11 315 VLAVLEAILL 11 332 QRIRIAIALL 11 384 QPQWLWA-SN 11 395 SSPGCEKVPI 11 413 HLVNSSCPGL 11 433 GUQRSVFNL 111 435 IQRSVFNLQI 11 439 VFNLQIYGVL 111 445 YGVLGLFWFL 11 449 GLFWTLNWVL. 11 503 AFGALILTLV 111 531 NPVARCIMCC 11 536 CIMCCFKCCL 11 539 CCFKCCLWCL 11 546 WCLEKFIKFL 11I 00 00 TableXXIXVI-HLA-B0702- 10mers-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 565 IYGKNFCV-SA 11 589 WLDKVTDLL 11 595 TDLLLFFGKL 11 616 FFFSGRIPGL 11 625 LGKDFKSPHL 11 630 KSPHLNY'tVIL 11 672 EDLERNNGSL I1I TableIeXV3-HLA-B0702.

l10murs-24P4C12 Each peptide is a portion of SEQ ID NO: 7; each start position is specifie, the length of peptide is 10 amino adds, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score FPW1TNITPPA 19 6 PWiTNITPPAL 12 1 LGRCFPWTNI 9 TableXX4.V-HLA-130702l0mers-24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position Is specified, the length of peptide is amino adids, and the end position for each peptide Is the start position pius nine.

Pos 1234567890 score 2 VLEAILLLVL 14 AILLLVLIFL 13 1 AVLEAILIIV 10 4 EAILLLVUF 10 3 LEA1LLLVLI 9 9 LVLIFLRQRI 7 TableX)OUX.V64iLA-B0702- 110mers-24P4C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids. and the end poisItion 'for each peptide Is t start position plus nine.

Pos 1234567890 sco re 9 IPRSVFNLQI 21 7 GLIPRSVFNL 12 TabIeXXIXV7-HL-BO702lomers-24P4Cl2 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 10 amino acids, end the end position for each peptide is the start position plus nine.

Pos 1234567890 score 9 AVGQMMSTMF 10 1 QSWYWILVAV 9 8 VAVGQMMSTM 8 4 YWILVAVGQIM 7 7 LVAVGQMMST 7 W1LVAVGQMM 6 TabeX)VUXV8-HLA-BO7O2- 110mers.2434C12 Each peptide is a portion of SEQ ID NO: 17; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position pius nine.

Pos 1234567890 score 111 NPITPTGHVF 17 14 TPTGHVFQTS 13 16 TGI4VFQTSIL 11 6 LPIMRNPITP 10 4 YWLPIMRNPI 9 7 PIMRNPITPT 9 21 QTSILGAYVI 9 10 RNPITPTGHV 8 13 ITPTGHVFQT 8 15 PTGHVFQTSI 8 18 HVFQTSILGA 8 TableXXXIX.V9.HLA-B0702- 10mers-24P4C12 Each peptide Is a portion of SEQ ID N0r 19; each start position is specified, the length of peptide is 10 amino adids, and the end position for each peptide Is the start position pius nine.

Pos 1234567890 score 9 YPI.PTQPATL 22 11 LPTQPATLGY 13 14 QPATLGWVLW 13 2 YWAMTALYPL 12 4 AMTALYPLPT 12 13 TQPATLGYVL 12 7 ALYPLPTQPA 11 TableXL-V14ILA.9B-10mers- 24P4C1 2 Pos 1234567890 score NoResultsFound.

TableXL-V3I-HILA-388.lmers- 24P4C12 Pos 1234567890 score NoResuftsFound.

TabloXL.V.HILA.B30810mers- 24P4C12 Pos 1234567890 score NoResuttsFound.

TableXIL-6HILA.B308-10mers- 24P4C12 Pos 1234567890 score NoResu~tsFound.

TableXL-.W.HLA.B08.l0mers- .24P4C12 Pos 1234567890 score NoResultsFound.

TableX.VO-HLA.B308-10mers* 24P4C12 Pos 1234567890 score INoResuttsFound.

TabeXL-VO-I4LA-B08- 10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TableXLIVI-A-81510-10mers- 24P4C12 Pos 1234567890 score NoResultsFeund.

TableXUL-V3-HLA-B31510- mers- 24P4C12 Pos 12345678190 Score NoResultFound.

TblIsX.V.HLA-B1510-10mers- 24P4C12 Pos 1234567890 score NoResuttaFound.

TableXLW.V.HLA.B31510l0mers-24P4C12 Pos 1234567890 score NoResultsFound.

TableXU.WV.HLA-81510-10mers- 2434IC12 Pos 1234567890 score TableXl.V3.HLA.B2709-l10mers- TabteXLIV.Vi-HLA-B4402- NoResultsFoufld. 24P4C12 10mems-24P4C12 Pos 1234567890 score Each peptide is a portion of SEQ ID TabIeXLI.V8.HLA.B1 510-10mrs- NoResuttsFound. NO: 3; each start position is 2402 specified, the length of peptlde Is Pos 1234587890 score TableXLIII.VS-HLA.2709-i~mers- amino acids, and the end position HoResubtFoufld. 24P4C12 for each pepfide is the start position Pos 1234587890 Sore plus nine.

TableXI-V9.HLA-1B1510-10mers- NoResuttsFound. Pos 1234567890 score 24P4C12 199 TTIQQGISGL 16 Pos 1234567890 score TableXLIl.V6-HLA-B2709-10mors- 203 QGISGLIDSL 16 NoResultsFound. 24P4C12 260 VULGVLGVL 16 Pos 1234567890 score 293 LGF1NLSAY 16 NoResultsFound. 307 ETWLAALIVL 16 TableXLIl.VI.HLA4S2705-1nlOTs- 316 LAVLEAILLL 16 24P34C12 TableXLIIIl.W.HLA-B2709-lofn1rs-24P4Cl2 380 ATSGOPQYVL 16 Pos 1234567890 score Pos 1234567890 Score 546 WCI.EKFIKFL 16 NoResults:oufd. NoResultsFound. 657 SVFGMCVDTL 16 34 DVICCVLFLL TabteXLII.V3.HLA.2705-10mers-2 4

P

4 Cl 2 TableioLlIII-HLA-B2709-l1fllers- 65 LYPRNSTGAY Pos 1234567890 Score 24P4C12 79 ENKDKPYLLY NoResuttsFound. Pos 1234567890 score 99 NIISVAENGL NoResults.Found. 104 AENGLQCPTP TableXL.V5-HLA-B32705-lO0ners- 138 EVFYTKNRNF 24PAIC12 TablXLIII-V9-HLA-B2709-10merOs- 213 NARDISVKIF Pos 1234567890 score 2413402 235 ALGVALVLSL NoResultFounkd. Pos 1234567890 score 239 ALVLSLLFIL NoResuttsFound. 278 EEYRVLRDKG TabteXLII.V6-HLA-275-10maTs- 284 RDKGASISQL 24P4C12 TableXLIV-Vi-HLA-B4402- 353 STMFYPLVTF Pos 1234567890 score l0mers-24P4C12 355 MFYPLVTFVL NoResuttaound. Each peptide Is a portion of SEQ I) 356 FYPLVTFVLL NO: 3; each start position Is 362 FVLLLUCIAY TableXLU.V7Mil-A-B2705.I0mers specified, the length of peptide is 10 383 VLWLCIAYW 24P4C12 amino acids, and the end position 370 AYWAMTALYL Pos 1234567890 score for each peptide is the start position 417 SSCPGLMCVF NoResultsFound. plus nine. 442 LQIYGVLGLF Pos 1234567890 score 451 FWTLNWVL.AL TableXLII.VS4ILA.92705-10tfers- 10 DEAYGKPVKY 23 482 IPTFPLISAF 24P4C12 78 GENKDKPYLL 22 561 IMIAIYGKNF Pos 1234567890 score 222 FEDFAQSWYW 21 596 DLLLFFGKLL NoResultsFound. 319 LEAILLLMLI 20 616 FFFSGRJPGL 47 GYIWGIVAW 19 637 YWVLPIMTSIL TabteXUIl.V9.HLA-B2705- Omers- 332 QRIRIAIALL 18 840 PIMTSILGAY 24P4C12 486 PUSAFIRTI 18 4 KQRDEDDEAY 14.

Pos 1234567890 score 502 LAFGALILTL 18 18 KYDPSFRGPI 14 NoResultsFound. 620 GRIPGLGKDF 18 80 NKDKPYLLYF 14 39 VLFLLFILGY 17 83 KPYLLYFNIF 14 TableXLIl.-VI.HLA-B2709.l0mers- 241 VLSLLILLL 17 130 NEFSQTVGEV 14 24P4C12 254 AGPLVLVUIL 17 131 EFSQTVGEVF 14 Pos 1234567890 Score 320 EAILLLMLIF 17 157 WITSLQQEL 14 NoResuttsFound. 321 AILU.MLIFL 17 164 OELCPSFLLP 14 476 FHKPODIPTF 17 173 PSAPALGRCF 14 TableXIll.V3.HL-A-B32709.l0mers- 512 VQIARVILEY 17 175 APALGRCFPW 14 24P4C12 699 NEAPPDNKKR 17 183 PWThVTPPAL 14 Pos 1234567890 *score 121 PEDPWTVGKN 16 220 KIFEDFAQSW 14 169 SFLL.PSAPAL 16 227 QSWYWILVAL 14 00 00 C0 00 TableXLIV.VI.HLA-B4402- 10mers.24P4Cl2 Each peptide is a portion of SEQ ID N0. 3; each start position is specified, dhe length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 scoe 231 WILVALGVAL 14 233 LVALGVALVL 14 240 LVLSIIFILL 14 243 SLLFILLLRL 14 250 LRLVAGPL VI 14 252 LVAGPLVLVI. 14 253 VAGPLVLVLI 14 262 ILGVLGVLAY 14 304 SVQETWL.AAL 14 331 RQRIRIAIAL 14 357 YPLVTFVLLL 14 431 SKGLIQRSVF 14 433 GLIQRSVFNL 14 467 GAFASFYWAF 14 542 KCCLWCLEKF 14 545 LWCLEKFKF 14 551 FIKFLNRNAY 14 569 HFCVSAKNAF 14 589 VVLDKVTDLL 14 595 TDLLLFFGKL 14 627 KDFKSPHLNY 14 629 FKSPHLNYYW 14 665 TLFLCFLEDL 14 686 YMSKSLLKIL 14 7 DEDDEAYGKP 13 31 SCTDVICCVL 13 32 CTDVICCVLF 13 VICCVIYLLF 13 49 IWGIVAWLY 13 56 WLYGDPRQVL 13 57 LYGDPRQVLY 13 87 LYFNIFSCIL 13 145 RNFCLPGVPW 13 153 PWNMTVITSL 13 188 NVTPPALPGI 13 237 GVALVLSLLF 13 248 LLLRLVAGPL 13 257 LV'LVLILGVL 13 271 YGIYYCWEEY 13 301 AYQSVQETWL 13 310 LAAUJVLAVI. 13 315 VLAVLEAILL 13 327 LIFLRQIRI 13 342 KEASKAVGOM 13 347 AVGQMMSTMF 13 405 NTSCNPTAI-L 13 425 VFQGYSSKGL 13 441 NLQIYGVLGL 13 TableXLIV.V1IHLA-B4402.

l0mers-24P4C1 2 Each peptide is a portion of SEQ ID NO:- 3; each start position is specified, the length of peptide is 10 amino acids, and the end posilion for each peptide is the start position plus nine.

Pos 1234567890 score 445 YGVLGLFWTL 13 447 WLGUWTLNW 13 449 GLFWTLNWVL 13 460 LGQCVLAGAF 13 478 KPQDIPTFPL 13 483 PTFPtISAFI 13 493 RTLRYHTGSL 13 495 LRYHTGSLAF 13 498 HTG&AGAL 13 500 GSLAFGALIL 13 517 V1LEYIDHKL 13 539 CCFKCCLWCL 13 557 RNAYIMIAJY 13 582 MRNIVRVVVL 13 590 VLDKVTDLLL 13 591 LDKVTDLLLF 13 592 DKVTDLIIFF- 13 606 WGGVGVLSF 13 659 FGMCVDTLFL 13 661 MCVDTLFLCF 13 662 CVDTLFLCFL 13 671 LEDLERNNGS 13 672 EDLERNNGSL 13 682 DRPYYMSKSL 13 33 TDVCCVLFL 12 37 CCVLFLLFIL 12 44 FILGYIWVGI 12 76 GMGENKDKPY 12 123 DPWrVGKNEF 12 132 FSQTVGEVFY 12 150 PGVPWNMTVI 12 163 QOELOPSFLL 12 216 DISVKIFEDF 12 223 EDFAQSWYWI 12 236 LGVALVLSLL 12 266 LGVLAYGIYY 12 274 YYCWEEYRVL 12 277 WEEYRVLRDK 12 286 KGASISQLGF 12 290 ISQLGFrTNL 12 300 SAYQSVQETW 12 306 QEV.VLMLIV 12 313 LM.LAVLEAI 12 318 VLEAILLLML 12 329 FLRQRIRIAl 12 350 QMMSTMFYPL 12 358 PLVTFVULLL 12 360 VrFVLLUCI 12 Tab~eXLIV.VI-HLA-B4402* l0mers-24PAIC12 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is amino acids, and the end position for each peptide Is the start positioni plus nine.

Pos 1234567890 score 368 CIAYWAMTAL 12 369 lAYWAMTALY 12 378 YLATSGGPOY 12 381 TSGQPQYVLW 12 395 SSPGCEKVPI 12 420 PGLMCVFQGY 12 436 QRSVFNLQIY 12 439 VFNLOIYGVI. 12 443 QWYGVLGLFW 12 456 WVLALGQCVL 12 463 CVLAGAFASF 12 464 VLAGAFASFY 12 488 ISAFIRTLRY 12 505 GALILTLVQI 12 509 LTLVQIARVI 12 510 TLVQIARVIL 12 548 LEKFIKFLNR 12 556 NRNAYIMIAI 12 571 CVSAKNAFML 12 572 VSAKNAFMLL 12 576 NAFMLLMRNI 12 588 VVVLOKVTDL 12 604 LLWGGVGVL 12 628 DFKSPHLNYY 12 630 KSPHLNYYWL 12 650 VlASGFFSVF 12 674 LERNNGSLDR 12 676 RNNGSLDRPY 12 677 NNGSLDRPYY 12 685 YYMSKSLLKI 12 14 GKPVXYDPSF 11 27 IKNRSCTDVI 11 40 LFLLFILGYI 11 48 YIWGIVAWL 11 77 MGENKDKPYL 11 116 CVSSCPEDPW 11 137 GEVFY11(NRN 11 161 SLQQELCPSF 11 162 LQQELCPSFL 11 208 LIDSU4ARDI 11 212 LNARDISVWI 111 221 IFEDFAQSWY 11 238 VALVLSLLFl 11 264 GVLGVLAYGI 11 305 VOETWLAALI 11 314 IVLAVLEAIL 11 348 VGQ4MSTMFY 11 413 HLVNSSCPGL 111 TableXLIV.V1IHLA-B4402lomers-24C2 Each peptide Is a porton of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and t end position for each peptide is the siar position plus nine.

Pos '1234587890 score 479 POIPTFPLI It 499 TGSI.AFGALI I I 519 LEYIDHKLRG III 526 GVQNPVARCI 11 532 PVARCINICCF 11 538 CIMCCFKCCL I11 537 IMCCFKCCLW I I 543 CCLWCLEKFI 11 552 IKFLNRNAYI 11 607 VGGVGVLSFF 11 608 GGVGVLSFFF 11 609 GVGVLSFFFF 11 625 LGKDFKSPHL 11 632 PHLNYYWLPI 11 642 ISILGAYVI 11 646 LGAYVIASGF 11 658 VFGMCVDTLF 11 683 RPYYMSKSLL 11 TableXLIV.V3-HLA.B4402-10mefS- 24P34CI12 Each peptide Is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 6 PWTNITPPAL 14 9 NITPPALPGI 13 1 LGRCFPWTNI 8 3 RCFPWTNITiP 7 8 TNITPPALPG 6 TableXULVV5.HILA-11MO42-11mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 3 LEAILILVII 21 4 EAILLLVLIF 18 AILLLVLIFL 17 2 .VLEAILILLVIL 13 9 LVUFLRQRI 10 TabIeXUvVVG4LA.4402.

l0mers-24P4C12 Each peptide is a portion of SEQ ID NCY. 13; each start position is spcified, the length of peptide is 10 amino adids, and the end position for each peptide is the start position plus nine.

Pos 1234587890 score 7 GLIPRSVFNL 17 5 SKGUPRSVF 14 10 PRSVFNLOIY 12 9 IPRSVFNLQI 10 TableXLIV.W-HLA.B4402.

111mers-24PAIC12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 9 AVGOMMSTMF 13 4 YWILVAVGQM 6 TabIeXLIV.V8-KLA.4402i~mers-244C12 Each peptide is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptidle is 10 amino acids, and the end position for each peplide Is the start position plus nine.

Poe 1234587890 score 11 NPITPTGHVF 17 4 YWLPIMRNPI 14 19 VFQTSILGAY 14 16 TGHVFQTSL 11 21 QISILGAYVI 11 15 -PTGHVFQTSI a TableXLIV.V9-HLA-B4402l10mers-24P4C112 Each peptide is a portion of SEQ ID NOr. 19; each start position Is specified, the length of peptide is 10 amino adds, and the end position for each peptide Is the start position plus nine.

Poe 1234567890 score 9 YPLPTQPATL 16 14 QPATLGYVI.W 13 11 LPTQPATLGY 12 13 TQPATLGYVL 12 2 YWAMTALYPL I11 18 LGYVLWASNI 9 16 ATLGYVLWAS 8 7 ALYPIPTOPA 7 TableXLV.VI.HLA-B5101.I0mers- 24P34C12 Pos 1234587890 score NoResultsFound.

