AU2007216892A1 - Nucleic acids and corresponding proteins entitled 273P4B7 useful in treatment and detection of cancer - Google Patents

Description

AUSTRALIA

FB RICE CO Patent and Trade Mark Attorneys Patents Act 1990 AGENSYS, INC.

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Nucleic acids and corresponding proteins entitled 2 73P4B 7 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:b NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 273P4B7 C USEFUL IN TREATMENT AND DETECTION OF CANCER This is a divisional of AU 2003258269, the entire contents of which are incorporated rherein by reference.

FIELD OF THE INVENTION 00 NThe invention described herein relates to genes and their encoded proteins, termed 273P4B7 and variants thereof, expressed in certain cancers, and to diagnostic Sand therapeutic methods and compositions useful in the management of cancers that express 273P4B7.

BACKGROUND OF THE INVENTION Cancer is the second leading cause of human death next to coronary disease.

Worldwide, millions 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 over a half-million people annually, with over 1.2 million new cases diagnosed per year.

While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer 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 S30,000 men die annually of this disease-second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate ,IC cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical 0 castration and chemotherapy continue to be the main treatment modalities.

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

NI On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et S al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et 1996, Proc. Natl.

Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 1445c 51), STEAP (Hubert, et Proc Nail Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter at al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

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

00 00 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 adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence 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 C1 carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

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 carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent In men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 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 patterns 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 muscle-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 increased 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 7- cancer.

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

C1 cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per S100,000 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, 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 00 00 mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking 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 CKl surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice: 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 cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An 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 reconstruction 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 and/or tamoxifen may reduce the chance of 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.

SSurgery, 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 will be removed, especially in young women who wish to have children. In 1) 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.

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 S past 20 years, there has been a slight but significant decrease in mortality rates among men (about per year) while 00 rates have increased slightly among women.

SSurgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival andlor 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.

SUMMARY OF THE INVENTION The present invention relates to a gene, designated 273P4B7, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northem blot expression analysis of 273P487 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (Figure 2) and amino acid (Figure 2, and Figure 3) sequences of 273P487 are provided. The tissue-related profile of 273P4B7 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 273P487 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary to all or part of the 273P487 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 273P4B7-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 acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 273P4B7-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 273P4B7 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 273P4B7 genes, mRNAs, or to 273P487-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 273P487.

Recombinant DNA molecules containing 273P487 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 273P4B7 gene products are also provided. The invention further provides antibodies that bind to 273P4B7 proteins and polypeptide fragments thereof, including polyclonal and monoclonal 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 nucleic acid sequence of Figure 2 Is encoded and/or the entire amino acid 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 273P4B7 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 273P4B7. A typical embodiment of this invention provides methods for monitoring 273P4B7 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 273P4B7 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 273P4B7 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 273P4B7 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 the production or function of 273P4B7. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 273P4B7 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The 00 00 antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as 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 273P4B7 and/or one or more than ri one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 273P4B7 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 273P4B7. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 273P4B7 antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 273P4B7 production) or a ribozyme effective to lyse 273P4B7 mRNA.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and 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 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 one must add the value °X 1" to each position in Tables VIII-XXI and XXII to XLIX 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 XLIX, or an oligonuceotide that encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide 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 Hydrophilicity profile of Figure ii) 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 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; SIlI) 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 Percent Accessible Residues profile of Figure 7; iv) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up CN 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 Sv) 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 Beta-turn profile of Figure 9.

Ci Moreover, the invention comprises 273P4B7 nucleic acid and amino acid sequences. Further, the invention comprises variants of 273P4B7, and fragments thereof. In an embodiment of the invention a protein fragment is: a subsequence of at least 158, or 262, or 420 contiguous amino acids of a protein of 273P4B7 v. 1; is an amino acid subsequence of a protein of 273P487 v. 1 with a proviso that 273P4B7 v. 1 protein is such that it does not include an valine or methionine at position 145; arginine or glycine at position 172; isoleucine or valine at position 889; or, lysine or arginine at position 989. An embodiment of an amino acid sequence of the invention is a fragment of a protein of 273P4B7 v. 1 with a proviso that it is not a protein of 273P4B7 v. 9, v. 10 or v.11. In an embodiment, an amino acid fragment of the invention is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 55,60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120,125,130, 135,140,145, 150,155, 156,157,158,159,160, 161, 162, 163, 164, 165, 170, 175, 180, 185, 190, 195, 200, 225,250, 260, 261, 262, 263, 264, 265, 270, 275, 300, 325, 350, 375,400, 418, 419, 420, 421,422, 423, 424,425,426, 427, 428, 429,430, 431, 432, 422, 434, 435, 450,475, 500, 525, 550, 575, 600, 650, 675, 700, 705, 710, 715, 716, 717, 718, 719, 720, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1127, 1150, 1175, 1200, 1025, or 1250 contiguous amino acids of a protein of Figure 2; in certain embodiments the fragment/subsequence comprises a functional or structural motif, as set forth herein, or comprises an immune system (antibody or T cell) epitope. Embodiments of a nucleic acid sequence of the invention comprise a sequence that encodes an amino acid sequence as set forth herein.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The 273P4B7 SSH sequence of 170 nucleotides.

Figure 2. A) The cDNA and amino acid sequence of 273P4B7 variant 1 (also called "273P487 v.1" or "273P4B7 variant is shown in Figure 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 3847 including the stop codon.

B) The cDNA and amino acid sequence of 273P4B7 variant 2 (also called "273P4B7 Is shown in Figure 2B.

The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 604-3987 including the stop codon.

C) 273P4B7 v.3 through v.8, SNP variants of 273P4B7 v.1. The 273P4B7 v.3 through v.8 are variants with single nucleotide difference from 273P4B7 v.1. 273P4B7 v.3, v.7, and v.8 code for the same protein as v.1. 273P4B7 v.4, v.5, and v.6 proteins differ from 273P4B7 v.1 by one amino acid. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above In Figures 2A and 2B.

D) The cDNA and amino acid sequence of 273P4B7 variant 9 (also called "273P4B7 is shown in Figure 2D.

The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 4-3324 including the S stop codon.

E) The cDNA and amino acid sequence of 273P4B7 variant 10 (also.called "273P4B7 v.10") is shown in Figure 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 688-1947 S including the stop codon.

F) The cDNA and amino acid sequence of 273P4B7 variant 11 (also called "273P4B7 v.11") is shown in Figure 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 114-1373 including the stop codon.

Figure 3.

0 A) The amino acid sequence of 273P4B7 v.1 is shown in Figure 3A; it has 1250 amino acids.

00 B) The amino acid sequence of 273P4B7 v.2 is shown in Figure 3B; it has 1127 amino acids.

C) The amino acid sequence of 273P4B7 v.4 is shown in Figure 3C; it has 1250 amino acids.

D) The amino acid sequence of 273P4B7 v.5 is shown in Figure 3D; it has 1250 amino acids.

E) The amino acid sequence of 273P4B7 v.6 is shown in Figure 3E; it has 1250 amino acids.

C F) The amino acid sequence of 273P4B7 v.9 is shown in Figure 3F; it has 1106 amino acids.

G) The amino acid sequence of 273P4B7 v.10 is shown in Figure 3G; it has 419 amino acids.

H) The amino acid sequence of 273P4B7 v.11 is shown in Figure 3H; it has 419 amino acids.

As used herein, a reference to 273P4B7 includes all variants thereof, including those shown in Figures 2, 3, and 11, unless the context dearly indicates otherwise.

Figure 4. Alignment of 273P487 with known homologs. Figure 4(A) Alignment of 273P4B7 with human unnamed protein (gi122760345). Figure 4(B) Alignment of 273P4B7 with human BJ-HCC-15 tumor antigen (gi122002580).

Figure 4(C) Alignment of 273P4B7 with Mouse Protein (gl|27706852).

Figure 5. Hydrophilidty amino acid profile of 273P4B7 v.1 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp Woods KR., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 6. Hydropathicity amino acid profile of 273P4B7 v.1 determined by computer algorithm sequence analysis using the method of Kyte 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-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 7. Percent accessible residues amino add profile of 273P4B7 v.1 determined by computer algorithm sequence analysis using the method of Janin (Janin 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 8. Average flexibility amino acid profile of 273P4B7 v.1 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 website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 9. Beta-turn amino acid profile of 273P4B7 v.1 determined by computer algorithm sequence analysis 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 (.expasy.ch/cgi-bln/protscale.pl) through the ExPasy molecular biology server.

Figure 10. Structures of transcript variants of 273P4B07. Variant 273P4B07 v.2 was identified as a transcript variant of 273P4B07 v.1. Variant 273P4B07 v.2 extended exon 1 by 22 bp as compared to v.1 and added an exon in between exons 1 and 2 of variant v.1. Variants v.9, v.10 and v.11 were part of the last exon of v.1 or v.2. Poly A tails and 0 SNP are not shown here. Numbers in underneath the boxes correspond to those of 273P4B07 v.1. Lengths of introns Sand exons are not proportional.

SFigure 11. Schematic alignment of protein variants of 273P4B07. Protein variants correspond to nucleotide variants. Nucleotide variants 273P4B07 v.3, v.7, and v.8 coded for the same protein as v.1. Variant v.2 coded a protein that was 123 amino acids shorter than v.1. Nucleotide variant 273P4B07 v.2 was a transcript variant of v.1, as shown in Figure C 10. Variants v.9 and v.10 were shorter and had some different amino acid as compared with v.1 in the corresponding positions shown in the figure. Variant v.11 was the same as the C-terminal part of v.1 and different from v.10 by one amino i acid at position 158. SNP in v.1 could also appear in v.2. Single amino acid differences were indicated above the boxes.

0\ Black boxes represent the same sequence as 273P4B07 v.1. Numbers underneath the box correspond to 273P4B07 v.1.

00 \0 Figure 12. Schematic alignment of SNP variants of 273P4B07. Variants 273P4B07 v.3 through v.8 were variants with single nucleotide differences as compared to variant v.1 (ORF:29-1858). Though these SNP variants were shown separately, they could also occur in any combinations and in any transcript variants that contained the base pairs, such as 0v.2 shown in Fig. 10. Numbers correspond to those of 273P4B07 v.1. Black box shows the same sequence as 273P4807 v.1. SNPs are indicated above the box.

Figure 13. Secondary structure and transmembrane domains prediction for 273P4B7 protein variant 1.

Figure 13A: The secondary structure of 273P4B7 protein variant 1 (Figure 13A) (SEQ ID NO: 134) was predicted using the HNN Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet Blanchet Geourjon C. and Del6age http://pbil.ibcp.fr/cgibinInpsa_automatpl?page=npsann.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (www.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed.

Figure 138: Schematic representation of the probability of existence of transmembrane regions of 273P4B7 variant I based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE Hofmann, W. Stoffel. TMBASE A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). Figure 13C: Schematic representation of the probability of the existence of transmembrane regions of 273P4B7 variant 1, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L.L. 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. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (www.expasy.chltools/).

Figure 14. 273P4B7 expression by RT-PCR. First strand cDNA was prepared from normal tissues (bladder, brain, heart,kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon and stomach), and from pools of patient cancer specimens (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, pancreas cancer pool, prostate cancer xenograft pool, prostate metastasis to lymph node, bone and melanoma cancer pool, cervical cancer pool, lymphoma cancer pool, stomach cancer pool, uterus cancer pool, and multi-xenograft pool). Normalization was performed by PCR using primers to actin.

Semi-quantitative PCR, using primers to 273P4B7, was performed at 22, 26 and 30 cycles of amplification. In (Figure 14A) picture of the RT-PCR agarose gel is shown. In (Figure 14B) PCR products were quantitated using the Alphalmager software. Results show strong of expression of 273P4B7 in prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, pancreas cancer pool, prostate cancer xenograft pool, prostate metastasis to lymph node, bone and melanoma cancer pool, cervical cancer pool, lymphoma cancer pool, stomach cancer pool, uterus cancer pool and multi-xenograft pool (prostate cancer, kidney cancer and bladder cancer xenograft pool). In normal tissues, 273P4B7 is predominantly expressed in testis and not in any other normal tissue tested.

Figure 15. 273P4B7 expression in normal tissues. Two multiple tissue northem blots (Clontech) both with 2 ug of C mRNA/anewere probed with the 273P4B7 sequence. Size standards In kilobases (kb) are indicated on the side. Results S show expression of an approximately 7kb 273P4B7 transcript in normal testis but not in the other normal tissues tested.

SFigure 16. Expression of 273P4B7 in pancreas, ovary, and testis cancer patient specimens. RNA was extracted S from normal pancreas (NPa), normal ovary normal testis (NTe), pancreas cancer patient specimen ovary cancer patient specimen (P2,P3,P4), and testis cancer patient specimen (P5,P6,P7). Northern blot with 10 ug of total RNA/lane was S probed with 273P4B7 SSH sequence. Size standards in kilobases (kb) are indicated on the side. 273P4B7 transcript was detected in the patient specimens, but not in the normal tissues.

00 O Figure 17. Expression of 273P4B7 In cervical cancer patient specimens. Figure 17(A): Total RNA was extracted from cervical cancer patient specimens (T1-T7), and HeLa cell line. Northern blot with 10 ug of total RNAlane was probed with 273P4B7 SSH sequence. Size standards in kilobases (kb) are indicated on the side. 273P4B7 transcript.was detected in all patient specimens tested as well as in the Hela cell line. Figure 17(B): First strand cDNA was prepared from a panel of cervical cancer patient specimens, normal cervix and HeLa cervical cell line. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles of amplification.

Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Expression was Srecorded as absent, low, medium or high. Results show expression of 273P4B7 in most of the cervical cancer tissues tested.

Figure 18. Expression of 273P4B7 in bladder cancer patient specimens. First strand cDNA was prepared from a panel of bladder cancer patient specimens, normal bladder and bladder cancer cell lines (UM-UC.3, TCCSUP, J82).

Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel (Figure and PCR products were quantitated using the Alphalmager software (Figure Expression was recorded as absent, low, medium or high.

Results show expression of 273P4B7 in most of the bladder cancer tissues tested, but not in the normal bladder tissues.

Figure 19. Expression of 273P4B7 in colon cancer patient specimens. First strand cDNA was prepared from a panel of colon cancer patient specimens, normal colon, and colon cancer cell lines (LoVo, CaCo-2, SK-C01, Colo205, and T284). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles 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 high. Results show expression of 273P4B7 in the majority of the colon cancer tissues tested, but not in the normal colon tissues. Expression was also detected in the cell lines LoVo, CaCo-2, SK-CO1, Colo205, but not in the T284 cell line.

Figure 20. Expression of 273P4B7 in ovary cancer patient specimens. First strand cDNA was prepared from a panel of ovarian cancer patient specimens, normal ovary and ovarian cancer cell lines (OV-1063, PA-1, SW626).

Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles 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 high. Results show expression of 273P4B7 in the majority of ovary cancer tissues tested as well as in the cell lines, but not in normal ovary.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections Definitions II.) 273P4B7 Polynucleotides II.A.) Uses of 273P4B7 Polynucleotides SII.A.1.) Monitoring of Genetic Abnormalities II.A.2.) Antisense Embodiments Primers and Primer Pairs II.A.4.) Isolation of 273P4B7-Encoding Nucleic Acid Molecules Recombinant Nucleic Acid Molecules and Host-Vector Systems III.) 273P4B7-related Proteins C III.A.) Motif-bearing Protein Embodiments III.B.) Expression of 273P4B7-related Proteins SIII.C.) Modifications of 273P4B7-related Proteins III.D.) Uses of 273P4B7-related Proteins IV.) 273P4B7 Antibodies 0 273P4B7 Cellular Immune Responses VI.) 273P4B7 Transgenic Animals VII.) Methods for the Detection of 273P4B7 VIII.) Methods for Monitoring the Status of 273P4B7-related Genes and Their Products IX.) Identification of Molecules That Interact With 273P4B7 Therapeutic Methods and Compositions Anti-Cancer Vaccines 273P4B7 as a Target for Antibody-Based Therapy 273P4B7 as a Target for Cellular Immune Responses X.C.1. Minlgene Vaccines X.C.2. Combinations of CTL Peptides with Helper Peptides 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 273P4B7.