TableXILV.V3LA05101-lmers- 244C12 Poe 1234587890 score NoResultsFound.

TableXLV-V.4LAB5101-10me's- 24P4C12 Pos 1234567890 score NoResultsFound.

TableXLV-V6-HLAB5I 24P4012 Pos 1234587890 score NoResuftsFound.

Tabi.XLVV7-HLA-1B5101.l1mers- 24P4Ci2 Pos 1234567890 score NoResuitsFound.

TabIL-8HL-A-B5101l4fllers.

24P4C12 Pos 1234567890 score NoResultFound.

TableXLV-VO-HLA-B5101.l0mers.

24P4C12 Pos 1234567890 score NoResutFound.

TableXLVVHLA.DR81.0101- 1 Smers-24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123458789012345 eco 227 QSWYWVILVALGVALV 39 206 SGLIDSLNARDISVK 33 247 ILLLRLVAGPLVLVL 33 313 LIVLAVLEAIU.LML 33 601 FGKLLWGGVGVLSF 33 246 FILLLRLVAGPLVLV 32 262 ILGVLGVLAYGIYY 32 353 STMFYPLVTFVLLLI 32 368 CIAYWAMTALYLATS 32 652 ASGFFSVFGMCVDTL 32 39 VLFUFILGYIWVGI 31 181 CFPWTNVTPPALPGI 31 277 WEEYRVLRDKGASIS 31 559 AYIMIAIYGKNFCVS 31 639 LPIMTSILGAYVIAS 31 YLLYFNIFSCILSSN 30 89 FNIFSCILSSNIISV 30 257 LVLVLILGVLGVLAY 30 259 LVLILGVLGVLAYGI 30 635 NYYWLPIMTSILGAY 30 646 LGAYV1ASGFFSVFG 30 235 ALGVALVLSLLFILL 29 345 SKAVGQMMSTMFYPL 29 LFLLFILGYIWGIV 28 242 LSLLFILLLRLVAGP 28 359 LVTFVLLLICIAYWVA 28 453 TLNWVLALGQCVLAG 28 812 VLSFFFFSGRIPGLG 28 640 PIMTSILGAYVIASG 28 167 CPSFLLPSAPALGRC 27 243 SLLILLLRLVAGPL 27 280 YRVLRDKGASISQLG 27 362 FVI±LICIAYWAMTA 27 423 MCVFQGYSSKGLIQR 27 501 SLA*GALILTLVQIA 27 575 KNAFMLMRNIVRWV 27 129 KNEFSQTVGEVFYTK 26 230 YWILVALGVALVLSL 26 254 AGPLVIVLILGVLGV 28 384 QPQYVLWASNISSPG 26 436 QRSVFNLQIYGVLGL 26 437 RSVFNLQIYGVLGLF 26 448 LGLFWRLNWVLALGQ 26 492 IRTLRYIITGSLAFGA 26 551 FIKFLNRNAYIMIAI 26 594 VTDLLLFFGKLLWVG 26 633 HUJYYWLPIMTSILG 26 688 SKSLU(ILGKKNEAP 26 44 FILGYIWVGIVAWLY 25 53 IVAWLYGDPRQVLYP 25 62 RQVINPRNSTGAYCG 25 NIFSCILSSNIISVA 25 228 SWYWILVALGVALVL 25 TableXLV-VI-HLADRB-IiOl5mers-24P4C1 2 Each peptide is a portion of SEQ ID N0r 3: each start position is specified, the length of peptide is amino adids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 eco 231 WILVALGVALVLSLL 239 ALVLSLLFILL.RLV 293 LGM1NLSAYQSVQE 299 LSAYQSVQETWLAAL 304 SVQTWLAUVLAV 319 LEAILLLMLIFLRQR 326 MLIFLRQRIRIAIAL 337 AIALLKEASKAVGQM 354 TMFYPLVtFVLLLUC 371 YWAMTALYLATSGQP 399 CEKVPINTSCNPTAH 451 FWTLNVNLALGOCVL 454 LNWVLALGQCVLAGA 471 SFYWAFHKPQDIPTF 482 IPTPPLISAFIRTLR 526 LRGVQNPVARCIMCC 683 RNIVRVLDKVTDL 603 KLLWVGGVGVLSFFF 51 VGIVAWLYGDPRQVL 24 97 SSNIISVAENGLQCP 24 229 WYWILVALGVALVLS 24 238 VALVLSLLFILLLRL 24 255 GPLVLVLILGVLGVL 24 256 PLVLVLILGVLGVLA 24 279 EYRV'LRDKGASISOL 24 307 ETWLAALIVLAVLEA 24 310 LAALIVLAVLEAILL 24 383 GQPQYVLWASNISSP 24 420 PGLMCVFQGYSSKGL 24 459 ALGQCVLAGAFASFY 24 506 AUJLTLVQIARV1LE 24 523 DHKLRGVONPVARCI 24 589 NFCVSAKNAFMLLMR 24 579 MLLMRNIVRVLDK 24 588 VVVLDKVTDLLLFFG 24 607 VGGVGVLSFFFISGR 24 644 SILGAYVLASGFFSV 24 680 GMCVDThFLCFLEDL 24 47 GYIWGIVAWLYGDP 23 59 GDPRQVLYPRNSTGA 23 165 ELCPSFLLPSAPALG 23 166 LCPSFLLPSAPALGR 23 241 VLSLLFILLLRLVAG 23 374 MTALYLATSGQPQYV 23 412 MiLVNSSCPGLMCVF 23 507 ULTLVQtARVI=EY 23 508 ILTLVQIARVILEYI 23 566 YGKNFCVSAKNAFML 23 604 LLWGGVGVLSFFFF 23 638 YYWLPIMTSILGAYV 23 33 TDV1CCVLFLLFILG 22 43 LFILGYIWVGIVAWL 22 86 LLYFNIFSCILSSNI 22 TabIXLVI-VI*HLA-DRBI -0101- 1 5mers-24P4C12 Each peptide is a portion of SEQ ID NO:, 3; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is Mhe start position plus fourteen.

Pos 123456789012345 scar e 160 TSLQQELCPSFLLPS 22 198 DITIQOGISGLIDSL 22 312 ALl VLA VLEAILLLM 22 316 LAVLEAII±LMLIFL 22 349 GQMMSTMFYPLVrFV 22 363 VLLLICIAYWAMTAL 22 419 CPGLMCVFQGYSSKG 22 439 VFNLQIYGVWGLFWT 22 4411 NLQIYGVLGLFWTLN 22 458 LALGOCWLAGAFASF 22 481 DIPTFPLISAFIRTL 22 511 LVQIARV1LEYIDH< 22 587 RVVVLDKVTDLLLFF 22 598 LLFFGKLLWVGGVGV 22 655 FFSWFGMCVDTLFLC 22 689 KSLLKILGKKNEAPP 22 138 EVFYTKNRNFCLPGV 21 151 GVY NMTVITSLQQE 21 153 PWNNTVTSLQQELC 21 203 QGISGLIDSLNARDI 21 300 SAYQSVOETWLAALI 21 329 FLRORIRiAIALi(E 21 331 RQRIRIAIALLKEAS 21 409 NPTAHLVNSSCPGIJJ 21 518 ILEYIDHKLRGVQNP 21 548 LEKFIKFLNRNAYIM 21 606 WVGGVGVLSFFFFSG 21 DEAYGKPVKYDPSFR 20 DPSFRGPIKNRSCTD 20 272 GIYYCWEEYRVLRDK 20 333 RIRIAJAU.KEASKA 20 449 GLFWILNWVLALGQC 20 476 FHKPQDIPTFPUSA 20 543 GCLWCLEKFIKFLNR 20 563 IAIGKNFCVSAKNA 20 599 LFFGKLLWGGVGVL 20 614 SFFFFSGRIPGLGKD 20 634 LNYYWhtPlMTSILGA 20 645 ILGAWVIASGFFSVF 20 656 FSVFGMCVDTLFLCF 20 657 SVFGMCVD1I.FLCFL 20 37 CCVILFILGYIVV 19 38 CVLFLLFILGYIWVG 19 82 DKPYLLYFNLFSCIL 19 122 EDPWTVGKNEFSQTV 19 179 GRCFPWTNVTPPALP 19 184 WTNVTPPALPGITND 19 245 LFILLLRLVAGPLVL 19 2711 YGIYYCWEEYRWLRD 19 317 AVLEAILLLMUFLR 19 323 LLLMLIFLRQRIRIA 19 336 IAIALLKEASKAVGQ 19 389 tAYWAMTAJ.YLATSG 19 TableXLVI-Vi-HLA.DRB1-0101.

l15mers-24114C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peplide Is the start position plus fourteen.

Pos 234578902345 scor Pos 23457890234 411 TAHLVNSSCPGLMCV 19 442 LQrI'GVLGLFWTLNW 19 450 LGQCVLAGAFASFYW 19 495 LRYHTGSLAFGALIL 19 503 AFGALILTLVQIARV 19 557 RNAYIM(AIYGKNFC f9 586 VRVVUKVTDLLLF 19 6853 RP'TYMSKSLLKILGK 19 6814 PYYMSKSLLKILGKK 19 TablenLV-3HLA-DRBI.0101.

l15mers-24P34C12 Each peplide is a portion of SEQ ID NO: 7; each start position is specified, the length of poptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 scare 9 CFPWTNITPPALPGI 31 7 GRCFPWTNITPPALP 19 12 WTNiTPPALPGITND 19 10 FPWTNITPPALPGIT 18 14 NITPPALPGITNDTT 16 TableXLVI.VS.HLA-ORBI-O101l16mers-24P14C12 Each peptide is a portion of SEQ ID NO: 11; each slart position is specified, the length of peptide is 15 amino adids, &nd the end position for each peptde is the start position plus fourteen.

Pos 123456789012345 scoe 2 UV1.AVLEAILLLVL 33 9 LEAiLLLVLIFLRQR 15 VLIFLRORIRIAIAL 1 ALl VLAVLEAILLLV 22 5 LAVLEAILLLVUJFL 22 6 AVLEAILLLVLIFLR 19 12 LLLVUFLRQRIRIA 19 13 LLVLIFLRQRIRAJ 18 7 VLEAJLULVLIFLRO 17 11 ILLLVUFLRQRIRI 17 14 LVLIFLRQRIRAJA 17 4 VLAVLEAILLLVLIF 16 10) AIU.LVUFLRQRIR 16 TabIeXLVIV6-HLA-DRB1-O1O1- I 5mers-24P34C12 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptidle is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 2 MCVFQGYSSKGLIPR 27 PRSVFNLQIYGVLGL 26 7 GYSSKGLIPRSVFNL 24 4 VFQGYSSKGLIPRSV 16 SKGLIPRSVFNLQIY 16 112 GLIPRSVFNLQIYGV 16 1 LMCVFQGYSSKGLIP 15 8 YSSKGLIPRSVFNLQ 15 TabteXLVVHLA-DRB1-01 01- I 5mers-24P4C12 Each peptide is a portion of SEQ ID NQr 15; each start position is specified, the length of peptide is 15 amino adds, and the end position for each peptide Is the start position plus fourteen.

Pos 123456789012345 score 6 OSWYWILVAVGQMMS 31 12 LVAVGQMMSTMFYPL 29 7 SWYWILVAVGQMMST 25 8 WYWILVAVGQMMSTM 24 9 YWILVAVGQMMSTN4F 24 1 FEDFAQSWYILVAV 18 AQSWYWVLVAVGQMM 16 11 ILVAVGQMMSTMFYP 15 TableXILVI.VIIItILA.DRBI.011.

l5niers-24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptidle is 15 amino acids, and the end position for each peptide Is the start position plus fourteen.

Pos 123456789012345 score 24 VFQTSILGAYViASG 28 7 NYYV&PIMRNPITPT 24 23 HVFQTSILGAYVIAS 23 6 LNYYiNLPIMRNPITP 20 HLNYYW..PIMRNPIT 18 21 TGHVFOTSILGAYVI 18 3 SPHLNYYWLPIMRNP 17 8 YYWLPIMRNPITPTG 17 13 IMRNPITPTGHVFQT 17 11 LPIMRNPITPTGHVF 16 12 PIMRNPITPTGHVFO 16 14 MRNPITPTGHVFQTS 16 26 QTSILGAWVIASGFF 16 9 YWLPIMRNPITPTGH 15 18 ITPTGHVFQTSILGA 15 19 TPTGHWFQTSILGAY 14 PTGHVFQTSILGAYV 14 TableXLVI49-HLA-DR311010I.

115mers;-24M Each peptidle Is a portion of SEQ ID NO: 19, each start position is specified, the length of peptide isi15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 4 CIAYWAMTALYPLPT 32 10 MTALYPLPTQPATLG 22 TLGYVLWASNISSPG 26 21 ATLGYVLWASNISSP 24 7 YWAMTALYPLPTQPA 23 1 LYPLPTOATLGYVL 23 5 IAYWAMTALYPLPTQ 19 2 LICIAYWAMTALYPL 17 1 LLICIAYWAMTALYP 16 16 LPTQPATLGYVLWAS 16 23 LGYVLWVASNISSPGC 16 24 GYVLWASNISSPGCE 16 9 AMTALYPLPTQPATI. TabteXILVII-VI-HLA-DR110301l5mers-24P4C112 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 54 VAWLYGDPRQVLYPR 36 586 VRWVLDKV1DLLLF 31 667 FLCFLEDLERNNGSL 29 312 ALIVL.AVLEAILLLM 28 97 SSNIISVAENGLQCP 27 155 NMTVITSLQIQELCPS 27 454 LNWVLALGOCVLAGA 27 549 EKFIKFLNRNAYIMI 27 136 VGEVFYTKNRNFCLP 26 508 ILTLVQIARVILEYI 26 622 IPGLGKDFKSPHLNY 26 376 ALYLATSGQPQYVLW 447 VWGLFWLNWVLALG 279 EYRVLRDKGASISQL 24 534 ARCIMCCFKCCLWCL 24.

567 GKNFCVSAKNAFM4LL 24 229 W'?WiLVALGVAI.VLS 23 238 VALVILSIJFI.RL 23 14 GKPVKYDPSFRGPIK 22 218 SVKIFEDFAQSWYWI 22 219 VKIFEDFAQSWYWIL 22 235 ALGVALVLSLLFILL 22 241 VLSLLFILLLRLVAG 22 360 VTFVLLLUCIAYWAM 22 515 ARVILEYIDHKLRGV 22 594 VTDLLLFFGKLLWG 22 33 TDVICCVLFLLFILG 21 167 CPSFLLPSAPALGRC 21 192 LPGrrNDTTIQQGIS 21 237 GVALVLSULFILLLR 21 239 ALVLSLLFILLLRLV 21 260 VULGVLGVLAYGIY 21 302 YQSVOEYVLAALIVL 21 319 LEAILLMFLRQR 21 431 SKGLIQRSVFNLQIY 21 TableXLVI.Vi .HLA-DRBI1-0301 I Smers-24P4C1 2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 461 GQCVLAGAFASFYWA 21 587 RWVU)KVTDLLLFF 21 590 VLDKVTDU.LFFGKL 21 596 TDLLLFFGKLLWVGG 21 658 VFGMCVDTLFLCFLE 21 32 CTDVICCVLFLLFIL 20 37 CCVLFLLFILGYIW 20 46 LGYIWVGIVAWVLYGD 20 47 GYIWGIVAWVLYGDP 20 74 YCGMGENKDKPYLLY 20 76 GMGENKDKPYLLYFN 20 231 WILVALGVALVLSLL 20 233 LVALGVALVLSLLPI 20 246 FILLLRLVAGPLVLV 20 250 LRLVAGPLVLVUILG 20 255 GPLWLVLILGVLGVL 20 258 VLVLILGVLGVLAYG 20 313 LIVLAVLEAILILML 20 316 LAVLEAILLLNILIFL 20 323 LLLMLIFLRQRIRIA 20 338 IALLKEASKAVGQMM 20 411 TAHLVNSSCPGLMCV 20 439 VFNLQIYGVLGLFWT 20 484 TFPLISAIRTLRYH 20 559 AYIMIAIYGKNFCVS 20 588 WVLDKVTDLLLFFG 20 602 GKLLWVGGVGVLSFF 20 604 LLWVGGVGVLSFFFF 20 691 LLKILGKKNEAPPDN 1158 MTVITSLQQELCPSF 19 159 ITSLQQELCPSFLLP 19 205 ISGUDSLNARDISV 19 335 RIAIALLKEASKAVG 19 348 VGQMMSTMFYPLVTF 19 366 LICIAYVWAMTALYLA 19 385 PQWVLWASNISSPGC 19 505 GALILTLVQIARVIL 19 576 NAFMLLMRNIVRWV 19 607 VGGVGVLSFFFFSGR 19 626 GKDFKSPIILNYYWLP 19 638 WIPIMTSILGAYViA 19 848 AYVIASGFFSVFGMC 19 663 VDTLRLCFLEDLERN 19 668 LCFLEDLERNNGSLD 19 684 P"~'SKSLLKILGKK 19 689 KSLILKILGKKNEAPP 19 3 GKORDEDDEAYGKPV 18 61 PRO VLYPRNSTGAYC 18 98 SNIISVAENGLQCPT 18 114 QVCVSSCPEDPWTVG 18 214 ARDISVKIFEDFAQS 18 243 SLLFILLLRLVAGPL 18 263 LGVLGVLAYGIYYCW 18 3127 UFIRCRIRIALALL 18 345 SKAVGQMMSTMFYPL 18 TabIeXLVII-V1 -HLA.DRBI-0301- 1 5mers-24P412 Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle Is.15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 462 QCVLAGAFASFWAF 18 530 QNPVARCIMCCFKCC 18 560 YIMLIYGI(NFCVSA 18 569 NFCVSAKNAFMLLMR 18 579 MLLMRNIVRVWVLDK 18 585 IVRVVVLDKVTDLLL 18 655 FFSVFGMCVDTLFLC 18 656 FSVFGMCVDTLFLCF 18 660 GMCVDTLFLCFLEDL 18 664 OTLFLCFLEDLERN~N 18 284 RDKGASISQLGFTN 17 290 ISOGFTTNLSAYOS 17 324 LLNLIFLRQRIRIAI 17 325 LMLIFLRQRIRIAIA 17 353 STMFYPLVTFVU.LI 17 423 MCVFQGYSSKGLIQR 17 437 RSVFNLQIYGVtGLF 17 485 FPLISAFIRTLRYHT 17 517 VILEYIDHKLRGVQN 17 519 LEYIDHKLRGVQNPV 17 523 DHKLRGVQNPVARCI 17 542 KCCLWCLEKFIKFLN 17 545 LWCLEKFIKFLNRNA 17 548 LEKFIKFLNRNAYIM 17 614 SFFFFSGRIPGLGKD 17 619 SGRIPGLGKDFKSPH 17 670 FLEDILERNNGSLDIRP 17 692 LKILGKKNEAPPDNK 17 TableXLVII.V3-HLADRB-0301-l5mers- 24P4C12 Each peplide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 12 WTNITPPALPGITND 12 3 APM..GRCFPWTNITP 9 CFPWTNITPPALPGI 7 GRCFPWTNITPPALP 8 6 LGRCFPWINITPPAL 7 TabIsXLV1-VM-LADRBI-0301l15mers244C12 Each peptide Is a poution of SEQ ID NO: 11; each start position is specified, the length of peptide Is 15 aino acids, and the end position for each peptide Is the start position plus fourteen.