XII.) Inhibition of 273P4B7 Protein Function XII.A.) Inhibition of 273P4B7 With Intracellular Antibodies XII.B.) Inhibition of 273P4B7 with Recombinant Proteins XII.C.) Inhibition of 273P4B7 Transcription or Translation XII.D.) General Considerations for Therapeutic Strategies XIII.) Identification, Characterization and Use of Modulators of 273P4B7 XIV.) KITSIArticles 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 clarity 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 r- 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 and/or parameters unless otherwise noted.

SThe 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 Association (AUA) system, stage Cl C2 disease under the Whitmore-Jewett system, and stage T3- T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not 00 recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the CK tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

"Altering the native glycosylation pattem" is Intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 273P4B7 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 273P4B7. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

The term "analog" refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule a 273P4B7-related protein). For example, an analog of a 273P4B7 protein can be specifically bound by an antibody or T cell that specifically binds to 273P4B7.

The term "antibody" is used in the broadest sense. Therefore, an "antibody" can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-273P4B7 antibodies comprise monoclonal and polyclonal 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-273P4B7 antibodies and clones thereof (including agoist, antagonist and neutralizing antibodies) and anti-273P4B7 antibody compositions with polyepitopic specificity.

The term "codon optimized sequences" refers to nucleotide 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 exon/intron 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 acids in every possible way for a given compound length the number of amino acids 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)).

O Preparation and screening of combinatorial libraries is well known to those of skill in the art Such combinatorial S chemical libraries include, but are not limited to, peptide libraries (see, U.S. Patent No. 5,010,175, Furka, Pept. Prot.

S Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptolds (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random blo- oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S.

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

rl USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara 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)), Cl analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), 00 oligocarbarnates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem.

IND 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, Stratagene, Cl Corp.), peptide nucleic acid 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, Uang et al., Science 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); isoprenolds, 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.

5,506, 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, Advanced Chem Tech, Louisville KY; Symphony, Rainin, 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 Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, 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 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 commercially available (see, ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).

The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocn, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinaiis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At 21 1131, 125, Y 9 0 Re86, Re 1 8 Sm15 3 Bi212or213 p32 and radioactive isotopes of Lu including Lu 77 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 acid 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 acid 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 acid 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 acid 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 Snucleic acids 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 discloses high throughput screening methods for Sproteins; U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding in arrays); while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.

00 IND 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. Fullerton, CA; Precision Systems, Inc., Natick, MA; etc.). These systems typically automate entire procedures, including all sample and 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, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

The term "homolog" 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.

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

The terms "hybridize", "hybridizing", "hybridizes" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1 SDS/100 .g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0I.1% SDS are above 55 degrees 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 polynucleotides that correspond or are complementary to genes other than the 273P4B7 genes or that encode polypeptides other than 273P487 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 273P4B7 polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical or chemical methods are employed to remove the 273P4B7 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 273P4B7 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term "mammal" refers to any organism classified as a mammal, including 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 S 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 CN prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

C1 The term "modulator" or test compound" or "drug candidate" or grammatical equivalents as used herein describe S any molecule, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the IN capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, a nucleic acid or C protein sequences, or effects of cancer sequences signaling, 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 0 is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of 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 nucleic acids 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 embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures Is run in parallel with 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 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 bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an Nterminal Cys to aid in solubility. In one embodiment, the C-termInus of the fragment Is kept as a free acid and the N-terminus is a free amine to aid in coupling, to cysteine. 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 acids to create a longer peptide/protein.

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

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

The term "monoclonal antibody" 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 "motif", as in biological motif of a 273P4B7-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function protein-protein interaction, proteln-DNA interaction, etc) or modification that is phosphorylated, glycosylated or amidated), or localization secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. 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 pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino 0acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically 00 different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

7 -A "pharmaceutical excipient" comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

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

The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides 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 "oligonucleotide". A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine as shown for example In Figure 2, can also be uracil this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil instead of thymidine The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".

An HLA "primary anchor residue" is an amino acid 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 class 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 class 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 supermbtif.

"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 See Thorium-229 (Th-229) (AC-225) S Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the 0 227 skeleton resulting from cancer breast and prostate cancers), and cancer (AC-2) radiolmmunotherapy Bismuth-212 (Bi-212) See Thorium-228 (Th-228) ismuth-213 S Bismh-21 3 See Thorium-229 (Th-229) (BI-213) N' Cadmium-109 S Cadmim109 Cancer detection (Cd-109) Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of r (Co-60) medical supplies 00 Copper-64 0 (Cu-64) A positron emitter used for cancer therapy and SPECT imaging Copper-67 Beta/gamma emitter used in cancer radloimmunotherapy and diagnostic studies breast (Cu-67) and colon cancers, and lymphoma) Dysprosium-166 S(Dy-166) Cancer radiolmmunotherapy Erblum-169 Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and (Er-169) toes Europium-152 (Eu-152) Radiation source for food irradiation and for sterilization of medical supplies Europium-154 (Eu-154) Radiation source for food irradiation and for sterilization of medical supplies Gadolinium-153 (Gd-153) Osteoporosis detection and nuclear medical quality assurance devices Gold-1i 98 (Au-198) Implant and intracavity therapy of ovarian, prostate, and brain cancers Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer radiolmmunotherapy, bone (Ho-166) marrow ablation, and rheumatoid arthritis treatment Osteoporosis detection, diagnostic Imaging, tracer drugs, brain cancer treatment, Iodine-125 radiolabeling, tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, (1-125) brachytherapy for treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as Iodine-131 other non-malignant thyroid diseases Graves disease, goiters, and hyperthyroidism), (1-131) treatment of leukemia, lymphoma, and other forms of cancer breast cancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (lr-192) arteriosclerosis and restenosis), and implants for breast and prostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries arteriosclerosis and (Lu-177) restenosis) Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver, lungs, heart, Molybdenum-99 and other organs. Currently, Tc-99m is the most widely used radioisotope used for diagnostic (Mo-99) imaging of various cancers and diseases involving the brain, heart, liver, lungs; also used In detection of deep vein thrombosis of the legs Osmium-194 (Os-194) Cancer radioimmunotherapy Palladium-103 (Pd-103) Prostate cancer treatment (Pd-i03) Platinum-195m (Pt-195m) Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug Phosphorus-32 Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer (P-32) Phosphorus-33 (P-33) Radium-223 (Ra-223) diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries arteriosclerosis and restenosis), and intracavity therapy Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of blocked arteries afteriosclerosis and restenosis) See Actinium-227 (Ac-227) Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of (Re-186) lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy Rhenium-188 (Re-188) Rhodium-105 (Rh-105) Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, treatment of rheumatoid arthritis, and treatment of prostate cancer Cancer radioimmunotherapy Samarium-145 (Sm-145) Ocular cancer treatment (Sm-153) Cancer radioimmunotherapy and bone cancer pain relief Scandium-47 (Sc-47) Cancer radioimmunotherapy and bone cancer pain relief (Sc-47) Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool Bone cancer detection and brain scans Strontium-89 (Sr-89) Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy Tecnetim-9mSee Molybdenum-99 (Mo-99) (Tc-99m) Thorium-228 (Th-228) Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimmunotherapy Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha (Th-229) emitters used in cancer radioimmunotherapy Thulium-170 (Tm-170) Gamma source for blood irradiators, energy source for implanted medical devices Tin-117m (Sn-117m) Cancer immunotherapy and bone cancer pain relief Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone (W-188) cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries arteriosclerosis and restenosis) Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, (Xe-127) and cerebral blood flow studies Ytterbium-175 Cancer radl (Yb-175) Cancer radloimmunotherapy Yttrium-91 (Y-91) Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers) By "randomized" or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides 0 (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, 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 bioactive proteinaceous agents.