Pos 123456789012345 score 1 ALl VLAVLEAILLLV 28 8 LEAJU.LVLIFLRQR 21 2 UIVLAVLEAILLLVL 200 00 00 TableXLVII*VSHLADRBIO0301l5mers.24P4C12 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 amino adids, and the end position for each peptide is the start position plus fourteen.- Pos 123456789012345 score LAVLEAILLLVLIFL 20 12 U.LVUIFLRQRIRIA 20 13 LLVUFLRQRIRLAI 17 14 LVLIFLRQRIRIAIA 17 4 VLAVLEAILLLVUIF 15 9 EAILLLVLIFLRQRI 15 AILLLVLIFLRQRIR 13 TabIeXLVII-V6-HLA-DRB1FO30Il15mers-24P4C112 Each peptide Is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each peptide Is the start position plus fourteen.

Pos 123456789012345 score SKGLIPRSVFNLQIY 22 2 'MCVFQGYSSKGLIPR 17 8 YSSKGLIPRSVFNLQ 16 11 KGLIPRSVFNLQIYG 12 1 LMCVFQGYSSKGLIP 11 PRSVFNLQIYGVLGL 10 TableXLVII.V-HLA-DRBI0301-15mers- 24P4C12 Each peptide is a portion of SEQ ID NO: each start position is specified, the length of peptide Is 15 amino adds, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 Score 9 YWILVAVGQMMSTMF. 18 12 LVAVGQMMSTMFYPL 18 I FEDFAQSWYWILVAV 16 8 wyW1LVAVGQMMSTM 13 WILVAVGQMMSTMFY 10 13 VAVGQMMSTMFYPLV 10 TableXLVI.V8.HLA-DRBI0301 -1 Smers- 24P4C12 Each peplide Is a portion of SEQ ID NO: 17; each start position Is specified, the length of peptide is 15 amiuno acids, and the end position for each peptide Is the start position plus fourteen.

Pos 123456789012345 score 22 GHVFQTSILGAYVIA 17 8 YYWLPIMRNPITPTG 16 RNPITPTGHVFQTSI 14 26 QTSILGAYVIASGFF 13 21 TGHVFQTSILGAYVI 12 WLPIMRNP[TPTGI{V 11 11 LPIMvRNPITPTGHVF 11 3 SPHLNYYWLPIMRNP 10 7 NYYWLPIMRNPI1PT 10 14 MRNPITPTGHVFQTS 9 19 rPTGHvFQTSILGAY 8 TableXLVI.V9HLADRBI.0301-15mors- 24P4C12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 2 LUCIAYWAMTALYPL 19 23 LGYVLWASNISSPGC 19 10 MTALYPLPTQPATLG 13 7 YWAMTALYPLPTQPA 12 112 ALYPLPTQPAT1.GYV 12 13 LYPLPTQPATLGYVL 12 20 PATLGYVLWASNISS 12 3 ICIAYWAMTALYPLP 14 YPLPTQPATLGYVIW 24 GYWLWASNISSPGCE 5 IAYWAMTALYPLPTQ 9 16 LPTQPATLGYVLWAS 9 T ableXLV1II-Vi -DRI -0401-1 Smers.

24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide isi1S amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 85 YI.LYFNIFSCILSSN 28 89 FNIFSCILSSNIISV 28 243 SLLFILLLRLVAGPL 28 353 STMFYPLVTFVLLLI 28 469 FASFYWAFHKPQOIP 28 548 LEKFIKFLNRNAY1M 28 575 KNAFMLLMRNIVRW 28 635 NYYWLPIMTSILGAY 28 54 VAWLYGDPRQVLYPR 26 98 SNIISVAENGLOCPT 26 153 PWNMTVITSLQQELC 26 189 PPALPGrrNDTTIQQ 26 192 LPGITNDTr1QQGIS 26 323 LLLMLIFLRQRIPIA 26 337 AJAW(KEASKAVGQM 26 385 PQYVLWASNISSPGC 26 419 CPGLMCVFQGYSSKG 26 454 LNWVLALGQCVLAGA 26 508 ILTLVQIARVILEYI 26 523 DHKLRGVQNPVARCI 26 579 MLLMRNIVRVWVLDK 26 16 PVKYDPSFRGPIKNR 22 38 CVLFLLFILGYIWG 22 82 DKPYIJ.YFNIFSCIL 22 86 LLYFNIFSCILSSNI 22 122 EDPWrVGKNEFSQTV 22 138 EVFYTKNRNFCLPGV 22 181 CFPWTNVTPPALPGI 22 219 VVIFEDFAQSWYWIL 22 227 QSWNVVLVALGVALV 22 228 SWYWILVALGVAL.VL 22 TableXLVIII-VI*DR1 -0401 A Smers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptie is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 272 GIYYCWEEYRVLRDK 22 277 WEEYRVLRDKGASiS 22 292 OLGFTTNLSAYQSVQ 22 299 LSAYOSVQETWLAAL 22 306 QE1WLAALI VIA VE 22 354 TMFYPLVrFVI±LIC 22 359 LVFFVLLLICIAYWA 22 384 QPQYWAWASNISSPG 22 423 MCVFOGYSSKGLIQR 22 442 LQIYGVLGLFWTLNW 22 448 LGUFWTLNWVLALGQ 22 453 TI.NWVLALGQCVLAG 22 488 ISAFIRTLRYHrGSL 22 501 SLAFGALILTLVQJA 22 557 RNAYIMIAIYGKNFC 22 633 HLNYYWLPIMTSILG 22 646 LGAYVLASGFFSVFG 22 652 ASGFFSVFGMCVDTL 22 667 FLCFLEDLERNNGSL 22 682 DRPYYMSKSLLKILG 22 14 GKPVKYDPSFRGPIK 20 39 VLFLLFILGYIWVGI 20 LFLLFILGYIWVGIV 20 43 IF(LGYIWVGIVAWL 20 97 SSNIISVAENGLQCP 20 133 SQTVGEVFYTKNRNF 20 146 NFCLPGVPWNMTVIT 20 149 LPGVPWNM1VITSLQ 20 155 NMIVITSLOQELCPS 20 156 MTVITSLQQELCPSF 20 198 D1TIQQGISGLIDSL 20 2D2 OQGISGUIDSLNARD 20 205 SGLIDSLNARDISVK 20 216 DISVKIFEDFAQSWY' 20 229 WYWILVALGVALVLS 20 230 YWILVALGVALVLSL 20 233 LVALGVALVLSLLFI 20 235 ALGVALVLSLLFILL .20 238 VALVLSIIFILLLRL 20 239 ALVLSULFIU.LRLV 20 241 VLSLLUL.IJRLVAG 20 242 LSLLFILLLRLVAGP 20 246 FILLLRLVAGPLVLV 20 247 ILLLRLVAGPLVLVL 20 254 AGPLVLVLILGVLGV 20 255 GPLVLVUILGVLGVI. 20 257 LVLVUILGVLGVLAY 20 259 LVLILGVL-GVLAYGI 20 262 ILGVLGVLAVGIYYC 20 279 EYRVURDKGAS1SOL 20 287 GASISC)LGFTTNLSA 20 290 ISQLGF1NLSAYQS 20 307 ETWLAALM.LAVLEA 20 310 LAALIVLAVLEAILL 20 311 AAUIVLAVI.EAJLLL 20 TabloXLVIII-VI-DRI-040 1.1 mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the lengthi of pepfide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 312 ALl VLAVLEAILULM 313 UVLAVLEAIUILML 315 VLAVIEAILLLMLIF 316 LAVLEAILLLMLIFL 319 LEAILLLMLIFLRQR 321 AILLLMLIFLRQRIR 324 LLMLIFLRORIRIAI 331 RQRIRIAIALLKEAS 333 RIRIAIAILKEASKA 335 RIAJALLKEASKAVG 356 FYPLVTFVWLICIA 363 VLLUCIAYWAMTAL 364 LLLICLAYWAMTALY 371 YWAMTALYLATSGQP 374 MTALYLATSGQPQYV 401 KVPINTSCNPTAHLV 420 PGLMCVFQGYSSKGL 438 QRSVFNLQIYGVLGL 444 IYGVLGLFWTLNWVL 445 YGVLGLFWTLNWVLA 447 VLGLFWTLNWVLALG 451 FWTLNWLALGQCV1. 479 PQDIPTFPLISAIR 484 TFPLISAFIRTLRYH 485 FPLISAFIRTLRYHT 505 GALILTLVQIARVIL 506 ALILTLVQtAR VILE 511 LVOIARVILEYIDHK 514 IARVILEYIDHKLRG 516 RVILEYIDHKLRGVQ 542 KCCLWCLEKFIKPL 545 LWCLEKFIKFLNRNA 549 EKFIIFLNRNAYIMI 558 NAYiMiAlYGKNFCV 582 MRNIVRVVVLDKVTD 583' RNIVRVLDKVTDL 586 VRVVVLDKVrDLLLF 588 WVLDKfDLLLFFG 594 VTDLLLFFGKLLWG 595 TDUILFFGKLLWVGG 601 FGKLLWGGVGVLSF 619 SGRIPGLGKDFKSPH 639 .LPIMTSILGAYVIAS 642 MTSILGAWVIASGFF 680 GMCVDTLFLCFLEDL 668 LCFLEDLERNNGSLD 688 SKSLLKILGKKNEAP 90 NIFSCILSSNIISVA 18 125 WrVGKNEFSQTVGEV 18 1152 VPWNMTVITSLQQEL 18 166 LCPSFLLPSAPALGR 18 195 ITNDTTIQQGISGLI 18 203 QGISGLIDSLt4ARol 18 210 DSLNARDISVKIFED 18 289 SISQGFTTNLSAYQ 18 TableXLVIII.Vi-DRi -0401 -1 Smers.

24P4IC12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 arrino adds, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 295 FTTNLSAYQSVQETW 18 342 KEASKAVGQMMSTMF 18 373 AMTALYLATSGQPOY 18 398 GCEKVPINTSCNPTA 18 428 GYSSKGLIORSVFNL -18 433 GUQRSVFNLQIYGV 18 476 FHKPQDIPTFPLISA 18 481 DIPTFPLISAFIRTL 18 502 LAFGALILTLVQIAR 18 527 RGVQNPVARCIMCCF 18 568 KNFCVSAKNAFMLLM 18 611 GVLSFFFFSGRIPGL 18 623 PGLGKDFKSPHLNYY 18 657 SVFGMCVDTIYLCFL 18 669 CFLEDLERNNGSLOR 18 DPSFRGPIKNRSCTI) 16 ILGYIWGIVAWLYG 16 53 IVAWLYGDPRQVLYP 16 AWLYGDPRQVLYPRN 16 63 QVLYPRNSTGAYCGM 16 144 NRNFCLPGVPWNMTV 16 151 GVPWNMTV1TSLQQE 16 167 CPSFLLPSAPALGRC 16 222 FEDFAQSWYWILVAL 16 226 AQSWYWILVALGVAL 16 271 YGIYYCWEEYRVLRD 16 326 MLIFLRQRIRIAIAL 16 368 CIAYWAMTALYLATS 16 369 IAYWAMTALYLATSG 16 375 TALYLATSGQPQVVL 16 387 YVLWASNISSPGCEK 16 437 RSVFNLQIYGVLGLF 16 449 GLFWTLNWVLALGOC 16 466 AGAFASFYWAFHKPO 16 470 ASFYWAFHKPQDIPT 16 471 SFYAFHKPQDIPTF 16 473 YWAFHKPQDIPTFPL 16 482 IPTFPLISAFIRTLR 16 518 ILEYIDHKLRGVQNP 16 543 CCLWCLEKFIKFLNR 16 563 IAJYGKNFCVSAKNA 16 598 LLFFGKLLWVGGVGV 16 612 VLSFFFFSGRIPGLG 16 613 LSFFFFSGRIPGLGK .16 614 SFFFFSGRIPGLGKD 16 634 UfrYWLPIMTSILGA 16 653 SGFFSVFGMCVDTLF 16 664 DTLFLCFLEDLERNN 16 62 ROVLYPRNSTGAYCG 15 325 LMUFLRQRIRIAIA 15 327 UFLRQRIRIAIALL 15 519 LEYIDHKLRGVONPV 15 587 RWVLDKVTDLLLFF 15 32 CTDVCCVLFLLFIL 14 33 TDVICCVLFLLFILG 14 TableXLVIIIVI-DR-0401-15mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 amino adids, and Mhe end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 36* ICCVLFLLFILGYIV 14 37 CCVLFLLFILGYIWV 14 42 LIFILGYIWGIVAW 14 46LGYIV VGIVAWLYGD 14 47 GYIWGI.VAWI.YGDP 14 48 YIWGIVAWLYGDPR 14 51 VGIVAWLYGDPRQVL 14 61 PRQVLYPRNSTGAYC 14 83 KPYLLYFNIFSCILS 14 84 PYU.YFNIFSCILSS 14 88 YFNIFSCILSSNIIS 14 92 FSCILSSNIISVAEN 14 93 SCILSSNIISVAENG 14 124 PWTVGKNEFSQTVGE 14 136 VGEVFYTKNRNFCLP 14 159 ITSLOQELCPSFLLP 14 163 QQELCPSFLLPSAPA 14 169 SFLLPSAPALGRCFP 14 175 APALGRCFPWTN'IW 14 184 WTNVTPPALPGITND 14 205 ISGLIDSLNARDISV 14 218 SVKIFEDFAQSWYWI 14 231 WILVALGVALVLSLI. 14 237 GVALVLSLLFliLLR 14 244 LLFILLI.RLVAGPLV 14 249 LLRLVAGPLVLVLIL 14 250 LRLVAGPLVLVLILG 14 258 PWLVLILGVLGVLA 14 258 VLVLILGVLGVLAYG 14 260 VULGVLGVLAYGIY 14 263 LGVLGVLAYGYYCW 14 296 TTNLSAYQSVQE1VJL 14 302 YQSVQETWLAALIVI. 14 322 ILLLMLIFLRQRIRI 14 338 tILKEASKAVGQMM 14 345 SKAVGQMMSTMFYPL 14 348 VGOMMSTMFYPLVTF 14 349 GQMMSTMFYPLVTFV 14 352 MSTMFYPLVTFVLLL 14 357 YPLVTFVW.IJCIAY 14 360 VTFVLWLCIAYWAM 14 361 TFVLLLICIAYWAMT 14 362 FVLLUCLANAMTA 14 366 UCIAYWAMTALYLA 14 376 ALYLATSGQPQWVLW 14 391 ASHISSPGCEKVPIN 14 399 CEKVPINTSCNPrAH 14 411 TMlLVNSSCPGL.MCV 14 412 AHLVNSSCPGLNCVF 14 422 LMCVFQGYSSKGLIQ 14 432 KGUQRSVFNLQIYG 14 439 VFNLQIYGVLGLFWT 14 441 NLQWYGVI.GLFWTLN 14 455 NWVLALGQCVLAGAF 14 457 VLALGOVLAGAFAS 14 TabIeXLVIII-V1 .DRI-0401-l 5mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3; each start position is specilied, the length of peplde is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456T89012345 score 462 QCVLAGAFASFYWAF 14 489 SAFIRTLRYHTGSLA 14 492 IRTLRYHTGSLAFGA '14- 499 TGSLAFGALILTLVQ 14 504 FGALILTLVQIARVI 14 509 LTLVQIARVILEYID 14 515 ARVILEYIDHKLRGV 14 526 LRGVQNPVARCIMCC 14 534 ARCIMCCFKCCLWCL 14 535 RCIMCCFKCCLWCLE 14 552 IKFLNRNAYIMLAIY 14 559 AYIMiAIYGKNFCVS 14 576 NAFMLLMRNIVRVWV 14 578 FMLLMRNIVRWVU) '14 585. IVRVVVLDKVTDLLL 14 591 LDKVTDU1LFPGKLL 14 596 DLLLFFGKLLWVGGV 14 602 GKLLWGGVGVLSFF 14 603 KLLWVGGVGVLSFFF 14 604 LLWGGVGVLSFFFF 14 607 VGGVGVLSFFFFSGR 14 609 GVGVLSFFFFSGRIP 14 610 VGVLSFFFFSGRIPG 14 622 IPGLGKDFKSPHLNY 14 631 SPHLNYYW&PIMTSI 14 636 YYtNLPIMTSILGAYV 14 647 (SAYVIASGFFSVFGM 14 655 FFSVFGMCVDTLFLC 14 -658 VFGMCVDTLFLCFLE 14 663 VDTLFLCFLEDLERN 14 665 TLFLCFLEOLERNNG 14 678 NGSLDRPYYMSKSLL 14 684 PYYMSKSLLKILGKK 14 689 KSLLKJLGKKNEAPP 14 TableXLVIII.V3-HLA-ORI-0401- 15mems-24P4C12 Each peptide is a portion of SEQ ID NO.