In one embodiment, a library is "fully randomized," with no sequence preferences or constants at any position. In CN another embodiment, the library is a "biased random" library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nudeotides or amino acid residues are CK randomized within a defined class, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or 0 large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for O SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

C A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

O Non-limiting examples of small molecules include compounds that bind or interact with 273P4B7, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 273P4B7 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, 273P4B7 protein; are not found In naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions "Stringency" of hybridization reactions is readily determinable 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 et 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 citrate/0.1% sodium dodecyl sulfate at 50oC; employ during hybridization a denaturing agent, such as formamide, for example, 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 oC; or employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42 oC, with washes at 4200C in 0.2 x SSC (sodium chloride/sodium, 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 et al., Molecular Cloning: A Laboratory Manual, New York: Cold 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 370C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardfs solution, 10% dextran sulfate, and 20 mglmL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50oC. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as S necessary to accommodate factors such as probe length and the like.

SAn HLA "supermotil" is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV The non- D) limiting constituents of various supetypes are as follows: A2: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207 CK A3: A3, All, A31, A*3301, A*6801, A*0301, A*1101, A*3101 87: B7, 8*3501-03, B*51, B*5301, B*5401, B*5501, 8*5502, 8*5601, B*6701, 8*7801, 8*0702, 8*5101, B*5602 CB 44: 8*3701, 8*4402, B*4403, B*60 (8*4001), 861 (8*4006) Al: A*0102, A*2604, A*3601, A*4301, A*8001 00 O A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003 CB 827: 8*1401-02, 8*1503, 8*1509, 8*1510, 8*1518, B*3801-02, B*3901, B*3902, 8*3903-04, 8*4801-02, B*7301, 8*2701-08 0B858: 8*1516, 8*1517, 8*5701, 8*5702, 858 B62: 8*4601, 852, 8*1501 (862), B*1502 (875), 8*1513 (B77) Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV As used herein "to treat" or "therapeutic" and 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 "transgenic 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, e.g.,an embryonic stage. A "transgene" is a bNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response "vaccine" is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, a minigene that encodes a polyepitopic peptide. 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, 26, 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 lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, dendritic cells.

The term "variant" refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein the 273P4B7 protein shown in Figure 2 or Figure 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.

The "273P487-related proteins" of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 273P487 proteins or fragments thereof, as well as fusion proteins of a 273P4B7 protein and a heterologous polypeptide are also Included. Such 273P4B7 proteins are collectively referred to as the 273P4B7-related proteins, the proteins of the invention, or 273P4B7. The term "273P4B7-related protein" refers to a polypeptide fragment or a 273P4B7 protein sequence of 4, S 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 amino acids; or, at least 30, 35, 40, (C 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, or 576 or more amino acids.

cj) II.) 273P4B7 Polynucleotides One aspect of the Invention provides polynucleotides corresponding or complementary to all or part of a 273P4B7 gene, mRNA, andlor coding sequence, preferably in isolated form, including polynucleotides encoding a 273P4B7-related protein and fragments thereof, DNA, RNA, DNAIRNA hybrid, and related molecules, polynuceotides or oligonucleotides complementary to a 273P4B7 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that 0O hybridize to a 273P487 gene, mRNA, or to a 273P4B7 encoding polynucleotide (collectively, "273P4B7 polynucleotides"). In all Instances when referred to in this section, T can also be U in Figure 2.

Embodiments of a 273P4B7 polynucleotide include: a 273P4B7 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 273P4B7 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a C polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 273P4B7 nucleotides 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; (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2A, from nucleotide residue number 95 through nucleotide residue number 3847, including the stop codon, wherein T can also be U, (111) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B, from nucleotide residue number 604 through nucleotide residue number 3987, including the stop codon, wherein T can also be U; (IV) a polynucleotide that encodes a 273P4B7-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-F; a polynucleotide that encodes a 273P4B7-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-F; (VI) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX; (VII) 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, 32, 33, 34, 35 amino acids of a peptide of Figures 3A and 3C-3E in any whole number increment up to 1250 that Includes 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 0.5 in the Hydrophilicity profile of Figure (VIII) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3A and 3C-3E in any whole number increment up to 1250 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 less than 0.5 in the Hydropathicity profile of Figure 6; (IX) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9,10,11,12,13,14,15,16,17,18, S19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a peptide of Figure 3A and 3C-3E in Q any whole number increment up to 1250 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 in the Percent Accessible Residues profile of Figure 7; 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3A and 3C-3E in 0, any whole number increment up to 1250 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15,16, 17, 18,19, 00 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 Average Flexibility profile of Figure 8; S(XI) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8,9,10,11,12,13,14,15,16,17,18, S19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29,30,31,32,33,34,35 amino adds of a peptide of Figure 3A and 3C-3E in any whole number increment up to 1250 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 in the Beta-turn profile of Figure 9; (XII) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3B in any whole number increment up to 1127 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 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure (XIII) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3B in any whole number increment up to 1127 that includes 1,2,3,4,5,6, 7,8, 9,r0, 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 Hydropathicity profile of Figure 6; (XIV) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 38 in any whole number increment up to 1127 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 amino acid position(s) having a value greater than 0.5 In the Percent Accessible Residues profile of Figure 7; (XV) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3B in any whole number increment up to 1127 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 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XVI) 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, 32, 33, 34, 35 amino acids of a peptide of Figure 3B in any whole number increment up to 1127 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 amino acid position(s) having a value greater than 0.5 in the Beta- Sturn profile of Figure 9; (XVII) a polynucleotide that is fully complementary to a polynuceotide of any one of l) (XVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XVII); (XIX) a peptide that is encoded by any of to (XVIII); and; (XX) a composition comprising a polynucleotide of any of (I)-(XVIII) or peptide of (XIX) together with a pharmaceutical excipient and/or in a human unit dose form; (XXI) a method of using a polynucleotide of any (I)-(XVIII) or peptide of (XIX) or a composition of (XX) in a 00 Smethod to modulate a cell expressing 273P487; S(XXII) a method of using a polynucleotde of any (I)-(XVIII) or peptide of (XIX) or a composition of (XX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 273P4B7; C (XXIII) a method of using a polynucleotide of any (I)-(XVIII) or peptide of (XIX) or a composition of (XX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 273P4B7, said cell from a cancer of a tissue listed in Table I; (XXIV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XIX) or a composition of (XX) in a method to diagnose, prophylax, prognose, or treat a a cancer; (XXV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XIX) or a composition of (XX) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and; (XXVI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XIX) or a composition of (XX) in a method to identify or characterize a modulator of a cell expressing 273P4B7.

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

Typical embodiments of the invention disclosed herein include 273P4B7 polynucleotides that encode specific portions of 273P4B7 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins andlor fragments thereof, for example: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 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, 625, 650, 675, 700, 725, 750, 775, 800, 825,850, 875,900, 925, 950, 975,1000,1025, 1050,1075,1100,1125,1150,1175,1200,1225,1235,1240,12451250 or more contiguous amino acids of 273P4B7 variant 1; the maximal lengths relevant for other variants are: variant 2, 1127 amino acids; variant 4, 1250 amino acids, variant 5, 1250 amino acids, variant 6, 1250 amino acids, variant 9, 1106 amino acids; variant 10, 419 amino acids; and variant 11,419 amino acids.

For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 273P4B7 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 273P4B7 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 273P4B7 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 273P4B7 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 273P487 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 273P487 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 273P4B7 protein shown In Figure 1 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 273P4B7 protein shown in Figure 0 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 273P487 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 273P4B7 protein shown in d) Figure 2 or Figure 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in Figure 2 or Figure 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 273P4B7 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

C^ Polynucleotides encoding relatively long portions of a 273P4B7 protein are also within the scope of the invention.

S For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 00 N or 50 etc.) of the 273P4B7 protein "or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 273P4B7 sequence as shown in Figure 2.

Additional illustrative embodiments of the invention disclosed herein include 273P4B7 polynucleotide fragments encoding one or more of the biological motifs contained within a 273P4B7 protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 273P4B7 protein "or variant" set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 273P4B7 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 273P4B7 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 VIII-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 VII.

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 acid position to calculate the position of that amino acid in the parent molecule.

II.A.) Uses of 273P4B7 Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 273P4B7 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 273P4B7." For example, because the 273P4B7 gene maps to this chromosome, polynucleotides that encode different regions of the 273P487 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g.

Krajinovic et Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 273P4B7 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 273P4B7 that may contribute to the malignant phenotype. In this context, these polynucleotides 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 et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 S (1994)).

0 Furthermore, as 273P4B7 was shown to be highly expressed in prostate and other cancers, 273P4B7 Cl polynucleotides are used in methods assessing the status of 273P4B7 gene products in normal versus cancerous tissues.

O- Typically, polynucleotides that encode specific regions of the 273P4B7 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific 0 regions of the 273P4B7 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays 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 S within the protein.

00 II.A.2.) Antisense Embodiments Other specifically contemplated nucleic acid related embodiments of the Invention disclosed herein are genomic DNA, S cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an altemative backbone, or including altemative bases, whether derived from natural sources or synthesized, and Include molecules capable of Inhibiting the RNA or S protein expression of 273P4B7. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid 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 acid molecules using the 273P4B7 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their Intracellular targets, 273P4B7. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 273P4B7 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (0-oligo) 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 0-oligos with 3H-1,2benzodithiol-3-one-l ,1-dioxide, which is a sulfur transfer reagent. See, lyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and lyer, R. P. et J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 273P4B7 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, Partridge et at., 1996, Antisense Nucleic Acid Drug Development 6: 169-175).

The 273P4B7 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 273P4B7 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 273P4B7 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 273P4B7 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 273P4B7 mRNA. Optionally, 273P4B7 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 273P487. Alternatively, the antisense molecules are modified to employ ribozymes in the Inhibition of 273P4B7 expression, see, L. A. Couture D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs Further specific embodiments of these nucleotides of the 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 specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radiolsotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a S 273P4B7 polynucleotide in a sample and as a means for detecting a cell expressing a 273P4B7 protein.

Examples of such probes include polypeptides comprising all or part of the human 273P4B7 cDNA sequence shown in 1 Figure 2. Examples of primer pairs capable of specifically amplifying 273P4B7 mRNAs are also described 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 C provided herein and used effectively to amplify andlor detect a 273P4B7 mRNA.

The 273P4B7 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their 00 I use as probes and primers for the amplification and/or detection of the 273P4B7 gene(s), mRNA(s), or fragments thereof; as C reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 273P4B7 polypeptides; as tools for modulating or inhibiting the expression of the 273P4B7 gene(s) and/or translation of the 273P4B7 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 273P4B7 or 273P4B7 related nucleic acid sequence from a naturally occurring 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 273P4B7-Encoding Nucleic Acid Molecules The 273P4B7 cDNA sequences described herein enable the isolation of other polynucleotides encoding 273P4B7 gene product(s), as well as the isolation of polynucleotides encoding 273P4B7 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 273P4B7 gene product as well as polynucleotides that encode analogs of 273P4B7-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 273P4B7 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems Lambda ZAP Express, Stratagene). Phage clones containing 273P4B7 gene cDNAs can be identified by probing with a labeled 273P4B7 cDNA or a fragment thereof. For example, in one embodiment, a 273P4B7 cDNA Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 273P4B7 gene. A 273P4B7 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 273P4B7 DNA probes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems The invention also provides recombinant DNA or RNA molecules containing a 273P4B7 polynucleotide, a fragment, analog or homologue thereof, Including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 273P4B7 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell.' Examples of suitable eukaryotic 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 prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary 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 polynucleotide comprising the coding sequence of 273P4B7 or a fragment, analog or homolog thereof can be used to generate 273P4B7 proteins or fragments thereof using any number of host-vector systems 0 routinely used and widely known in the art.

C A wide range of host-vector systems suitable for the expression of 273P4B7 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector 0pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 273P4B7 can be expressed in several prostate cancer and non-prostate cell lines, 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 273P4B7 protein or fragment thereof. Such host-vector systems Scan be employed to study the functional properties of 273P4B7 and 273P4B7 mutations or analogs.

00 Recombinant human 273P4B7 protein or an analog or homolog or fragment thereof can be produced by

\O

S mammalian cells transfected with a construct encoding a 273P4B7-related nucleotide. For example, 293T cells can be c- transfected with an expression plasmld encoding 273P4B7 or fragment, analog or homolog thereof, a 273P4B7-related Sprotein is expressed in the 293T cells, and the recombinant 273P4B7 protein is isolated using standard purification methods affinity purification using anti-273P4B7 antibodies). In another embodiment, a 273P4B7 coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 273P4B7 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 273P4B7 coding sequence can be used for the generation of a secreted form of recombinant 273P4B7 protein.

As discussed herein, redundancy in the genetic code permits variation in 273P4B7 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% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/-nakamuralcodon.html.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or 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 cell.

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. Cell BioL, 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively 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)).

IIl.) 273P4B7-related Proteins Another aspect of the present invention provides 273P4B7-related proteins. Specific embodiments of 273P4B7 proteins comprise a polypeptide having all or part of the amino acid sequence of human 273P4B7 as shown in Figure 2 or Figure 3. Alternatively, embodiments of 273P487 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 273P4B7 shown in Figure 2 or Figure 3.

Embodiments of a 273P4B7 polypeptide include: a 273P4B7 polypeptide having a sequence shown in Figure 2, a peptide sequence of a 273P4B7 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides 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 273P4B7 peptides comprise, without limitation: a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in c Figure 2A-F or Figure 3A-H; (II) a 273P4B7-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to San entire amino acid sequence shown in Figure 2A-F or 3A-H; (III) a 273P4B7-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an Sentire amino acid sequence shown in Figure 2A-F or 3A-H; 00 (IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso IN that it is not an entire protein of Figure 2; S(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is Salso 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 acid 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 acids of a protein of Figure 3A, 3C-3E in any whole number increment up to 1250 respectively that includes 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 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, 3C-3E, in any whole number increment up to 1250 respectively that includes at least 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 less than 0.5 in the Hydropathlcity profile of Figure 6; (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 acids of a protein of Figure 3A, 3C-3E, In any whole number increment up to 1250 respectively that includes at least 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 0.5 in the Percent Accessible Residues profile of Figure 7; (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, 3C-3E, in any whole number increment up to 1250 respectively that includes at least 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 pgreater than 0.5 in the Average Flexibility profile of Figure 8;

O

(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, amino acids of a protein of Figure 3A, 3C-3E in any whole number increment up to 1250 respectively that includes at least 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 Beta-turn profile of Figure 9; (XIV) 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, C^ 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3B, in any whole number increment up to 1127 respectively that includes at least 1,2,3,4,5,6,7,8,9, 10,11,12,13,14,15,16,17,18,19,20,21,22, 00 0 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 SHydrophilicity profile of Figure 0 (XV) 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 3B, in any whole number increment up to 1127 respectively that includes at least 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 less than 0.5 in the Hydropathicity profile of Figure 6; (XVI) a polypepide 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 3B, in any whole number increment up to 1127 respectively that includes at least 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 0.5 in the Percent Accessible Residues profile of Figure 7; (XVII) 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 3B, in any whole number increment up to 1127 respectively that includes at least 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 0.5 in the Average Flexibility profile of Figure 8; (XVIII) 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, amino acids of a protein of Figure 3B In any whole number increment up to 1127 respectively that includes at least 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 0.5 in the Beta-tum profile of Figure 9; (XIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively; (XX) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively; (XXI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively; (XXII) a peptide that occurs at least five times In Tables VIII-XXI and XXII to XLIX, collectively; (XXIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XXIV) a peptide that occurs at least once in Tables VIII-XXI, and at least twice In tables XXII to XLIX; (XXV) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XXVI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX; (XXVII) a peptide which comprises one two, three, four, or five of the following characteristics, or an CKl ollgonucleotide encoding such peptide: 1) 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 Sgreater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure ii) 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 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; 00 0 iii) 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 Percent Accessible Residues profile of Figure 7; Cl iv) 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 Average Flexibility profile of Figure 8; or, 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;; (XXVIII) a composition comprising a peptide of (I)-(XXVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.