7; each start position Is specified, the length of peptide is 15 amino acids, and the end position for each peptide Is the start position plus fourtee.

Pos 1234W789012345 score 9 CFPWTNITPPALPGI 22 3 APALGRCFPWrNITP 14 12 WTNITPPALPOITND 14 4 PALGRCFPWTNITPP 12 ALGRCFPWTNITPPA 12 8 RCFPWTN[TPPALPG 12 13 TNITPPALPGITNDT 12 14 NITPPALPGITNDTT 12 7 GRCFPWTNITPPAI.P 10 TableXLVIII-V5DRI-M4015lmers- 2412402 Each peptide is a portion of SEQ ID NO, 11; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each poplide is the start position plus fourteen.

Pos 123456789012345 score 12 U.LVLIFLRQRIRIA 26 I ALVLAVLEAILLLV 2 LIVLAVLEAILLLVA 4 VLAVLEAIW.LVLIF 5 LAVLEAILLVIFL 8 LEAILLLVLIFLROR 10 AJWNLLIFLRQRIR 13 LLVUFLRQRIRIAI 15 VLIFRRIRIAIAL 16 14 LVUFLRQRIRIAIA 9 EAILLLVLIFLRQRI 14 11 IUJ.VLIFLRQRIRI 14 3 IVLAVLEAILLLVLI 12 6 AVLEAILLLVUIFLR 12 TableXLVIII-V6-HLADR-I.415mers- 24C2 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 15 amino acids. and the end position for each peptida Is the start position plus fourteen.

Pos 123456789012345 score 2 MCVFQGYSSKGLIPR 22 15 PRSVFNLQIYGVLGL 12 GLIPRSVFNLQrI'GV 18 1 LMCVFQGYSSKGLIP 14 11 KGLIPRSVFNLOIYG 14 7 GYSSKGLIPRSVFNL 12 8 YSSKGLIPRSVFNLQ 12 9 SSKGLIPRSVFNLQI 12 TableXLVIII-W7-HLA-DRI-0401-15mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 15 amio acids, and the end position for each peptide Is tho start position plus fourteen.

Pos '123456789012345 score 9 YWILVAVGQMMSTMF 26 6 QSWiYINILVAVGQMMS 22 7 SWYWILVAVGQMIMST 22 8 WYWILVAVGQMMSTM 1 FEDFAQSWYW1lLVAV 16 5 AQSWYWILVAVGQMM 16 10 W1LVAVGOMMSTMFY 14 '12 LVAVGQMMSTMFYPL 14 TabhaXLMII-V8-HLA4OR1.041-15mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3: each start position is specified, the length of peptide is 17 amino acids, anid the end position for each peptidie is the start position plus fourteen.

Pos 123456189012345 score 00 00 TableXLVIII.V8.HLA-DRI-0401-i5mers- 24P4C12 Each peptde is a porton of SEQ ID NO:, 3; each start position is specified, the length of peptide is 17 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 7 NYYWLPIMRNPITPT 28 HLNYYWLPIMRNPIT 22 8 YYW1.PIMRNPITPTG 20 RNPITPTGHVFQTSI 20 26 QTSILGAYVIASGFF 20 18 ITPTGHVFQTSILGA 18 19 TPTGHVFQTSILGAY 18 3 SPHLNYYWLPIMRNP 14 WLPIMRNPITPTGHV 14 11 LPIMRNPITPTGHVF 14 21 TGHVFQTSILGAYVI 14 TabIXLVIl-V9-HLADRI.0401.

i 5mers-24P4CI2 Each peptide, Isa portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score MTALYPLPTQPATLG 28 23 LGYVLWASNISSPGC 26 11 TALYPLPTQPATLGY 22 22 TLGYVLWASNISSPG 22 7 YWAMTALYPLPTQPA 20 PATLGYVLWASNISS 20 IAYWAMTALYPLPTQ 16 2 UICLAYWAMTALYPL 14 3 ICIAYWAMTALYPLP 12 PLPTQPATLGYVLWVA 12 21 ATGYVLWASNISSP 12 TableXLIX-VI-DRB13101.15mers- 24P4C12 Each peptide is a portion of SEQ ID NCVr 3; each start position is specified, the length of peplide is 15 amino acids, and the end position for each pepide Is the start position plus fourteen.

Pos 123456789012345 score 243 SLLFILLLRLVAGPL 31 DEAYGKPVKYDPSFR 26 DPSFRGPIKflRSCTD 26 668 LCFLEDLERNNGSLD 26 575 KNAFMLLRNIVRW 25 613 LSFFFFSGRIPGLGK 25 226 AQSWYWILVALGVAL 23 228 SWYWILVALGVALVL 23 277 WEEYRVL-RDKGASIS 23 359 LVTFVU.LICIAYWA 23 448 LGLFWTLNWVLALGO 23 579 MLLMRNIVRVVVtDK 23 598 LLFFGKLLWGGVGV 22 633 HLNYYWLPIMTSILG 22 TableXUX-Vi-DRBI.1i101.-1 24P4C1 2 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 276 CWEEYRVLRDKGASI 21 338 IAWEASKAVGQMM 21 508 ILTLVQIARV1LEYI 21 516 RVILEYIDHKLRGVQ 21 542 KCCLWCLEKFIKFLN 21 585 IVRVWLDKVTDLLL 21 685 YYMSKSW(ILGKKN 21 17 LPSAPALGRCFPWTN 334 IRIAIALLKEASKAV 371 YWAMTALYLATSGQP 549 EKFIKFLNRNAYIMI 591 LDKVrDLU.FFGKLL 619 SGRIPGLGKDFKSPH 689 KSIIKILGKKNEAPP 36 ICCVLFLLFILGYIV 19 122 EOPWrVGI(NEFSQTV 19 256 PLVLVLILGVLGVLA 19 259 LVLILGVLGVLAYGI 19 310 LAAIJVLAVL-EAJLL 19 353 STMFYPLVTFWLLLI 19 523 DHKLRGVQNPVARCI 19 567 GKNFCVSAKNAFMLL 19 612 WLSFFFFSGRIPGLG 19 838 YYWLPIMTSILGAYV 19 16 -PVKY'0PSFRGPIKNR 18 48 YIWVGIVAWLYGDPR 18 85 YLLYFNIFSCILSSN 18 137 GEVFYTKNRNFCLPG 18 181 CFPWTNVTPPALPGI 18 227 QSWYWILVALGVALV 18 244 LLFILLLRLVAGPLV 18 326 MLIFL.RQRIRLAIAL 18 419 CPGLMCVFQGYSSKG 18 469 FASFYWAFHKPQDIP 18 470 ASFYWAFHKPQOIPT 18 488 ISAFIRTLRYlITGSL 18 489 SAFIRTLRYHTGSLA 18 597 U±LFFGKLLWGGVG 18 41 FLLFILGYIWVGIVA 17 45 ILGYIVVGIVAWLYG 17 .71 TGAYCGMGENKDKPY 17 86 LLYFNIFSCILSSNI 17 306 QETWLAALIVLAVLE 17 325 LMUFLRQRIRIAI 17 354 TMFYPLVTFVLWLC 17 369 IAYWAMTALYLATSG 17 384 QPQYWLWASNISSpG 17 442 LQIYGVLGLFWTLNW 17 482 IPTFPUJSAFIRTLR 17 501 SLAFGALILTLVQiA 17 548 LEKFIKFLNRNAYIM 17 615 FFFFSGRIPGLGKDF 17 635 NYYW'LPIMTSILGAY 17 652 ASGFFSVFGMCVDTL 17 82 DKPYLLYFNIFSCIL 16 TabIeXLIX.V1 DRBI1-1 101-1 5mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 3, each start position is specified, the lengjth of peptide is 15 amino acids, and the enid position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 89 FNIFSCILSSNIISV 16 179 GRCFPWTNVTPPAI.P 16 253 VAGPLVLVLILGVLG 16 299 LSAYQSVQETWLMAL 16 323 LLLMLIFLRQRIRIA 16 368 CLAYWAMTALYLATS 16 387 YVLWASNISSPGCEK 16 490 AFIRTLRYIITGSLAF 16 494 TLRYHTGSLAFGALI 16 506 ALILTLVQIARVILE 16 517 V1LEYIDHKLRGVQN 16 557 RNAYIMIAIYGKNFC 16 563 IAJYGKNFCVSAKNA 16 583 RNIVRVVVLOKVTOL 16 646 LGAYVIASGFFSVFG 16 43 LFILGYIVVGIVAWL 15 44 FILGYIWVGIVAWLY 15 47 GYrSYGIVAWLYGOP 115 54 VAWLYGDPRQWLYPR 15 73 AYCGMGENKDKPYU. 15 153 PWNMTVITSLQQELC 15 156 MTViTSLQQELcPsr 15 195 ITNDTTIQQGISGLI 15 207 GLIDSU'JARDISVKI 242 LSLLFILLLRLVAGP 15 357 YPLVTFVI.LICIAY 15 429 YSSKGLIQRSVFNLQ 15 485 FPUSAFIRTLRYHT 15 519 LEYIDIIKLRGVQNPV 15 527 RGVQNPVARCIMCCF 15 545 LWCLEKFIKFLNRNA 15 595 rDLLLFFGKI-LWGG 15 600 FFGKLLWGGVGVLS 15 603 KLLWVGGVGVLSFFF 15 681 LDRPYYMSKSLLKIL 15 TableXLIX-V3HLADRBi-1101-15mera- 24P14C12 Each peptide is a portion of SEQ ID NO: 7; each start posiboon is specified, the length of peptide Isi15 amino acids, and the end positio for each peptide is the start position pius fourteen.

Pos 123456789012345 score 9 CFPWTNITPPALPGI 18 7 GRCFPwrNrrPPALP 16 112 WTNITPPALPGITND 8 TableXLIX-V5-HLA.DRBI.1101.lfmers.

24P4C12 Each pepfide is a portion of SEQ ID NO: 11; each start position Is specified, the length of peptide i 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 15 VIIFIRORIRIAIAL 14 LVLIFLRQRIRIAIA 112 LLLVUFLRQRIRIA 10 AILLLVLIFLRQRIR 2 LIVLAVLEAILLLVL 8 LEAIUJ.VLIFLRQR 113 LLVLIFLRQRIRIAI 1 ALl VLAVLEAI LLLV 5 LAVLEAILLLVLIFL 9 EAILLLVLIFLRQRI 11 ILLLVUJFLRQRIRI score 18 17 16 14 14 14 13 13 13 13 TableXUX-V6HILA.DIRBI.1 101- I Smers-24P4C12 Each peptide is a portion of SEQ ID NO-.

13; each start position is specified, the length of peptid is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 8 YSSKGLIPRSVFNLQ 1 INC VFQGYSSKGLIP 14 15 PRSVFNLQIYGVLGL 13 2 MCVFOGYSSKGLIPR 5 FQGYSSKGLIPRSVF 3 CVFQGYSSKGLIPRS 9 11 KGLIPRSVFNLOIYG 9 6 QGYSSKGLIPRSVFN 8 4 VFQGYSSKGUPRSV 7 7 GYSSKGLIPRSVFNL 7 TabIeXLIX.VF.HLA-DRBI-1 101.l5mers- 24P4C12 Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide isi15 amidno adds, and the end position for each pept is the staut position plus fourteen.

Pos 123456789012345 score 5 AQSWflNILVAVGQMM 23 6 QSWYWILVAVGQlMS 18 9 YWILVAVGQMMSTMF 18 7 SVAVJILVAVGQMMST 16 12 LVAVGOMMSTMFYPL 12 I1 FEDFAQSWYWILVAV I11 TableXLIX-V8-HLA-DRBI -110141 Smers- 24P4C12 Each peptide Is a portion of SEQ ID NQr 17; each start position is specified, the length of peptide is 15 annino acids, and the end position for each pepide Is the start position plus fourteen.

Pos 123456789012345 score NYYWLPIMRNPITPT 24 5 HLNYYWLPIMRNPIT 18 6 LNYYWLPIMRNPiTP 17 115 RNPITPTGHVFQTSI '16 8 YYWLPIMRNPITPTG 13 21 TGHVFQTSLGAYV1 13 TabIeXLIX-W9HLA.DRBI.1 1O1-15mers- 00 442 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the r~l length of peptide is 15 amino acids, and the end position for each peptide is the start positon plus fourteen.

Pos 123456789012345 score 004 CIAYWAMTALYPLPT 22 0010 MTALYPLPTQPATLG 18 22 TLGYVLWASNISSPG 17 7 YWATALYPLPTQPA 14 13 LYPLPTQPATLGYVL 13 00 20 PATLGWVLWASNISS 12 Nl 23 LGYVLWASNISSPGC 12 24 GYVLWASNISSPGCE 12 IAYWAMTALYPLPTQ 11 TALYPLPTQPATLGY 00 Table L: PropertIes of 24P4C12 Bioinformatic URL Outcome Program ORF ORF finder 6 to 2138 Protein length 710aa Transmcmbrane region TM Prod httpi/www.ch.anbnct.org/ I I TM, 39-59, 86-104, 231-250,252-273, 309- 330, 360-380, 457-474, 497-515. 559-581.604- 626, 641-663 HMMTop http:llwww.nzim.hu/hmmtop/ 1 ITM, 35-59 84-104 231- 250 257-277 308-330 355- 377 456-475 500-19 550- 572 597-618 649-671 Sosui httpJ/www.genome-ad.jp/SOSui/ 13TM, 34-65, 86-108, 145- 167, 225-247, 307-329, 357-379,414-436.447-469, 501-523, 564-586, 600-622, 644-666 TMHMM httpi/www.cbs.dtu.dk/swviioelTMHMM 1IM, 36-58,228-250, 252- 274, 308-330, 356-378, 454-476,497-519, 559-581, 597-619 Signal Peptide Signal P http:/www.cbs.dtu.dk/serviocwJSigna1P/ no p1 p1MW tool http:t/www.xpasy.chtools/ 8.9 pi Molecular weight p/MW tool httpl/www.expasy.ch/tools/ 79.3 kiD Localization PSORT httpi/psornibb.acjp 80% Plasma Membrane, Golgi PSORT If httpt/psortnibbacjp/ 65% Plasma Membrane, 38% endoplasmic reticulum MotifS Pfarn http:/Aww.sanger.acuk/Pm/ DUPS80, uknown function Prints httpJ/ww.biochcnLuci.a.uk/ Blocks httpil/www.blocks.flcrc.orgl Anion exchanger family 313-359 Prosite http:/www.prositmorg/ CYS-RICH 536- 547 Table LI. Exon oaopositiona of 24P4C12 v.1 Exon number Start End Length I 1 45 2 46 94 49 3 95 16B 74 4 169 247 79 248 347 100 6 348 473 126 7 474 534 61 8 535 622 88 9 623 706 84 707 942 236 11 943 1042 100 12 1043 1135 93 13 1136 1238 103 14 1239 1492 254 1493 1587 16 1586 1691 104 17 1692 1765 74 18 1766 1836 71 19 1837 1931 1932 2016 21 2017 2573 557 00 00 Table LIZ. Nualeotid6 gagccatggg gggaaagcag acgacccctc ctttcgaggc tcctcttcct gctcttcatt gagacccccg gcaagtcctc agaacaaaga taagccg tat acatcatctc agttgctgag cctgcccgga ggacccatgg tcttctatac aaaaaacagg tcacaagcct gcaacaggaa ggcgctgctt iccatggacc ccaccataca gcaggggatc ttaagatctt tgaagattit tgatgtctac catgttctac actgggccat gactgctctg catccaacat cagctccccc cggcccacct tgtgaactcc ccaaaggcct aatccaacgt tctggaccct taactqggta ccttctactg ggccttccac tcatccgcac actccgttac ttgtgcagat agcccgggtc accCtgtagc ccgctgcatc ttatcaagtt cctaaaccgc gtgtctcagc caaaaatgcg tggacaaagt cacagacctg qqqtCctgtc cttctttttt gcccccacct caactattac tcgccagcgg cttcttcagc tqgaagacct ggaqcgqaac ttctaaagat tctgggcaag gacagctccg gccctgatcc acttcgcctt acaggtctcc tcacgcctgt aatccaacac tcgagaccag cctggccaac ccgagagtgg tggcatgcac gcttgaaccc gggaggcaga gggtgacaga ctctgtctcc tttgttaact cagtaaaaaa cgggacgagg cccatcaaga ctaggttaca taccccagga ctcctgtact aacggcctac actgtgggaa aacttttgtc ctctgcccca aacgttactc agcggtctta gcccagtcct ccaciggtca tacctggcta ggctgtgaga tcgtgcccag tctgtcttca ctggccctgg aagccccagg cacactgggt atcttggagt atgtgctgt aatgcataca ttcatgctac ctgctgttct ttctccggtc tggctgccca gttttcggca aacggctccc aagaacgagg aggactgcac attttgtggt tttgagaggc atggtgaaac ctgtcatccc ggttgcagtg aacaaaac aaaaaaaaaa atgacgaggc acagaagctg tcgtggtggg actctactgg tcaacatctt agtgccccac aaaacgagtt tgccaggggt gtttcctCct caccggcgct ttgacagcct ggtat tggat cctttgtcct catcgggca aagtgccaat ggctgatgtg atctgcaaat gccaatgcgt acatccctac cattggcatt atattgacca tcaagtgctg tcatgatcgc tcatqcgaaa ttgggaagct qca tcccggg tcatgacctc tgtgtgtgga tggaccggcc cgcccccgga cccaccccca aaaaaaaggt tgaggcgggc ctccgtctct ctacgggaag ccagtcaaat cacagatgtc atctgctgcg gattgtggcc tggttgtatg ggcctactgt ggcatggggg cagctgcatc ctgtccagca accccaggtg tgtgtgtcct ctcacagact gttggggaag accctggaat atgacqgtga cccctctgct ccagctctgg cccagggatc accaatgaca caatgcccga gacatcagtg tcttgtggct gtggqacaga cctcctcatc tgcattgcct accccagtat gtgctctggg aaatacatca tgcaacccca cgtcttccag ggctactcat ctatggqqtc ctggggctct cctcgctgga gcctttgcct cttcccctta atctctgcct tggagccctc atcctgaccc caagctcaga ggagtgcaga cctctggtgt ctggaaaaat catctacqgg aaqaatttct cattgtcagg gtggtcgtcc gctggtggtc ggaggcgtgg gctgqgtaaa gactttaaga catcctgggg gcctatgtca cacgctcttc ctctqcttcc ctactacatg tccaagagcc caacaagaag aggaagaagt ccgtccagcc atccaacctc tttaggccag gcgccgtggc ggatcacctg agtcaggagt attaaaaata caaaaattag 120 180 240 300 360 420 480 540 600 660 720 780 640 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2251 sequence of transcript variant 24P4C12 v.7 (SEQ ID NO: 94) agctactcgg gaggctgagg caggagaatc agccgagatc gcgccactgc actccaacct aaacaaacaa aaagatttta ttaaagatat Table LIII. Nucaotide sequence alignment of 24P4C12v.1 v.1 (SEQ ID 24P4C12 v.7 (SEQ ID NO: 96).