(XXIX) a method of using a peptide of (I)-(XXVII), or an antibody or binding region thereof or a composition of (XXVIII) in a method to modulate a cell expressing 273P4B7,; (XXX) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 273P4B7; (XXXI) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 273P4B7, said cell from a cancer of a tissue listed in Table I; (XXXII) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer; (XXXIII) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and; (XXXIV) a method of using a a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to identify or characterize a modulator of a cell expressing 273P4B7 As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 273P4B7 polynucleotides that encode specific portions of 273P4B7 mRNA sequences (and those which are complementary to such sequences) such as those that encode S the proteins and/or fragments thereof, for example: (S 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 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, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025,1050,1075,1100,1125,1150,1175,1200,1225,1235,1240,1245, and 1250, or l more contiguous amino acids of 273P487 variant 1; the maximal lengths relevant for other variants are: variant 2, 1127 amino acids; variant 4, 1250 amino acids, variant 5, 1250 amino acids; variant 6, 1250 amino acids; variant 9, 1106 amino C] acids; variant 10, 419 amino acids; and variant 11,419 amino acids..

00 In general, naturally occurring allelic variants of human 273P4B7 share a high degree of structural identity and IND homology 90% or more homology). Typically, allelic variants of a 273P4B7 protein contain conservative amino acid CI substitutions within the 273P4B7 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 273P4B7. One class of 273P4B7 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 273P4B7 amino add sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine valine and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice 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, glycine and alanine can frequently be interchangeable, as can alanine and valine Methionine which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine and arginine are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments (see, e.g. Table III herein; pages 13-15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford University); Henikoffet al., PNAS 1992 Vol 89 10915-10919; Lel et al., J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 273P4B7 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 273P487 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Sitedirected mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. 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 cloned DNA to produce the 273P4B7 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 acids. Such amino acids include alanine, glycine, serine, and cysteine.

Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the betacarbon 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 acid can be used.

As defined herein, 273P4B7 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that Is "cross reactive" with a 273P4B7 protein having an amino acid sequence of Figure 3. Asused in this 1 sentence, "cross reactive" means that an antibody or T cell that specifically binds to a 273P4B7 variant also specifically binds to a 273P4B7 protein having an amino acid sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein CN shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that Q00 specifically binds to the starting 273P487 protein. Those skilled in the art understand that antibodies that recognize proteins IN bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded C( as a typical number of amino acids in a minimal epitope. See, Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et J Immunol (1985) 135(4):2598-608.

SOther classes of 273P4B7-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of Figure 3, or a fragment thereof. Another specific class of 273P4B7 protein variants or analogs comprises one or more of the 273P4B7 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 273P4B7 fragments (nucleic or amino acid) that have altered functional (e.g.

Immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of Figure 2 or Figure 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 273P4B7 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 273P4B7 protein shown in Figure 2 or Figure 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 273P4B7 protein shown In Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 273P4B7 protein shown In Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 273P4B7 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 273P487 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 273P4B7 amino acid 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 273P4B7 protein shown in Figure 2 or Figure 3 ae 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.

273P4B7-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Altematively, recombinant methods can be used to generate nucleic acid molecules that encode a 273P4B7-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 273P4B7 protein (or variants, homologs or analogs thereof).

O Moreover the invention comprises 273P487 nucleic acid and amino acid sequences. Further, the invention Scomprises variants of 273P4B7, and fragments thereof. In an embodiment of the invention a protein fragment is: a subsequence of at least 158, or 262, or 420 contiguous amino acids of a protein of 273P4B7 v, 1; is an amino acid subsequence of a protein of 273P4B7 v. 1 with a proviso that 273P4B7 v. 1 protein is such that it does not include an valine or methionine at position 145; arginine or glycine at position 172; isoleuclne or valine at position 889; 1 or, lysine or arginine at position 989. An embodiment of an amino acid sequence of the invention is a fragment of a protein of 273P4B7 v. 1 with a proviso that it is not a protein of 273P4B7 v. 9, v. 10 or v.11. In an embodiment, an amino C acid fragment of the invention is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 00 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,170, 175,180, 185,190,195,200, 225, 250, 260, 261,262, 263, 264, 265, 270, 275,300, 325, 350, 375, 400, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,432, 422, 434, 435, 450, 475, 500, 525, 550, 575, 600, 650, 675, 700, 705, 710, 715, 716,717, 718,719, 720, 725, 750, 775, 800, 825, 850, 875,900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1127, 1150, 1175, 1200, 1025, or 1250 contiguous amino acids of a protein of Figure 2; in certain embodiments the fragment/subsequence comprises a functional or structural motif, as set forth herein, or comprises an immune system (antibody or T cell) epitope. Embodiments of a nucleic acid sequence of the invention comprise a sequence that encodes an amino acid sequence as set forth herein.

III.A.) Motif-bearing Protein Embodiments Additional illustrative embodiments of the invention disclosed herein include 273P487 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 273P4B7 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 publicly available Intemet sites (see, URL addresses: pfam.wust.edul; searchlauncher.bcm.tmc.edulseqsearch/struc-predict.html; psort.ims.u-tokyo.ac.jpl; cbs.dtu.dkl; ebi.ac.uklinterpro/scan.html; expasy.ch/toolslscnpsitl.html; EpimatrixT M and EpimerTM, Brown University, brown.edu/Research/TB-HIVLab/epimaepimatpimatrix.html; and BIMAS, bimas.dcrt.nih.gov.).

Motif bearing subsequences of all 273P4B7 variant proteins are set forth and identified in Tables VIIl-XXI and XXII-

XLIX.

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

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 included if the motif is relevant for location.

Polypeptides comprising one or more of the 273P4B7 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 273P4B7 motifs discussed above are associated with growth dysregulation and because 273P4B7 is overexpressed in certain cancers (See, Table Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel 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 Biochem.

Biophys. Acta 1473(1):21-34 (1999); Raju et 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 al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified Sin accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 273P4B7 protein that are capable of optimally binding to specified HLA alleles Table IV; Epimatrix m and Epimer T M Brown University, URL brown.edulResearch/TB- HIVLab/epimarix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have C sufficient 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 CK epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

00 Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For S example, one begins with an epitope that bears a CTL or HTL motif (see, the HLA Class I and HLA Class II CN motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid 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 SIV, 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 et al.; Sette, Immunogenetics 1999 50(3-4): 201- 212; Sette et J. Immunol. 2001 166(2): 1389-1397; Sidney et Hum. Immunol. 1997 58(1): 12-20; Kondo et a., Immunogenetics 1997 45(4): 249-258; Sidney et J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et 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 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3)1 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et PMID: 7895164, UI: 95202582; O'Sullivan et al., J.

Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 751-761 and Alexander et 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 XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVl-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 include 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, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

273P4B7-related proteins are embodied in many forms, preferably in isolated form. A purified 273P4B7 protein molecule will be substantially free of other proteins or molecules that impair the binding of 273P4B7 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 273P4B7related proteins include purified 273P4B7-related proteins and functional, soluble 273P4B7-related proteins. In one embodiment, a functional, soluble 273P4B7 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 273P4B7 proteins comprising biologically active fragments of a 273P4B7 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 273P4B7 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 273P4B7 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically S bind to the starting protein.