NO: 95) and Score =1358 bits (706), Expect =0.Oldentities 706/706 (100%) Strand Plus/ Plus 24P4C12v.l: I 24P4Cl2v.7: 1 gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat acgacccctcctttcgaggccccatcaagaacagaagctgcacagatgtcatctgctgcg 120 acgacccctcctttcgaggccccatcaaqaacagaagctgcacagatgtcatctgctgcg 120 24P4Cl2v.l: 24P4C12v.7: 24 P4 Cl2v. 1: 24P4C12v.7: 24P4Cl2v.l: 24P4Cl2v.7: 121 tcctcttcctgctcttcattctagttacatcgtggtggggattgtggcctggttgtatg 180 121 tcctcttcctgctcttcattctaggttacatcgtggtggggattgtggcctggttgtatg 180 181 gagacccccqgcaagtcctctaccccaggaactctactggggcctactgtggcatggggg 240 181 gagacccccggcaagtcctctaccccagqaactctactggggcctactgtggcatggggg 240 2424C12v.l: 241 agaacaaagataagccgtatctcctgtacttcaacatcttcagctgcatcctgtccagca 300 00 00 24P4C1.2v.7: 241 24P4C12v.l: 301 24P4C12v.7: 301 24P4Cl2v.l: 361 24P4C12v.7: 361 24P4C12v.1: 421 24P4Cl2v.7: 421 24P4C12v.l: 481 24P4C12v.7; 481 24P4C12v.l: 541 24P4C12v.7: 541 24P4C12v.l: 601 24P4C12v.7: 601 24P4C12v.l: 661 24P4Cl2v.7: 661 agaacaaagataagccgtatctcctgtacttcaacatcttcagctgcatcctgtccagca aca tca tctCagt tgctgagaacggcctacagtgccccacaccccaggtgtgtgtg tcct I1 IIII IIII IIII I IllIllI I I11 I I 1 1 I tI 1 1 1 1 11 1 1 1 1IIII11 IIII IIII acatcatctcagttgctgagaacggcctacagtgccccacaccccaggtgtqtgtgtcct cctgcccggaggacccatggactgtgggaaaaaacgagt tctcacagactgttggggaag cctgcccggaggacccatggactgtgggaaaaaacgagttctcacagactgttggggaag tcttctatacaaaaaacaggaacttttgtctgccaggggtaccctggaatatgacggtga tc ttctatacaaaaaacaggaacttttgtctgccaggggtaccctggaatatgacggtga tcacaagcctgcaacaggaactctgccccagtttcctcctcccctctgctccagctctgg tcacaagcctgcaacaggaactctgccccagtttcctcctcccctctgctccagctctgg ggcgctgctttccatggaccaacgttactccaccggcgctcccagggatcaccaatgaca ggcgctgctttccatggaccaacgttactccaccgqcgctcccagggatcaccaatgaca ccaccatacagcaggggatcagcggtcttattgaCagcctcaatgcccgagacatcagtg ccaccatacagcaggggatcagcggtcttattgacagcctcaatgcccgagacatcagtg ttaagatctttgaagattttqcccagtcctggtattggattcttgt 706 ttaagatctttgaagatttrgcccagtcctggtattggattcttgt 706 Score 2971 bits (1545), Expect O.Oldentities 1545/1545 (100%) Strand PlusI Plus 24P4Cl2v.l: 1043 24P4Cl2v. 7: 24 P4C12v. 1: 24P4Cl2v.7: 24P4C12v.1: 24P4C12v. 7: 24P4C12v. 1: 24P4Cl2v. 7: 24P4Cl2v. 1: 24P4Cl2v. 7: 24P4Cl2v. 1: 24P4Cl2v.7: 707 1103 767 1163 827 1223 887 1263 947 1343 1007 ggctgtgggacagatgatgtctaccatgttctacccactggtcacctttgtcctcctcct 1102 ggctgtgggacagatgatgtctaccatgttctacccactggtcacctttgtcctcctcct 766 catctgcattgcctactgggccatgactgctctgtacctggctacatcggggcaacccca 1162 11111111111111111111 111111111111111111111 1 1111111 111111111 11 catctgcattgcctactggccatgactgctctgtacctggctacatcgggqcaacccca 826 gtatgtgctctgggcatccaacatcagctcccccggctgtgagaaagtgccaataaatac 1222 gtatgtgctctgggcatccaacatcagctcccccggctgtgagaaagtgccaataaatac 886 atcatgcaaccccacggcccaccttgtgaactcctcgtgcccagggctgatgtgcgtctt 1282 atcatgcaaccccacgqcccaccttqtgaactcctcgtgcccagggctqatgtgcgtctt 946 ccagggctactcatccaaaggcctaatccaacgttctgtcttcaatctgcaaatctatgg 1342 ccagggctactcatccaaaggcctaatcczaacgttctgtcttcaatctgcaaatctatg 1006 ggtcctggggctcttctggacccttaactgggtactggccctgggccaatgcgtcctcgc 1402 ggtcctggggctcttctggacccttaactgggtactggccctgggccaatgcgtcctcgc 1066 24P4C12v.1: 24P4C12v.1t 24 P4C12v. 1: 24P4Cl2v.7: 24P4C12v. 1: 24P4Cl2v. 7: 24P4Cl2v.l: 24P4C12v.7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4C12v.7: 24P4C12v.1: 24P4C12v.7: 24P4C12v. 1: 24P4Cl2v.7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4C12v.7; 24P4C12v. 1: 24P4C12v.7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v.1: 24P4C12v.7: 1403 1067 1463 1127 1523 1187 1583 1241 1643 1307 1703 1367 1763 1427 1823 1487 1883 1547 1943 1607 2003 1667 2063 1727 2123 1787 2183 1847 t qqcc tttgcctccttc tactgggcc ttccacaagccccagga catccctacct tccc ItI ill tII IIIIII 111111 ii1111111 111111111111111 t tggagcctttgcctccttctactgggccttccacaagccccaggacatccctaccttccc cttaatctctgccttcatccgcacactccgttaccacactgggtcattggcatttggagc 1111 IIIIII111111 i 111111111 i i 11111111 t111111111 l11111111 cttaatctctgccttcatccgcacactccgttaccacactgggtcattggcatttggagc cctcatcctgacccttgtgcagatagcccgggtcatcttqgagtatattgaccacaagct cctca tcctgaccct tgtgcaga tagcccgggtcatcttggagta ta ttgaccacaagct cagaggagtgagaacctgtagcccctgcatcatgtgctgtttcaagtgctgcctctg cagaggagtgcgaaccctgtagcccgctgcatcatgtgctgtttcaagtgctgcctctg gtgtctggaaaaatttatcaagttcctaaaccgcaatgcatacatcatgatcgccatcta gtgtctggaaaaatttatcaagttcctaaaccqcaatgcatacatcatgatcgccatcta cgggaagaatttctgtgtctcagccaaaaatgcgttcatgctactcatgcgaaacattgt cgggaagaatttctgtgtctcagccaaaaatgcg ttcatgctactcatgcgaaacattgt cagggtggtcgtcctggacaaagtcacagacctgctgctgttctttgggaaqctgctggt cagggtggtcgtcctggacaaagtcacagacctgctqctgttctttgggaagctgctggt ggtcggaggcgtgggggtcctgtccttcttttttttctccggjtcgcatcccqgqctggg ggtcgqaggcgtgggggtcctgtccttcttttttttctccggtcgcatctcggggctggg iaaagactttaagagcccccacctcaactattactggctgcccatcatgacctccatcct taaagactttaagagcccccacctcaactattactggctgcccatcatgacctccatcct gggggcctatgtcatcgccagcggcttcttcagcgttttcggcatgtgtgtggacaCgCt gggggcctatgtcatcgccagcggcttcttcagcgttttcggcatgtgtqtqgacacgct cttcctctgcttcctggaagacctggagcggaacaacggctccctggaccggccctacta cttcctctgcttcctggaagacctggagcggaacaacggctccctggaccggccctacta catgtccaagagccttctaaagattctgggcaagaagaacgaggcgcccccggacaacaa catgtccaagagccttctaaagattctgggcaagaagaacgaggcgcccccggacaacoa gaagaqgaagaagtgacagctccggccctgatecaggactgcaccccacccccaccgtcc gaagaggaagaagtgacagctccqgccctgatccaggactgcaccccacccccaccgtcc agccatccaacctcacttcgccttacaggtctccattttgtggtaaaaaaaggttttagg agccatccaacctcacttcgccttacaggtctccattttgtggtaaaaaaaggttttagg 1462 1126 1522 1186 1582 1246 1642 1306 1702 1366 1762 1426 1822 1486 1882 1546 1942 1606 2002 1666 2062 1726 2122 1786 2182 1846 2242 1906 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4Cl2v.7: 24P4C1Zv. 1: 24P4C12v.7: 24P4C12v. 1: 24P4C12v. 7: 24P4C12v. 1: 24P4Cl2v. 7: 2243 ccaggcgccgtggctcacgcctgtaatccaacactttgagaggctgaggcgggcggatca 2302 1907 ccaggcgccgtggctcaCgCCtgtaatCCaacaCttgagaggctgaggcgggcggatca 1966 2303 cctgagtcaggagttcgagaccagcctggccaacatggtgaaacctccgtctctattaaa 2362 1967 cctgagtcaggagttcgagaccegcctggccaacatggtgaaacctccgtctctattaaa 2026 2363 aatacaaaaattagccgaqagtggtggcatgcacctgtcatcccagctactcgggaggct 2422 2027 aatacaaaaattagccgagagtggtggcatgcacctgtcatcccagctactcgggaggct 2086 2423 gaggcaggagaatcgcttgaacccgggaggcagaggttgcagtgagccgagatcgcgcc-a II IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Ill l i Iii I I I I Ii 1 1 1 1 1 1 1 1 11 1 1 1 1 1 I I 2087 gaggcaggagaatcgcttgaacccgggaggcagaggttgcagtgagccgagatcgcgcca 2483 ctgcactccaacctgggtgacaqactctgtctccaaaacaaaacaaacaaacaaaaagat 2147 ctgcactccaacctgggtgacagactctgtctccaaaacaaaacaaacaaacaaaaagat 2543 tttattaaagatattttgttaactcagtaaaaaaaaaaaaaaaaa 2587 2207 tttattaaagatattttgttaactcagtaaaaaaaaaaaaaaaaa 2251 2402 2146 2542 2206 Table LIV.

.MGGKQRDEDD

PRQVLYPRNS

PEDPWTVGQ4 C FPWT NVT PP

STMFYPLVTF

HLVNSSCPGL

YWAEFiKPQD1

VARCIMCCE'K

KVTDLLLFFG

SGFFSVF'GMC

Peptide sequences of protein coded b~y 24P4C12 v.7 (SEQ F.AYGRPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIV VGIVAWLYGD rcAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQC PTPQ)VCVSSC EFSQTVGEVF YTJKNRNFCLP GVPWNMTVIT SLOQEL.CPSF LLPSAPALGIR ALPGITNDTT IQQGISGLID SLNARDISV< IFEDFAQSWY WILVAVGQMM VLLLICIAYW AMTALYLhTS GOPOYVLWAS NISSPGCEKV PINTSCNPTA MCVFQGYSSK GLIORSVFNL QIYGVLGLEW TLNWVLALGQ CVLAGAFASF PTFPLISAFI RTLRYHTGSL AFGALILTLV QIARVILEYI DHKLRGVQNP CCLWICLEKFI KE'LN1RNAYIM IAIYGKNFCV SA1KNAFMLLM RNIV'JTVVLD KLLVVGGVGV LSFFFFSGRI PGLGKDFXSP HLNYYWLPIM TSILGAYVIA VDTLELCFLE DLERNNGSLD RPYY14SKSLL KILGKKI4EAP PONKKPXI( ID NO: 97) 120 180 240 300 360 420 480 540 598 Table LV. Amino acid sequence alignment of 24P4C12v. I v. I (SEQ ID NO: 98) and 24P4CI2 v. 7 (SEQ ED NO: 99).

Score =1195 bits (3091), Expect .OIldentities 598/710 Positives 598/710 Caps 112/710 24P4C12v.1- I.

24P4Cl2v.7: I 24P4C12v.1: 6 24P4Cl2v.7 6 24P4C12v.1: I 24P4C12v.7: 1 24P4C12v.1: 1 24P4C12v.7: 1 24P4C12v.1: 2

NGGKQRDEDDEAYGKPVKYDSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD

MGGK0RDEDDEAYGKPVYDPSF1RGPIKNRSCTDVICCVLFLLFrLGYIVVGIVAWLYGD

MGGQRDEDDQSTAYKGENDPYLINSCDICLSSNISVAGLQCIVVVSSCY

PRQVLYPRNSTGAYCGNGNKDPYLLYFNIFSCILSSNIISVAENGL4QCP>TPQVCVSSC PRQVLYPRNSTGAYCGMGENKDKPYLLYFwrFSCILSSNIISVAENGLOCPTPQVCVSSC PRQVOPRGNSACvGEVFKLYTIIFLPGVRILSNITSLQENLCPLPSAPALGR PEDPWTVGKNEFSOTVGEVFYTKI4RNFCLPGVPWNMTVITSLOQELCPSFLLPSAPALGR PEDPWTVGKNEFSQTVGEVFYTKNqRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR

CFPWTVNVTPPALPGENDTIIQGISLIDLGNRIVITSFELAQSWLLA

CFPWTNVTPPALPGITNDTTIQOGISGLIDSLNAP.DISVKIPEOFAQSWYWILVALGA

CFPWTNVTPPALPGITNDTTIQOGISGLIDSLNARDISVKIFEDASWY4ILVA-

VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS

24P4C12v.7: 235 24P4C12v.1: 301 24P4Cl2v./1: 236 24P4CI2v.1: 361 24P4Cl2v.7: 249 24P4CI2v.1: 421 24P4C12v.7: 309 24P4Cl2v.1: 481 24P4Cl2v.7: 369 24P4C12v.1: 541 24P4C12v.7: 429 24P4C12v.1; 601 24P4Cl2v.7: 489 24P4C12v.I: 661 24P4Cl2v.7: 549 AYQSVQETWLAALIVLAVLEAILLUL1LI FLRQRIRIAIALLKEASKAVGQMHSTMFYPLV 360

VGQMMSTMFYPLV

VGQHMSI14EYPLV 248 TFVLLLICIAY4AMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCN4PTAJILVNSSCP 420 TEVLLLICIAYWAMTALYLATSGPQYVLWASNISSPGCEpVPINTSCNPTAHLVNJSSCP TEVLLLICIAYW'AMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 308 GLMCVFPQGYSSXGLIQRSVFlJLQIYGVGLFWTLNWVLALGQCvLAGAFASFYWAFHKPQ 480

GUMCVFOGYSSKGLIRSVFLIYGVLGLETLWVLALGOCVAGAFASYWAFHKPQ

GU4CVFOGYSSKGLIQRSVFNLQIYGVLGLEVTLNWVj.ALGQCVI.AGA'ASFYWAFHKpQ .368 DIPTFPLrSAFIRTLRYHTGSLAFALILTLVIARVILEYIDHKLRGVNPVARCIMCC 540 DIPT FPLISAFITLRYHTGSLAFG1ALILTLVQ1ARVILEYI DHKLRGVQNPVARCIt4CC DIPTFPLISAFIRTLRYHiTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 428 FKCCLWCLEKFI!(FLNRNAYIMIAIYGKNFCVSA(NAFNLLMRNIVRVVVLDKVTDLLLF 600 EKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSACNAF?4LLMRNIVRVVVLDKVmLLLF FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAdLLKRNIVRVVVLDKVTDLLLF 488 FGKLLVVGGVGVLSFFFFSGRIPGI.GKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFG 660 FGKLLVVGGVGVLSFFFFSGRI PGLGKDSPHiLNYYWLPIlTSILAVIASGFF'SVEG FGKLLVVCGVGVLSFFFESGRXPGLGKDFKSPHLNYwLPIMTSILGAYVIASGFFSVFG 548 MCVDTLFLCFLEDLER14NGSLDRPYYM4SKSLLKILGKIQ4EAPPDNKKaKK 710 MCVDTLFLCFLEDLERNNGSLDRPYYMSKSLIKILGKKNAPP1IKKRKKi MCVDTLFLCFLEDLER1NGSLORPYYSKSLLKILGKK4EAPPDNKKikKK 598 Table LVI. Nueleotide sequence of transcript variant 24P4C12 v.8 (SEQ gagccatggg gggaaagcag cgglgacqagg atgacgaggc ctacgggaag ccagtcaaat acgacccctc ctttcgaggc cccatcaaga acagaagctg cacagatgtc atctgctgcg tcctcttcct gctcttcatt ctaggttaca tcgtggtggg gattgtggcc tggttgtatg gagacccccg gcaagtcctc tacecccagga actctactgg ggcctactgt ggcatgggqg agaacaaaga taagccgta t ctcctgtact tcaacatctt cagctgcatc ctgtccagca ocatcatcic agttgctqag aacggcctac agtgccccac accccaggtg tgtgtgtcct cctgcccgga ggacccatgg actgtgggaa aaaacgagtt ctcacagact gttggggaag tcttctatac aaaaaacagg aacttttgtc tgccaggggt accctggaat atgacggtga tcacaagcct gcaacaggaa ctctgcccc& gtttcctcct cccctctgct ccagctctgg ggcgctgctt tccatggacc aacgttactc caccggcgct cccagggatc accaatgaca ccaccataca gcaggggatc agcggtctta ttgacagcct caatgcccga gacatcagtg ttaagatctt tgaagatttt gcccagtcct ggtattggat tcttgttgcc ctqggggtgg ctctggtctt gagcctactg tttatcttgc ttctgcgcct ggtggctggg cccctggtgc tggtgctgat cctgqgagtg ctgggcgtgc tggcatacgg catctactac tgctgggagg agtaccgagt gctgcgggac aagggcgcct ccatctccca gctgggtttc accaccaacc tc-agtgccta ccagagcgtg caggagacct qgctggccqc cctgatcgtg ttqqcggtgc ttgaagccat cctgctgctg atgctcatct tcctgcggca gcggattcgt attgccatcg ccctcctgaa ggaggccagc aaggctgtgg gacagatgat gtctaccatg ttctacccac tggtcacctt tgtcctcctc ctcatctgca ttgcctactg ggccatgact gctctgtacc tggctacatc ggggcaaccc cagtatgtgc tctgggcatc caacatcagc tCCCCCggct gtgaqaaagt gccaataaat acatcatgca accccacggc ccaccttgtg aactcctcgt gcccagggct gatgtgcgte ttccagggct actcatccaa aggcctaatc caacgttctg tcttcaatct gcaaatctat ggggtcctgg ggctcttctg gacccttaac tgggtactgg ccctgggcca atgcgtcctc gctggagcct ttgcctcctt ctactgggcc ttccacaagc cccaggacat ccctaccttc cccttaatct CtgccttCat ccgcacactc Cqttaccaca ID NO: 100) 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1060 1140 1200 1260 1320 1380 1440 1500 1560 L620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 ctgggtcatt tggagtatat gctgtttcaa catacatcat tgctactcat tgttctttgg ccqgtcgcat tgcccatcat gggcctatgt tcctctgctt tgtccaagag agaggaagaa ggcatttgga gccctcatcc tgacccttgt tgaccacaag ctcagaggag tgcagaaccc gtgctgcctc tggtgtctgg aaaaatttat gatcgccatc tacgggaaga atttctgtgt Qcgaaacatt gtcagggtgg tcgtcctgga qaagctgctg gtggtcggag gcgtgggggt cccggggctg ggtaaagact ttaagagccc gaggaaceca ataaccccaa cgggtcatgt catcgccagc ggcttcttca gcgttttcgg cctggaagac ctggagcgga acaacggctc ccttctaaag attctgggca agaaqaacga gtgacagctc cggccctgat ccaggaCtgc gcagatagcc tgtagcccgc caagttccta cteagccaaa Caaagtcaca CCtgtCCttc ccacctcaac cttccagacc catgtgtgtg cctggaccgg ggCgcccccg &cccC8CCCC cgggtcatct tgcatcatgt aaccgcaatg aatgcgttca gacctgctgc ttttttttct tattactggc tccatcctgg gacacgctct ccctactaca gacaacaaga caccgtccaq ccatcaacc tcacttcgcc ttacaggtct ccattttgtg gtaaaaaaag gttttaggcc aggcgccgtg gctcacgcct-gtaatccaac actttgagag gctgaggcgg gcggatcacc tgagtcagga gttcgagacc agcctggcca acatggtgaa acctccgtct ctattaaaaa tacaaaaatt agccgagagt ggtggcatgc acctgtcatc ccagctactc gggaggctga ggcaggagaa tcgcttgaac ccgggaggca gaggttgcag tgagccgaga tcgcgccact gcactccaac ctgggtgaca gactctgtct ccaaaacaaa acaaacaaac aaaaagattt tattaaagat attttgttaa ctcagtaaaa aaaaaaaaaa aaa 2280 2340 2400 2460 2520 2580 2623 Table Nuclectide sequence alignment of 24P4C12v.1 v.1 (SEQ ID NO: 101) and 24P4C12 v.8 (SEQ ID NO; 102) Score 3715 bits (1932), Expect 0.Oldentities 1932/1932 (100%) Strand Plus/ Plus 24P4C12ir.1: I 24P4Cl2v.O: 1 24P4Cl2v.1: 61 24P4C12v.B: 61 24P4Cl2v.1: 121 24P4Cl2v.8: 121 24P4C12v.1: 191 24P4Cl2v.B: 181 24P4Cl2v.1: 241 24P4Cl2v.8: 241 24P4Cl2v.1: 301 24P4C12v.8: 301 24P4C12v.1; 361 24P4Cl2v.B: 361 24P4C12v.l: 421 24P4C12v.8: 421 24P4C12v.l: 491 24P4Cl2v.6: 461 24P4Cl2v.1: 541 24P4C12v.8: 541 24P4C12v.l: 601 24P4C12v.8: 601 gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat acgacccctcctttcgaggccccatcaagaacagaagctgcacagatgtcatctgctgcg 120 acgacccctcctttcgaggccccatcaagaacagaagctgcacagaLg tcatctgctgcg 120 tcctcttcctgctcttcattctaggttacatcgtggtggggattgtggcctggttgtatg 180 tcctcttcctgctcttcattctaggttacatcgtggtggggattgtggcctggttgtatg 180 gagacccccggcaagtcctctaccccaggaactctactggggcctactgtqgcatggggg 240 gagacccccggcaagtcctctaccccaggaactctactggggciactgtggcatggggg 240 agaacaaagataagccgtatctcctgtacttcaacatcttcagctgcatcctgtccagca 300 agaacaaagataagccgtatctcctgtacttcaacatcttcagctgcatcctgtccagca 300 acatcatctcagttgctgagaacggcctacagtgccccacaccccaggtgtgtgtgtcct 360 acatcatctcagttgctgagaacggcctacagtgcccacacccaggtgtgtgtgtcct 360 cctgcccggaggacccatggactgtgggaaaaaacgagttctcacagactgttggggaag 420 cctgcccggaggacccatggactgtgggaaaaaacgagttctcacagactgttggggaag 420 tcttctatacaaaaaacaggaacttttgtctgccagggtaccctggaatatgacggtga 460 tcttctatacaaaaaacaggaacttttgtctgccaggggtaccct9atatgacggtg 480 tcacaagcctgcaacaggaactctgccccagtttcctcctcccctctgctccagctctgg 540 tcacaagcctgcaacaggaactctgccccagtttectcctCccctctgetccagctctgg 540 ggcgctgctttccatggaccaacgttactccaccggcgctcccagggtctccaatgaca 600 gcgctgctttccatggaccaacgttactccaccggcgctcccagggatcaccatgac 600 ccaccatacagcaggggetcagcggtcttattgacagcctcaatgccCgagacatcagtg 660 ccaccatacagcaggggatcagcggtcttattgacagcctcaatgcccgagacatcagtg 660 00 00 24P4C12v. 1: 24P4Cl2v.8: 24P4C12v. 1: 24P4Cl2v.B: 24 P4Cl2v 1: 24P4C12v.8: 24P4C12v.l: 24P4C12v.B: 24P4C12v. 1: 24P4Cl2v.8: 24P4C12v. 1: 24P4C12v. 8: 24P4C12v. 1: 24P4Cl2v. 8: 24P4C12v. 1: 24P4Cl2v.8: 24P4Cl2v. 1: 24P4C12v. 8: 24P4C12v. 1: 24P4Cl2v. 8: 24P4C12v. 1: 24P4C12v. 8: 24P4Cl2v. 1: 24P4Cl2v. 8: 24P4C12v. 1: 24P4C12v. 8: 24P4C12v. 1: 24P4C12v. 8: 661 661 721 721 781 781 841 841 901 901 961 961 1021 1021 1081 1081 1141 1141 1201 1201 1261 1261 1321 1321 1381 1381 1441 1441 ttaagatctttgaagattttgcccagtcctggtattggattcttgttgccctgggggtgg 720 ttaegatctttgaagattttgcccagtcctggtattggattcttgttgccctgggggtgg 720 ctctggtctqagcctactgtttatCttgCttCtgcgCCtggtggCtgggccCctggtgc 780 ctctggtcttgagcctactgtttatcttgcttctgcgcctggtggctgggcccctggtgc 780 tggtgctgatcctgggagtgctgggcgtgctggcatacggcatctactactgctgggagg 840 .tggtgctgatcctgggagtgctgggcgtgctggcatacggcatctactactgctgggagg 840 agtaccgagtgctgcgggacaagggcqcctccatctcccagctggqtttcaccaccaacc 900 agtaccgagtgctgcgggacaagggcgcctccatctcccagctgggtttcaccaccaacc 900 tcagtgcctaccagagcgtgcaggagacctggc-tggccgccctgatcgtgttggcggtgc 960 tcagtgcctaccagagcgtgcaggagacctggctggccgccctgatCgtgttggcgg-gC 960 ttgaagccatcctgctgctgatgctcatcttcctgcggcagcggattcgtattgccatcg 1020 ttqaagccatcctgctgctgatgctcatcttcctgcggcagcggattcgtattgccatcg 1020 ccctcctgaaggaggccagcaaggctgtgggacagatgatgtctaccatgttctacccac 1080 ccctcctgaaggaggccagcaaggctgtgggacagatgattctaccatgttctacccac 1080 tggtcacctttgtcctcctcctcatctgcattgcctactgggccatgactgctctgtacc 1140 tggtcacctttgtcctcctcctcatctgcattgcctactgggccatgactgctctgtacc 1140 tggctacatcggggcaaccccagtatgtgttggcatccaacatcagctcccccggct 1200 tggctacatcggggcaaccccagtatgtgctctgggcatcaacatcagctcccccggct 1200 gtgagaaagtgccaataaataca tcatgcaaccccacggcccaccttgtgaactcctcgt 1260 gtgagaaagtgccaataaatacatcatgcaaccccacggcccaccttgtgaactcctcgt 1260 gcccaggqctgatgtgcgtcttccagggctactcatccaaaggcctaatccaacgttctg 1320 gcccagggctgatgtgcgtcttccagggctactcatccaaaggcctaatccaacgttctg 1320 tcttcaatctgcaaatctatggggtcctggggctcttctggacccttaactgggtactgg 1380 tcttcaatctgcaaatctatggggtcctgggctttctgacccttaactgggtactgg 1380 ccctgggccaatgcgicctcgctggagcctttgcctccttctactgggcttccacaagc 1440 *ccctgggccaatgcgtcctcgctggagcctttgcctcCttCtaCtgggccttccaa~gc 1440 ccaggacatccctaccttccccttaatctctgccttcatccgcacatccgttaccaca 1500 cccaggacatccctaccttccccttaatctctqccttcatccgcacactccgttaccaca 1500 00 00 24P4C12v. 1: 24P4Cl2v.8: 24 P4 C2v. 1: 24P4C12v. 8: 24 P4Cl2v. 1: 24P4C12v.8: 24P4Cl2v.l: 24P4Cl2v.8: 24P4Cl2v. 1: 24P4Cl2v.B: 24P4Cl2v.1: 24P4CI2v.8: 24P4Cl2v. 1: 24P4Cl2v.8: 1501 1501 1561 1561 1621 1621 1681 1681 1741 1741 1801 1801 1861 1861 ctgggtcattggcatttggagccctcatcctgacccttgtqcagatagcccgggtcatct ctgggtcattggcatttggagccctcatcctgacccttgtgcagatagcccgggtcatct tggagtatattgaccacaagctcagaggagtgcagaaccctgtagcccgctgcatcatgt tggagta tattgaccacaagctcagaggagtgcagaaccctgtagcccgctgcatcatgt gctgtttcaagtgctgcctctggtgtctggaaaaatttatcaagttcctaaaccgcaatg gctgtttcaagtgctgcCtctggtgtctggaaaaatttatcaagttcctaaaccgcaatg catacatcatgatcgccatctacgggaagaati&ctgtgtctcagccaaaaatgcgttca catacatcatgatcgccatctacgggaagaatttctgtgtctcagccaaaaatgcgttca tgctactcatgcgaaacattgtcagggtggtcgtcctggacaaagtcacagacctgctgc tgctactcatgcgaaacattgtcagggtggtcgtcctggacaaagtcacagacctgctgc tgttctttgggaagctgctggtggtcggaggcgtgggggtcctgtccttcttttttttct tgttctttgggaagctgctggtggtcggaggcgtgggggtcctgtccttcttttttttct ccggtcgcatcccggggctgggtaaagactttaagagccccacctcaactattactggc ccggtcgcatcccggggctgggtaaagactttaagagcccccacctcaactattactggc 1560 1560 1620 1620 1680 1680 1740 1740 1800 1800 1860 1860 1920 1920 24P4C12v.1: 1921 tgcccatcatga 1932 111111111111 24P4C12v.8: 1921 tgcccatcatga 1932 score 1263 bits (657), Expect 0.Oldentities 657/657 (100%) Strand Plus/ Plus 24P4C12v. 1: 1931 gacctccatcctgggggcctatgtcatcgccagcggcttcttcagcgttttcqgcatgtg 1990 24P4C12v. 8: 1967 gacctccatcctgggggcctatgtcatcgccagcggcttcttcagcgttttcggcatgtg 2026 24P4C12v. 1: 1991 tgtggacacgctcttcctctgcttcctggaagacctggagcggaacaacggctcctgga 2050 24P4C12v. 8: 2027 tgtggacacgctcttcctctgcttcctggaagacctggagcggaacaacggctccctgga 2086 24P4C12v. 1: 2051 ccggccctactacatgtccaagagccttctaaagattctgggcaagaagaacgaggcgcc 2110 24P4Cl2v. 8: 2087 ccggccctactacatgtccaagagccttctaaagattctgggcaagaagaacgaggcgcc 2146 24P4Cl2v. 1: 2111 cccggacaacaagaagaggaagaagtqacagctccggccctgatccaggactgcacccca 2170 24P4C12v. 8: 2147 cccggacaacaagaagaggaagaagtgacagctccggccctgatccaggactgcacccca 2206 24P4C12v. 1: 2171 ccccc-accgtccagccatccaacctcacttcgccttacaggtctccattttgtggtaaaa 2230 24P4C12v. 8: 2207 cccccaccgtccagccatccaacctcacttcgccttacaggtctccattttgtggtaaaa 2266 24P4Cl2v. 1: 2231 aaaggttttaggccaggcgccgtggctcacgcctgtaatccaacactttgagaggctgag 2290 24P412v9: 267aaaggttttaggccaggcgccgtggctcacgcctgtaatccaacactttgagaggctgag 2326 24P4C12v.8: 2267 24P4C12v.1: 24P4C12v. 8: 24P4C12v. 1: 24P4C12v. 6: 24P4C12v.1: 24P4C12v.8: 24P4C12v.1: 24P4C12v.B8: 24P4C12v. 1: 24P4C12v.8: 2291 gcgggcggatcacctgagtcaggagttcgagaccagcctggccaacatggtgaaacctcc 2350 2327 gcgggcggatcacctgagtcaggagttcgagaccagcctqgccaacatggtgaaacctcc 2386 2351 gtctctattaaaaatacaaaaattagccgagagtggtggcatgc-acctgtcatcccagct 2410 2387 gtctctattaaaaatacaaaaattagccgagagtggtggcatgcacctgtcatcccagct 2446 2411 actcgggaggctgaggcaggagaatcgcttgaacccgggaggcagaggttgcagtgagcc 2470 2447 actcggqaggctgaggcaggagaatcgcttgaacccgggaggcagaggttgcagtgaqgcc 2506 2471 gagatcgcgccactgcactccaacctgggtgacagactctgtctCcaaaacaaaacaaac 2530 2507 gagatcgcgccactgcactccaacctgggtgacaqactctgtctccaaaacaaaacaaac 2566 2531 aaacaaaaagattttattaaagatattttgttaactcagtaaaaaaaaaaaaaaaaa 2587 2567 aaacaaaaagattttattaaagatattttgttaactcagtaaaaaaaaaaaaaaaaa 2623 Table LVIII. Peptide, sequences of protein coded by 24P4CI2 v.6 (SEQ MGGKQRDEDD EAYGKPVKYD PSFRPPKNR SCTDVICCVL FLLFILGYIV VGIVAWLYGD PRQVL'YPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQC PTPQVCVSSC PEDPWTVGKN EFSQTVGEVF YTKNR1NFCLP GVPWNMTVIT SLQQELCPSF LLPSAPALGR CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAOSWY WILVALGVAL VLSLLF'ILLL RLVAGPLVLV LILGVLGVLA YGIYYCWEEY RVLROKGASI SQLGFLTNLS AYQSVQETWL AALIVLAVLE AILLILIF RQRIRIAIAL LKEASKAVGQ MMSTMFYPLV TFVLLLICIA YWAI4TALYLA TSGOPQYVLW ASNISSPGCE KVPINTSCNP TARLVNSSCP GLMCVFQGYS SKGLIQRSVF NLQIYGVLGL FWTLNWVLAL GQCVLAGAFA SFYWAFHKPQ DIFTFPLISA FIRTLRYHTG SLAFGALILT LVQIAP.VILE YIDHKLRGVQ NPVARCII4CC FKCCLWCLEK FIKFLNRNAY IMIAIYGKNF CVSA1KNAFl4L LMRI4IVRVVV LDKVTDLLLF FGKLLWVGGV GVLSFI'FFSG RI PGLrI(OFK SPHLNYYW4LP IMRNPITPTG HVFQTSILGA YVIASGFFSV FGMCVDTLFL CFLEDLERNN GSLDRPYYMS KSLLKI LGKK NEAPPDNCK