C 273P4B7-related polypeptides that contain particularly interesting structures can be predicted and/or Identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte- Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-273P4B7 antibodies or T cells or in identifying cellular factors that bind to 273P487. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles r can be generated, and Immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, 1982, J.

00 Mol. Biol. 157:105-132. Percent Accessible Residues proples 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 r immunogenic peptide fragments identified, using the method of Bhaskaran Ponnuswamy 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-tum profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, Roux 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a 273P4B7 protein that are capable of optimally binding to specified HLA alleles by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmiheidelberg.com/; the listings in Table Epimatix T and EpimerTM, Brown University, URL (brown.edu/Research/TB- HIVLab/epimatrixepimatrix.html); and BIMAS, URL bimas.dcrtnih.gov/). Illustrating this, peptide epitopes from 273P4B7 that are presented in the context of human MHC Class I molecules, HLA-A1, A2, A3, All, A24, B7 and B35 were predicted (see, Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 273P4B7 protein and relevant portions of other variants, for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutationor exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Blolnformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmiheidelberg.coml.

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 HLA-A2 (see, Falk et Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, 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 binding peptides are 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine or methionine at position 2 and a valine or leucine at the C-terminus (see, Parker et al., J. Immunol. 149:3580-7 (1992)).

Selected results of 273P4B7 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII- XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, 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 targets for T-cell recognition.

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 et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be appreciated that every epitope predicted by the BIMAS site, EpimerTM and Epimatrix T sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.coml, or BIMAS, bimas.dcrt.nih.gov/) are to be "applied" l to a 273P4B7 protein in accordance with the invention. As used in this context "applied" means that a 273P4B7 protein is evaluated, visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art.

C Every subsequence of a 273P4B7 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a 00 subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

Cr- III.B.) Expression of 273P4B7-related Proteins In an embodiment described in the examples that follow, 273P4B7 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 273P4B7 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, 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 273P4B7 protein in transfected cells. The secreted HIS-tagged 273P4B7 in the culture media can be purified, using a nickel column using standard techniques.

II.C.) Modifications of 273P4B7-related Proteins Modifications of 273P4B7-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 273P4B7 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 273P4B7 protein. Another type of covalent modification of a 273P487 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 273P4B7 comprises linking a 273P4B7 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 273P4B7-related proteins of the present invention can also be modified to form a chimeric molecule comprising 273P4B7 fused to another, heterologous polypeptide or amino acid 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. Altematively, a protein in accordance with the invention can comprise a fusion of fragments of a 273P487 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of 273P4B7. A chimeric molecule can comprise a fusion of a 273P487-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a 273P4B7 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 273P4B7-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 273P4B7 polypeptide in place of at least 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 regions of an IgGI molecule. For the production of immunoglobulin fusions see, O U.S. Patent No. 5,428,130 issued June 27,1995.

0 III.D.) Uses of 273P4B7-related Proteins SThe proteins of the invention have a number of different specific uses. As 273P4B7 Is highly expressed in prostate and other cancers, 273P4B7-related proteins are used in methods that assess the status of 273P4B7 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 273P4B7 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in Cthose regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 00 273P4B7-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 273P4B7 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or CK to elicit an immune response to the epitope. Alternatively, 273P4B7-related proteins that contain the amino acid residues of one or more of the biological motifs in a 273P4B7 protein are used to screen for factors that interact with that region of S273P4B7.

273P4B7 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies antibodies recognizing an extracellular or intracellular epitope of a 273P4B7 protein), for identifying agents or cellular factors that bind to 273P4B7 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 273P4B7 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 273P4B7 gene product Antibodies raised against a 273P4B7 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 273P4B7 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 273P4B7-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 273P4B7 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 273P4B7-expressing cells in radioscintigraphic imaging methods). 273P4B7 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 273P4B7 Antibodies Another aspect of the invention provides antibodies that bind to 273P4B7-related proteins. Preferred antibodies specifically bind to a 273P4B7-related protein and do not bind (or bind weakly) to peptides or proteins that are not 273P4B7related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCI; or normal saline NaCI); 4) animal serum such as human serum; or, 5) a combination of any of 1) through these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 40C to 370C. For example, antibodies that bind 273P4B7 can bind 273P4B7-related proteins such as the homologs or analogs thereof.

273P4B7 antibodies of the invention are particularly useful in cancer (see, Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of S other cancers, to the extent 273P487 is also expressed or overexpressed in these other cancers. Moreover, intracellularly O expressed antibodies single chain antibodies) are therapeutically useful in treating cancers in which the expression of 273P4B7 is involved, such as advanced or metastatic prostate cancers.

D The invention also provides various immunological assays useful for the detection and quantification of 273P4B7 and mutant 273P4B7-related proteins. Such assays can comprise one or more 273P4B7 antibodies capable of recognizing and 1 binding a 273P4B7-related protein, as appropriate. These assays are performed within various Immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked Immunosorbent assays (ELISA), C enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or 00 IN stimulatory) as well as major histocompatibility complex (MHC) binding assays.

SIn addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 273P4B7 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 273P4B7 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 273P4B7 expressing cancers such as prostate cancer.

273P4B7 antibodies are also used in methods for purifying a 273P4B7-related protein and for isolating 273P4B7 homologues and related molecules. For example, a method of purifying a 273P4B7-related protein comprises incubating a 273P4B7 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 273P4B7-related protein under conditions that permit the 273P4B7 antibody to bind to the 273P487-related protein; washing the solid matrix to eliminate impurities; and eluting the 273P4B7-related protein from the coupled antibody. Other uses of 273P4B7 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 273P4B7 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 273P4B7-related protein, peptide, or fragment in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 273P4B7 can also be used, such as a 273P4B7 GST-fusion 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 273P4B7-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 273P4B7-related protein or 273P4B7 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 273P4B7 protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 273P4B7 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 273P4B7 amino acid sequence are used to identify hydrophilic regions in the 273P4B7 structure. Regions of a 273P4B7 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schult or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, 1982, J. Mol. Blol. 157:105- 132. Percent Accessible Residues profiles can be generated using the method of Janin 1979, Nature 277:491-492.

Average Flexibility profiles can be generated using the method of Bhaskaran Ponnuswamy 1988, Int. J. Pept.

Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, Roux 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 273P4B7 antibodies are further illustrated by way of the examples provided herein.

Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are Cr methods for preparing immunogenic conjugates of a protein with a carrer, such as BSA, KLH or other carrer protein. In some circumstances, direct conjugation using, for example, carbodilmide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 273P4B7 immunogen Is often conducted O by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the Immunization schedule, tilers of antibodies can be taken to determine adequacy of antibody formation.

273P4B7 monoclonal antibodies can be produced by various means well known In the art For example, Immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and 00 Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 273P4B7-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

SThe antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 273P4B7 protein can also be produced in the context of chimeric or complementaritydetermining region (CDR) grafted antibodies of multiple species origin. Humanized or human 273P4B7 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones ef al., 1986, Nature 321: 522-525; Riechmann et 1988, Nature 332: 323-327; Verhoeyen et 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies Include phage display and transgenic methods (for review, see Vaughan et 1998, Nature Biotechnology 16: 535-539). Fully human 273P4B7 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries phage display) (Griffiths and Hoogenboom, Building an in vitro immune 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 libraries. Id., pp 65-82). Fully human 273P4B7 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described In PCT Patent Application W098/24893, Kucheriapati and Jakobovits etal., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.

Drugs 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114598 issued 5 September 2000). This method avoids the in vfro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 273P4B7 antibodies with a 273P4B7-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 273P4B7-related proteins, 273P4B7-expressing cells or extracts thereof. A 273P4B7 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radiolsotope, a fluorescent compound, a bioluminescent compound, chemilumlnescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 273P4B7 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known In the art Wolff et al., Cancer Res. 53: 2560-2565).