KK

ID NO: 103) 120 180 240 300 360 420 480 540 600 660 720 722 Table LIX. Rmino acid sequence alignment of 24P4Cl2v.2. v.1 (SEQ IDNO: 104) and 24P4C12 v.8 (SEQIDNO: 105) Score 1438 bits (3722), Expect 0.Oldentities 710/722 Positives 710/722 Gaps 12/722 (it) 24P4C12v.1: 24P4C12v.8: 24P4C12v.1; 24P4C12v.8: 24P4C12v.1: 24P4C12v.8: 24P4C12v. 1: 24P4Cl2v. 8: 24P4C12v. 1: MGGKOREDAYGKPV1KYDPSFRGPI1ORSCTDVICCVLFLLFILGYIVVGIVAWLYGD

MGGKQRDEDDEAYGKPVKDPSFRGPIKNRSCTDVICCVLFLLFILGYIVGIVAWLYGD

NGGKQTDEDDF.AYGKPVKYDPSFRGPIKlNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD PRQVLYPRNSTGAYCGtGENKDKPYLLYFNIFSCILSSNIISVAENGLQ)CPTPQVCVSSC PRQVLYPRNSTGAYCGM4GENKDKPYLLYflIFSCILSSNIISVAENGLQCPTPQVCVSSC PRQVLYPRNSTGAYCGMGENKDKPYLLYFlIFSCILSSNIISVAENGLQCPTPQVCVSSC PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPW1NTVITSLQQELCPSFrLLPSAPALGR PEDPWTVGTKNE FSQTVGEVEYTKWRNFCLPGVPWN4TVITStQQELCPS FLLPSAPALGR

PEDPWTVGKNEFSQTVGEVFYKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR

CFPWTNVTPPALPGITNDTTIOOGISGLI DSLNARDI SVKIFEDE'AQSWYI4ILVALGVAL CFPWTNVTPPALPGITNrr1QOGISGLIDSLNADISVKIFEDFAQSWYWqILVALGVAL

CFPWYVTPPALPGITDTTQQGISLIDSLARDISVKIFEFASWYWILVALGVAL

VLSLLFILLLRLVAGPLVLVLILGLGVLAYGIYYCWEEYRVLRDKGASISQLGF-rTNLS

VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS

00 00 24P4Cl2v. 8: 241 VLSLLFILLLRLVAGPLVLVLTLGVLGVLAYGIYYCW4EEYRVLRDKGASISOLGFTTNLS 300 24P4C12v.1: 301 24P4C12v.8: 301 AYQSVQETWLAALIVLAVLEAILLLMLIFLRORIRIAIALLKASKAVGQMNSTMFYPLV 360 AYQSVQETWLAALIVLAVLEAILLLMLI FLRQRIRIAIALLKEASKAVGQMMSTMFYPLV AYQSVQETWLAALIVLAVLEAI LLU4LI FLRQRIRIAIALLKEASKAVGQt4MSTMFXPLV 360 TEVLLLICIAYWAMTALYLATSGQPQYVLWASISSPGCEKVPINTSCNTALSSCP 420 TIVLLLICIAYWA±4TALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTA4LVNSSCP TFVLLLICIAYWMTAYLATSGPQVLWASNISSPGCPINTSCNPAHLJNSSCP 420 24P4C1.2v. 1: 24P4Cl2v.B: 24P4C12v.1: 421 24P4C12v.8: 421 24P4C12v.1: 481 24P4C12v.8: 461 24P4C12v.1: 541 24P4C12v.8: 541 24P4C12v.I: 601 24P4C12v.8: 601 24P4Cl2v.1: 649 24P4C12v.8: 661 24P4C12v.l: 709 GLMCVFQGYSSKGLIQRSVFLQIYGVLGLFTLWVLALG7QCVLAAFASFYWAFRKPQ 480 GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWILALGQCVLAGAF-ASFYW1AFHKPO GLNCVF0GYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALOCVLAGAFASYAFHKPQ 480 DIPTFPLISAFIRTLRYHTGSLAFALILTLVIA(VILEYIDHKLRGVQNPVARCIMCC 540 DI PTFPLISAFIRTLRYHTGSLAFGALlLTLVQIARVILEYIDH1U.RGVQNPVARCIMCC DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 540 FKCCLWCLEKFIKFLNRNAYIMAIYGKFCVSANALL IVRVWVLDVDLLLF 600 FKCCLWCLEKFIKFLNNAYIMIAIYGKFCVSAKNAFMLI2RNIVR1VVLDKVTDLLLF FKCWLKIFNNYMAYKFCSKAKURIRVLKTLL 600 FGKLLVVGGVGVLSFFFSGRIPGLGKDKSPHLNYLPIM TSILGA 648 FGKLLWVGGVGVLSE'FFFSGRI PGLGKDFKSPHLNYY1QLPIM

TSILGA

FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNWLPIRNPITPTGHVFQTSILGA 660 YVIASGFFSVFMCVTLFLCFLEDLERNGSLDRPYYMSKSLLKIKNEAPPNK 708

YVIASGFFSVFGMCVDLFLCFLEDLERNNGSLDRPSKSLLKIGKKEAPPDNKKR

YVAGFVGCDLLFELRNSDPYSSLIGKFAPNK 720 KI( 710

KK

24P4C12v.8: 721 KK 722 Table LX. 1Ruceotide sequence of transcript variant 24P4C12 v.9 (SEQ MD NO: 106) gagccatggg gggaaagcag cgggacgagg acgacccctc ctttcgaggc cccatcaaga tcctcttcct gctcttcatt ctaggttaca gagacccccg gcaagtcc taccccagga agaacaaaga taagccgtat ctcctgtact acatcatctc agttgctgag aacggcctac cctgcccgga ggacccatgg actgtgggaa tcttctatac aaaaaadagg aacttttgtc tcacaagcct gcaacaggaa ctctgcccca ggcgctqctt tccatggacc aacgttactc ccaccataca gcaggggatc aqcggtctta ttaagatctt tgaagatttt gcccagtcct ctctggtctt gagcctactg tttatcttgc tggtgctgat cctqggagtg ctgggcgtgc agtaccgagt gctgcgggac aagggcgcct .tcagtgccta ccagagcgtg caggagacct ttgaagccat cctgctgctg atgctcatct ccctcctgaa ggaggccagc aaggctgtgg tggtcacctt tgtcctcctc ctcatctgca ctctgcccac gcagccagcc actcttggat ccggctgtga gaaagtgcca ataaatecat cctcgtgccc agggctgatg tgcgtcttcC gttctgtctt caatctgcaa atctatgggg tactggccct gggccaatgc gtcCtCgctg acaagccca gqacatccct accttcccct accacactgg gtcattggca tttggagccc tcatcttgga qtatattgac cacaagctca tcatgtgctg tttcaagtgc tgcctctggt gcaatgcata catcatgatc gccatctacg cgttcatgct actcatgcga aacattgtca tgctgctgtt ctttgggaag ctgctggtgg ttttctccgg tcgcatcccg gggctgggta actggctgcc catcatgacc tccatcctgg gcgttttcgg catgtgtgtg gacacgctct atgacgaggc ctacgggaag acagaagctg cacagatgtc tcgtggtggg gattgtgCC actctactgg ggcctactgt tcaacatctt cagctgcatc agtgccccac accccaggtg aaaacgagtt ctcacagact tgccaggggt accctggaat gtttcctcct CCCCtctgCt caccggcgct cc-cagggatc ttgacagcct caatqcccga ggtattggat tcttgttgcc ttctgcgcct ggtggctggg tggcatacgg cfltctactac ccatctccca gctgggtttc ggctggccgc cctgatcgtg tcctgcggca gcggattcgt gacagatgat gtctaccatg ttgcctactg ggccatgact atgtgctbtg ggcatccaac catgcaaccc cacggcccac 3,tctgctgcg tggttgtatg ;gcatggggg -tgtccagca tgtgtgtcct gttggggaag Btgacggtga ccagctctgg accaatgaca gacatcagtg Ctgggggtgg cccctggtgc tqctgggagg accaccaacc ttggcggtgc attgccatcg ttctacccac gctctgtatc atcagctccc cttgtgaact ctaatccaac cttaactggg tgggccttcc acactccqtt atagcccggg gcccgctgca ttcctaaacc gccaaaaatg gtcacagacc tccttctttt ctcaactatt ggcttcttca ctggagcgga 120 180 240 300 360 420 480 540 600 660 720 780 640 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 agggctacte tcctggggCt gagcctttgc taatctctgc tc-atcctgac gaggagtgr-a gtctggaaaa ggaagaattt gggtggtcgt tcggaggCgt aagactttaa gggcctatgt tcctctgctt atccaaaggc cttctggacc ctccttctac cttcatccgc ccttgtgcag gaacectgta atttatcaag ctgtgtctca cctggacaaa gggggtCCtg gagcccccac catcgccagc cctggaagac acaacggctc cctggaccgg ccctactaca tgtccaagag ccttctaaag attctgggca 2100 agaagaacga ggcgcccccg gacaacaaga agaggaagaa gtgacagctc cggccctgat 2160 ccaggactgc accccacccc caccgtccag ccatccaacc tcacttcgcc ttacaggtCt 2220 ccattttgtg gtaaaaaaag gttttaggcc aggcgccgtg gctcacgcct gtaatccaac 2280 accttgagaq gctgaggcgg gcggatcacc tgagtCagga gttcgagacc agcctggcca 2340 acatggtgaa acctccgtct ctattaaaaa tacaaaaatt agccgaqagt ggtggcatqc 2400 acctgtcatc ccagctactc gggaggctga ggcaggagaa tcgcttgaac ccgggaggca 2460 gaggttgcag tgagccgaga tcgcgccact gcactcc-aac ctgggtgaca gactctgtct 2520 ccaaaacaaa acaaacaaac aaaaagattt tattaaagat attttgttaa ctcagtaaaa 2580 aaaaaaaaaa aaa 2593 Tabl.e LXI. Nucleotide fequonoe alignment of 24P4C12v.l v.1 (SEQ ID NO: 107) and 24P4C12 v.9 (SEQ ID NO: 108) Score 2188 bits (1138), Expect 0.Oldentities 1138/1138 (100%) Strand Plus/ Plus 24P4Cl2v.l: I 24P4C12v.9; 1 24P4C12v.1: 6: 24P4C12v.9: 6: 24P4Cl2v.1: 1: 24P4C12v.9, 1: 24P4Cl2v.1: 11 24P4C12v.9; 11 24P4C12v.l: 2Z 24P4C12v.9: 2.

24P4Cl2v.l: 31 24P4C12v.9: 34 24P4C12v.1: 3 24P4C12v.9: 3 24P4CI2v.1: 4 24P4Cl2v.9: 4 24P4C12v.1: 4 24P4Cl2v.9: 4 24P4C12v.1: 5 24P4C12v.9: 5 24P4CI2v.1: 6 gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat gagccatggggggaaagcagcgggacgaggatgacgaggcctacgggaagccagtcaaat acgacccctcctttcgaggccccatcaagaacagaagctgcacagatgtcatctgctgcg acgacccctcctttcgaggccccatcaagaacagaagctgcacagatgtcatctgctgcg tcctcttcctgctcttcattctaggttacatcgtggtggggattgtggcetggttgtatg tcctcttcctgctcttcattctaqttacatcgtggtggggattgtggcctggttgtatg gagacccccggcaagtcctctaccccaggaactctactggggcctactgtggeatgggqg gagacccccggcaagtcctctaccccaggaactctactgggqcctactgtggcatggg agaacaaagataagccgtatctcctgtacttcaacatcttcagctgcatcctgtccagca agaacaaagataagccgtatctCctgtacttcaacatcttcagctgcatcctgtccagca acatcatctcagttgctgagaacggcctacagtgccccacaccccagg tgtgtgtgtcct acatcatctcagttgctgagaacggcctacagtgccccacaccccaggtgtgtgtgtcct cctgcccggaggacccatggactgtgggaaaaaacgagttctcacagactgttggggaag cctgcccggaggacccatggactgtgggaaaaaacgagttctcacagactgttggggaag tcttctatacaaaaaacaggaacttttgtctccaggggtaccctggaatatgacggtga tcttctatacaaaaaacaggaacttttgtctgccaggggtaccctggaatatgacggtga tcacaagcctgcaacaggaactctgccccagtttcctcctcccctctgctccagctcgg tcacaagcctgcaacaggaactctgccccagtttcctcctcccctctgctccagctctgg ggcgctgctttccatggacCaacgttactccaccggcgctcccagggatcaccaatgaca gqcgctgctttccatggaccaacgttactccaccggcgctcccagggatcaccaatgaca ccaccatacaqcaggggatcagcggtcttattgacagcctcaatgcccgagacatcagtg 0024P4C12v. 9: 601 ccaccatacagcaggggatcagcggtcttattgacagcctcaatgcccgagacatcagtg 660 rl24P4C12v. 1: 661 ttaagatctttgaagattttgccCagtcctggtattggattcttgttgccctgggggtgg 720 24F4C12v. 9: 661 ttaagatctttgaagattttgcccagtcctggtattggattcttgttgccctgggggtgg 720 00 24P4Cl2v. 1: 721 ctctggtcttgagcctactgtttatcttgcttctgcgcctggtggctgggcccctggtgc 760 24P4Cl2v. 9: 721 ctctggtcttgagcctactgtttatcttgcttctgcgcctggtggctgggcccctggtgc 780 00 24P4C12v. 1: 781 tggtgctgatcctgggtgctgggcgtgctggcatacggcatctactactgctgggagg 840 IND24P4C12v.'9: 781 tggtgctgatcctgggagtgctgggcgtgctggcatacggcatctactactgctgggagg 840 (N ~24P4C12v. 1: 841 agtaccgagtgctgcgggacaagggcgcctccatctcccagctgggtttcaccaccaacc 900 24P4Cl2v. 9: 841 agtaccgagtgctgcgggacaagggcctccatctcccagctgggtttcaccaccaacc 900 rl24P4C12v. 1: 901 tcggcacggggaggctgcgcgctacttgcgg 960 24P4C12v. 9: 901 tcggcacggggaggctgcgcgctac~tgcgg 960 24P4Cl2v.l: 961 ttgaagccatcctgctgctgatgctcatcttcctgcggcagcggattcgtattgccatcg 1020 24P4C12v. 9: 961 ttgaagccatcctgctgctgatgctcatcttcctgcggcagcggattcgtattgccatcg 1020 24.P4C12v.1: 1021 cctcgaggcacagttggcgtagcacttcaca 1080 24 P4Cl2v.9: 1021 cctcgaggcacagttggcgtagcacttcaca 1080 24P4Cl2v. 1: 1081 tggtcacctttgtcctcetcctcatctgcattgcctactgggccatgactgctctgta 1138 24P4C12v. 9: 1081 tggtcacctttgtOCtcctcctcatctgcattgcctactgggccatgactgctctgta 1138 Score 2738 bits (1424), Expect 0.Oldentities- 1424/1424 (100%) Strand -Plus/ Plus 24P4C12v. 1: 1164 taggttgctcaacgtcccgttaaatcatatc 1223 24P4C12v. 9: 1170 taggttgctcaacgtcccgttaaatcatatc 1229 24P4C12v. 1: 1224 tcatgcaacccacggcccaccttgtgaactcctcgtgcccagggctgatgtgcgtcttc 1283 24P4C12v. 9: 1230 tctcacccgcacttactcctccggtagggct 1289 24P4C12v. 1: 1284 cagggctactcatccaaagcctaatccaacgttctgtcttcaatctgcaaatctatggg 1343 24P4C12v. 9: 1290 caggtactcatccaaaqgcctaatccaacgttctgtcttcaatctgcaaatctatggg 1349 24P4Cl2v.l1: 1344 gtcggccttgcctatggatgctgcatctccc 1403 24P4C12v. 9: 1350 gtcggccttgcctatggatgctgcatctccc 1409 24P4C12v. 1: 1404 gggctgcctcatgctcccaccagctctctcC 1463 24P4C12v. 9: 1410 ggagcctttgcctccttctactgggccttccacaagccecaggacatccctaccttcccc 1469 24P4C12v. 1: 24P4c12v. 9: 24P4Cl2v. 1: 24P4cl2v. 9: 24P4Cl2v. 1: 24P4C12v. 9: 24P4C12v. 1: 24P4C12v. 9: 24P4C12v. 1: 24P4Cl2v. 9: 24 P4Cl2v. 1: 24P4Cl2v. 9: 24 P4Cl2v.1: 24P4Cl2v. 9: 24 P4Cl2v 1: 24P4C12v.9: 24P4Cl2v. 1: 24P4Cl2v. 9: 24P4C12v. 1: 24P4Cl2v. 9: 24P4Cl2v.1: 24P4Cl2v.9: 24P4Cl2v.1: 24P4Cl2v.9: 1464 1470 1524 1530 1584 1590 1644 1650 1704 1710 1764 1770 1824 1830 1884 1890 1944 1950 2004 2010 2064 2070 2124 2130 ttaatctctgccttcatccgcacactccgttaccacactgggtcattggcatttggagcc I ttaatctctgccttcatccgcacactccgttaccacactgggtcattggcatttggagcc I ctcatcctgacccttgtgcagatagcccgggtcatcttggagtatattgaccacagctc 1 ctcatcctgacccttgtgcagatagcccgggtcatcttggagtatattgaccacaagctc1 agaggagtgcagaaccctgtagcccgctgcatcatgtgctgtttcaagtgctgcctctgg

I

agaggagtgcagaaccctgtagcccgctgcatcatgtgctgtttcaagtgctgcctctgg1 tgtctggaaaaatttatcaagttcctaaaccgcaatgcatactctgatcgccatctac tgtctggaaaaatttatcaagttcctaaaccgcaatgcatacatcatgatcgccatctac gggaagaatttctgtgtctcagccaaaaatgcgttcatgctactcatgcgaaacattgtc gggaagaatttctgtgtctcagccaaaaatgcgttcatgctactcatgcgaaacattgtc agggtggtcgtcctggacaaagtcacagacctgctgctgttctttgggaagctgctggtg agggtggtcgtcctggacaaagtcacagacctgctgctgttctttgggaagctgctggtg gtcgqaggcgtgggggtcctgtccttcttttttttctccggtcgcatcccgqgggctgggt gtcggaggcgtgggggtcctgtccttcttttttttctccggtcgcatcccggggctgggt aaagactttaagagcccccacctcaactattactggctgcccatcatgacctccatcctg aaagactttaagagcccccacctcaactattactggctgcccatcatgacctccatcctg ggggcctatgtcatcgccagcggcttcttoagcgttttcggcatgtgtgtggacacgctc ggggcctatgtcatcgccagcggcttcttcagcgttttcggcatgtgtgtggacacgctc ttcctctgcttectggaagacctggagcggaacaacggctccctggaccggccctactac ttcctctgcttcctggaagacctggagcgqgaacaacggctccctggaccggccctactac atgtccaagagccttctaaagattctgggcaagaagaacgaggcgcccccggacaacaag atgtccaagagccttctaaagattctggcaagaagaacgaggcgcccccggacaacaag aagaggaagaagtgacagctccggccctgatccaggactgcaccccacccccaccgtcca aagaggaagaagtgacagctccggccctgatcaqgactgaccccacccccaccgtcca gccatccaacctcacttcgccttacaggtctccattttgtggtaaaaaaaggttttaggc gccatccaacctcacttcgccttacaggtctccattttgtggtaaaaaaaggttttaggc caggcgccgtggctcacgcctgtaatccaacactttgagaggctgaggcggcggatcac Icaggcgccgtggctcacgcctgtaatccaacactttgagaggctgaggcggcggatcac .523 .529 ~583 L589 1643 1649 1703 1709 1763 1769 1823 1829 1883 1889 1943 1949 2003 2009 2063 2069 2123 2129 2183 2189 2243 2249 2303 2309 24P4C12v.l: 2184 24P4C12v. 9: 24P4C12v. 1: 24P4C12v. 9: 2190 2244 2250 24P4CI2v. I: 24P4C12v. 9: 24P4C12v. 1: 24P4C12v. 9: 24P4C12v. I: 24P4C12v. 9: 24P4C12v. 1: 24P4C12v.9:.

24P4C12v. 1: 24P4C12v. 9; 2304 ctgagtcaggagttcqagaccagcctggccaacatggtgaaacctccgtctctattaaaa 2363 2310 ctgagtcaggagttcgagaccagcctggccaacatggtgaaacctccgtctctattaaaa 2369 2364 atacaaaaattagccgagagtggtqgcatgcacctgtcatcccagctactcgggaggctg 2423 2370 atacaaaaattagccgagagtggtggcatgcacctgtcatcccagctactcgggaggctg 2429 2424 aggcaggagaatcgcttgascccgggaggcagaggttgcagtgagccgagatcgcgccac 2483 2430 aggcaggaqaatcgcttgaacccgggaggcagaggttgcagtgagccgagatcgcgccac 2489 2484 tgcactccaacctgggtgacagactctgtctccaaaacaaaacaaacaaacaaaaagatt 2543 2490 tgcactccaacctgqtgacagactctgtctccaaaacaaaacaaacaaacaaaaagatt 2549 2544 ttattaaagatattttgttaactcagtaaaaaaaaaaaaaaaaa 2587 2550 ttattaadgatattttgttaactcagtaaaaaaaaaaaaaaaaa 2593 Table LXII. Peptide, sequences of HGGKQRDEDD EAYGKPVKYD PSFRGPIKNR PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN PEDPWTVGKN EFSQTVGEVE' YTKNRNFCLP CFPWTNVTPP ALPGITNDTT IQQGISGLID VLSLLFILLL RLVAGPLVLV LILGVLGVLA AYQSVQETWL AALIVLAVLE AILLLHLIFL TFVLLLICIA YWANTALYPL PTQPATLGYV CPGU4CVEVQG YSSKGLIQRS VFNLQIYGVL PQDIPTFPLI SAFIRTLRYH TGSLAFGALI CCFKCCLWCL EKFIKFLN&N AYIMIAIYG< LFFGKLLVVG GVGVLS FFFF SGRI PGLGKD FGMCVDTLFL CFLEDLERIW GSLDRPYY1IS Table LXIII. Amino acid sequence 24F4C12 v. 9 (SEQ ED NO: 111) Score 1424 bits (3686), Expect 705/713 Gaps 4/713 protein coded by 24P4C12 v.9 (SEQ SCTDVICCVL FLLFILGYIV VGIVAWLYGD IFSCILSSk4I ISVAENGLQC PTPQVCVSSC GVPWN14TVIT SLQOELCPSF LLPSAPPIIGR SU4ARDISVI( IFEDFAQSWY WILVALGVAL YGIY'YCWEEY RVLRDKGASI SQLGETTNLS RQRIRIAIAL LKEASKAVGQ k4NSTMFYPLV LWASNISSPG CEKVPINTSC NP'rAILVNSS GLEWTLNWVL ALGQCVLACA FASEYWAFHK LTLVQIARVI LEYIDHKLRG VQNPVARCIM NFCVSAKNAF !4LL4RNIVRV VVLDKVTDLL FKSP1ILNYYW LPIM1TSILGA YVIASGFFSV KSLLKILGKK NEAPPDNKKR KK ID NO.- 109) 120 180 240 300 360 420 480 540 600 660 712 alignment of 24P4Cl2v. I v. 1 (SEQED NO: 110) and .Oldentities 704/713 Positives= 24P4C12v.1: 1 24P4C12v.9: 1 24P4C12v.1: 6 24P4CI2v.9; 6 24P4CI2v.I: I 24P4C12v.9: I.

24P4C12v.l: 1 24P4C12v.9; 1 24P4Cl2v.l: 2 24P4C12v.9: 2 24P4C12v.1: 3 24P4C12v.9: 3 MGG!(QRDEDDEAYGKPVKYDPS FRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD

MGGKQRDEDDEAYGKPVKYIPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD

?4GGKQfDEDEAYGPKYD)PSFRGPKNRSCDVICCVLFLLILGYIVVGIVAWLYGD PRQVLYPRNSTGAYCG4GENlKDKPYLLYFNIFSCILSSNIISVAENGLOCPTPQVCVSSC PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLoCPTP0VCVSSC PEDPWTVGKNEFSOVGEVT1ONCLPGVPWNTVITSLQQELCPSFLLPSAPALGP, PEDPWTVGKNEFSQ'rVGEVFYT1ONRNFCLPGVPWNMT4VITSLQQELCPSFLLPSAPALGR PEDPWTVGKNFSQTVGEVFYTKNRNFCLPGVPWN4TVITSLQQELCPSFLLPSAPALGR

CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFASW'WILVALGVAL

CFPWTNVTPPALPGITNDTTIOGISGLIDSLIARDISVKI'EDFAQSWYhILVALGVAL CFPWTI4VTPPALPGITt4DTTIQQGISGLIDSLNARDISVKIEEDFAQSWYILVA.GVAL VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQljGFTTNLS VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISoQWF'rTNLS

VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQWGFTTNLS

AYOSVQETWLAALIVLAVLEAI LLLMLI FLRQRIRIAIALLKEASKAVGQHNSTMFYPLV AYQSVOETWLAALIVLAVLEAILLLMLIFLRORXRIAIALLKEASKVGQNNST4FYPLV AYQSVQETWLALIVLAVLEAILLI4LIFLRQRIRIAIALLKEASKAVGQNSTMFYPLV 24P4C12v. 1: 24P4Cl2v. 9: 24P4Cl2v. 1: 24P4C12v. 9: 24P4Cl2v. 1: 24P4C12v. 9: 24P4Cl2v.l: 24P4C12v.9: 24 P4Cl2v. 1: 24P4Cl2v.9: 24 P4Cl2v. 1: 24P4Cl2v.9: 361 TEVLLtICIAYWAMTALYLATSGQPQ YVLWASNISSPGCEKVPINTSCPTAHLVNS TEVLLLICIAYWA14TALY OP. YVLWASNISSPGCEI{VPINTSCNPTAIILVNS 361 TFVLLLICIAYWAMTALYPLPT-QPATLGYVLWASNISSPGCEKPINTSCNPTAHLVNS 418 SCPGL14CVFQGYSSKGLIORSVFlJLOIYGVLGLFWTLNWVIALGQCVLAGAFASFYWAFH

SCPGLMCVFOGYSSKGLIQ)RSVFNQIYGVLGLWTLNVLALGOCVLAGAFASFYWAFH

420 SCPGLMCVFQGYSSKGLIQRSVNLQIYGVLGLWTLNWVLAIAQCVLAGAFAS

E"YAFH

4-78 KPQDIPTFPLIAFIRTLRYHTSLAF'GALILTVOIARVILYID4KLRGVQNPVARCI KPODIPTFPLISAFIRTLRYHTGSLAFlGALI LTLVQIA1RVILEYIDHKLRGVQNPVARCI 480 KPODIPTFPLISAFIRTLRYHTGSAFGALILTLVIARVILEYIDHKLRGVQNPVARCI 538 MCCFKCCLWCLEKFKFLNRNAYIMIAIYGK SAK AE4LLRNIVRVVLDVDL MCCFKCCLWCLEKFIKFLN{NAYIMIAIYGKNFCVSAKN~AFMLI24RN

IVRVWVLDKVTDL

540 MCCFKCCLWCLEKFIKFLNRNAYIIAIYGKNCVsAkNAE4LLMRNIVRVVVLDKVTDL 598 LLFFGKLLVVGG;VGVLSFFFSGRIPGLKDFKSPLYYWLPIMTSIL.GAYVIASGFFS LLFF)GKLLVVGGVGVLSFFFFSGRIPGLGKDE1(SPHLNYYWLPIMTSILGAYVIASGFFS 600 LLFFGKLLVVGGVGVLSFFFFSGRIPGWGKDFKSPHLNYYLPIMTSILGAYVIASGFFS 658 VFGNCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGK1QEAPPDNKKRKK 710 VFGMCVDTLFLCFLEDLEPJ4NGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 660 VFGMCVDTLFLCFLEDLERNNGSLDRPYYtSKSLLKILGKKIEAPPDNKKRKK 712

Claims (9)

1. A composition comprising a substance that a) modulates the status of a protein of Figure 2, or. b) a molecule that is modulated by a protein of Figure 2, 00whrbthsttsoabldeoay raLueuOstmccaltaexrseaprtiofFgr2ismdltd w0rb h ttso ldeoay rat trs rsoahcRta xrse rti fFgr smdltd

2. A composition which comprises: 00 a) a peplide of Tables Vlll-XXI; INO b) a peptide of Tables XXII to &LV, or c) a peptide of Tables XLVI to XLIX 00 A composition whlic omprises a peptide region of at least 5amino acids of Figure 2 i any whole number increment up to the end of said peptide. wherein the amino acid position selected from: ria) an amino acid position havig a value greater tha 0.5 In the Hydrophidty, profile of Figure b) an amino acid position having a value lens than 0.5 In the Hydropathicity profile of Figure 6; c) an amino acid position having a value greater than 0.5 in the Percent Acessible Residues profile of Figure 7; d) an amino add position having a value greater than 0.5 In the Average Flexibility profile of Figure 8; e) an amino add position having a value greater than 0.5 in the Beta-turn profile of Figure 9; Sa combination of at least two of a) through e); g)a combination of at least three of a) through e); h) a combination of at least four of a) through or, ia combination of five of a) through e).

4. A composition of claim 3 further limited by a proviso that the at least 5 amino acids is not the entire amino acid sequence of Figure 2- A method of inhibitig growth of bladder, ovary, breastL uterus, or stomach cancer cells that express a protein of Figure 2Z the method comprising: administerfing to ft ellns mhe composition of claim 1.

6. A method of claim 5 wherein the administering step comprises administering to said cells an antibody or fragment thereof, either of whui specifically binds to a 24P4012 protein.

7. A mnethod of claim 5 wherein the administering step comprises adrninistein to said cells a 24P4C12 protein.

8. A method of claim 5 wherein the administering step comprises administerin to said cells a polynuicleotide comprising a coding sequence fo a 24P34C1 2 protein or comprising a polynucloide complementary to a coding sequence for a24P4C2 protein.

9. A method of claim 5 wherein Die administering step comprises adminste*n to said cells a ribozyme that cleaves a polynudleotide that encodes a protein of Figure Z 00 A method of claim 5 wherein the administering step comprises administering human T cells to said cancer cells, CK1 wherein said T cells specifically recognize a peptidle subsequence of a protein of Figure 2 while the subsequence is in the context of the particular HLA moleue. 00 11. A method of clim 5 wherein Vhe admninistering step comprises administering a vector that delivers a nucteotide tiat encodes a single chain monoclonal antibody, whereby the encoded single chain antibody is expressed intraceularly within the bladder, ovary, breast uterus, or stomach cancer cells that express a protein of Figure 2 00 N12. A method of generating a mammalian immune response in a mammal having a bladder, ovary, breast uterus, or stomach cancer, the response directed lo a protein of Figure 2, the imethod omVMisng: providing a mammal having a malignancy of a bladder, ovary, breast -uterus, or stomach tIssue; 00 exposing cells of the mammal's Immune system to a portion of a) a protein of Figure 2 and/or ci b) a nudleotide sequence that encodes said protein, whereby an Immiune response is generated to said protein.

13. A method of generating an immune respose of claim 12, said method comprising: providing a 24P4C1 2 protein that comprises at least one T cell or at least one B cell eptop; and, contacting the epitope with a miammalian immune system T cell or B cell respectively, whereby the T cell or B cell Is activated.

114. A method of dlaim 13 wherein the immune system call is a B cell, whereby the Induced B cell genierates antibodies tat specificaly bind to the 24134C12 protein. A method of claimn13 wherein the Immune system cell is a T cell that is a cytotoxic: T cell (CTI), whereby the activated CfIL kills an autologous cell that expresses the 24P34C1 2 protein. 16. A method of dam 3 wheren IM immune system cellIs aT cell thats ahelper T ca(HTL), whereby the activated M~ seciretes cytoldnes that facilitate the cytotodc activity of a cytotoxc T cell (CTIL) or the antibody-producing activity of a B cell. 17. A imethod for detecding, In a sape frombladder, ovary, breast uterus, or stomachtfissue, the presence of a 24P4C1 2 protein or a 24P4C1 2 pollynudeootide, comprising steps of~ contacting the sample with a substa of claim I that specifially binds to the 2MPCI2 protein or to the 24P4C1 2 pollynudeotlide, respectively; and, determining that thiere is a complex of the substanc with the 24P4C1 2 protein or the substanc with the 24P4C12 polynudeofide, respectively. 1s. A method of caim17 comprising steps of~ cointacting the sample with an antibody or fragmient thereof either of which specifically bind to the 24P4C1 2 protein; and, 00 determining that there is a complex of the antibody or fragment thereof and the 24P4C12 protein. l 19. A method of daim 17 further comprising a step of: .C taking the sample from a patient who has or who is suspected of having the cancer. 00 20. A method of daim 17 for detecting the presence of a protein of Figure 2 mRNA in a sample comprising: 0 producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using 24P4C12 polynudeotides as sense and antisense primers, wherein the 24P4C12 00 polynudeotides used as the sense and antisense primers serve to amplify a 24P4C12 cDNA; and, N detecting the presence of the amplified 24P4C12 cDNA. \O 21. A method of claim 17 for monitoring one or more 24P4C12 gene products in a biological sample from a patient who 00 has or who is suspected of having bladder, ovary, breast uterus, or stomach cancer, the method comprising: Sdetermining the status of one or more 24P4C12 gene products expressed by cells in a tissue sample from an Sindividual; comparing the status so determined to the status of one or more 24P4C12 gene products in a corresponding normal sample; and, identifying the presence of one or more aberrant gene products of 24P4C12 in the sample relative to the normal sample. 22. The method of claim 21 further comprising a step of determining if there are one or more elevated gene products of a 24P4C12 mRNA or a 24P4C12 protein, whereby the presence of one or more elevated gene products in the test sample relative to the normal tissue sample indicates the presence or status of a bladder, ovary, breast uterus, or stomach cancer. DATED this EIGHTH day of FEBRUARY 2008 Agensys, Inc. by Patent Attorneys for the Applicant: F.B. RICE CO.