273P4B7 Cellular Immune Responses 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 world- S wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells 1) (Buus, S. et al., Cell47:1071, 1986; Babbitt, B. P. et al., Nature 317:359,1985; Townsend, A. and Bodmer, Annu. Rev.

Immunol. 7:601, 1989; Germain, R. Annu. Rev. Immunol. 11:403,1993). Through the study of single amino acid C substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, Southwood, et J. Immunol. 160:3363,1998; Rammensee, et Immunogenetics 41:178,1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A.

00 NO and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H.

Curr. Opin. Immunol. 4:79,1992; Sinigaglia, F. and Hammer, J. Curt. Biol. 6:52,1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immuno. 45:79-93,1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding ceft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, et Immunity4:203, 1996; Fremont et Immunity 8:305, 1998; Stem et al., Structure 2:245,1994; Jones, E.Y. Curt. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C.

et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. Bjorkman, P. J. and Wiley, D. J. Mol. Biol. 219:277, 1991.) Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA 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 confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenlcity.

Various strategies can be utilized to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal individuals (see, Wentworth, P. A. et Mol. Immunol.

32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sc. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796,1997; Kawashima, I. etal., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T.cells specific for the peptide become activated during this time and are detected using, a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.

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

A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.

Peptide-specific T cells are detected using, a 5 1 Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated CK and/or from chronically ill patients (see, Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. etal., Immunity 7:97, 1997; Bertonl, R. et al., J. Clin. 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 O that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells. At the end of the culture period, T cell activity is detected using assays including 5 1 Cr release involving peptide-sensitized 00 targets, T cell proliferation, or lymphokine release.

VI.) 273P4B7 Transgenic Animals SNucleic acids that encode a 273P4B7-related protein can also be used to generate either transgenic animals or CN "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 273P4B7 can be used to clone genomic DNA that encodes 273P4B7. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 273P4B7. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the 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 273P4B7 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 273P4B7 can be used to examine the effect of increased expression of DNA that encodes 273P4B7. 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 273P4B7 can be used to construct a 273P4B7 "knock out" animal that has a defective or altered gene encoding 273P487 as a result of homologous recombination between the endogenous gene encoding 273P4B7 and altered genomic DNA encoding 273P4B7 introduced into an embryonic cell of the animal. For example, cDNA that encodes 273P4B7 can be used to clone genomic DNA encoding 273P4B7 in accordance with established techniques. A portion of the genomic DNA encoding 273P4B7 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 Capecchi, 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, Li et al., Cell, 9:915 (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 Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric 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, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 273P4B7 polypeptide.

V1I.) Methods for the Detection of 273P487 Another aspect of the present invention relates to methods for detecting 273P4B7 polynucleotides and 273P4B7-related proteins, as well as methods for identifying a cell that expresses 273P4B7. The expression profile of 273P4B7 makes it a C/3 diagnostic marker for metastasized disease. Accordingly, the status of 273P4B7 gene products provides information useful for C predicting a variety of factors incuding susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 273P487 gene products in patient samples can be analyzed by a C variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including In situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Westem blot analysis 00 and tissue array analysis.

C More particularly, the invention provides assays for the detection of 273P4B7 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 273P4B7 polynucleotides include, for example, a 273P4B7 gene or fragment thereof, 273P4B7 mRNA, altemative splice variant 273P4B7 mRNAs, and recombinant DNA or RNA molecules that contain a 273P4B7 polynucleotide. A number of methods for amplifying and/or detecting the presence of 273P4B7 polynucleotides 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 273P4B7 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 273P487 polynucleotides as sense and antisense primers to amplify 273P4B7 cDNAs therein; and detecting the presence of the amplified 273P4B7 cDNA. Optionally, the sequence of the amplified 273P4B7 cDNA can be determined.

In another embodiment, a method of detecting a 273P4B7 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 273P4B7 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 273P487 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 273P4B7 nucleotide sequence (see, Figure 2) and used for this purpose.

The invention also provides assays for detecting the presence of a 273P487 protein In a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 273P4B7-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Westem blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 273P487-related protein In a biological sample comprises first contacting the sample with a 273P4B7 antibody, a 273P4B7-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 273P4B7 antibody; and then detecting the binding of 273P4B7-related protein in the sample.

Methods for identifying a cell that expresses 273P4B7 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 273P4B7 gene comprises detecting the presence of 273P487 mRNA in the cell.

Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 273P4B7 riboprobes, Northem blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 273P4B7, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 273P4B7 gene comprises detecting the presence of 273P4B7-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 273P4B7-related proteins and cells that express 273P4B7-related proteins.

O 273P4B7 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 273P4B7 gene CN expression. For example, 273P4B7 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 273P4B7 expression or overexpression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that 0 quantifies 273P4B7 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 273P4B7-related Genes and Their Products Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and 00 cells progress from a normal physiological state to precancerous and then cancerous states (see, Alers et Lab

NO

Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23:19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 273P4B7 expression in cancers) allows for early detection of 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 273P4B7 in a biological sample of interest can be compared, for example, to the status of 273P4B7 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 273P4B7 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 273P4B7 status In a sample.

The term "status" in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 273P4B7 expressing cells) as well as the level, and biological activity of expressed gene products (such as 273P4B7 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 273P4B7 comprises a change in the location of 273P4B7 and/or 273P4B7 expressing cells andlor an increase in 273P4B7 mRNA and/or protein expression.

273P4B7 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ 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 273P4B7 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northem Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 273P4B7 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southem analysis (to examine, for example perturbations in a 273P4B7 gene), Northern analysis and/or PCR analysis of 273P4B7 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 273P4B7 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 273P4B7 proteins and/or associations of 273P4B7 proteins with polypeptide binding partners). Detectable 273P4B7 polynucleotides include, for example, a 273P487 gene or fragment thereof, 273P4B7 mRNA, alternative splice variants, 273P4B7 mRNAs, and recombinant DNA or RNA molecules containing a 273P4B7 polynucleotide.

The expression profile of 273P4B7 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 273P4B7 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 273P4B7 status and diagnosing cancers that express 273P4B7, such as cancers of the tissues listed in Table I. For example, because 273P4B7 mRNA is so highly expressed in prostate and other 0 cancers relative to normal prostate tissue, assays that evaluate the levels of 273P4B7 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 273P4B7 dysregulation, and can provide prognostic information useful Cd in defining appropriate therapeutic options, The expression status of 273P4B7 provides information including the presence, stage and location of dysplastic, Cprecancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease.

SConsequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 273P4B7 in biological samples such as those from individuals suffering from, or suspected of suffering from a 00 O pathology characterized by dysregulated cellular growth, such as cancer.

SAs described above, the status of 273P4B7 in a biological sample can be examined by a number of well-known I> procedures in the art. For example, the status of 273P4B7 in a biological sample taken from a specific location in the body 0can be examined by evaluating the sample for the presence or absence of 273P4B7 expressing cells those that express 273P4B7 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 273P4B7-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 273P4B7 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 et 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 273P4B7 gene products by determining the status of 273P4B7 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 273P4B7 gene products in a corresponding normal sample. The presence of aberrant 273P4B7 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 273P4B7 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 273P4B7 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 273P4B7 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 273P4B7 mRNA or express it at lower levels.

In a related embodiment, 273P4B7 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 273P4B7 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 273P4B7 expressed in a corresponding normal sample. In one embodiment, the presence of 273P4B7 protein is evaluated, for example, using immunohistochemical methods. 273P4B7 antibodies or binding partners capable of detecting 273P4B7 protein expression are used in a variety of assay formats well known In the art for this purpose.

In a further embodiment, one can evaluate the status of 273P4B7 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nudeotide and amino acid sequences are C observed in a large number of proteins associated with a growth dysregulated phenotype (see, Marrogi et 1999, J.

Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 273P4B7 may be indicative of the presence or Cpromotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation In 273P4B7 indicates a Spotential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nudeotide and amino acid sequences are well known In the art.

For example, the size and structure of nucleic acid or amino acid sequences of 273P487 gene products are observed by the Northern, Southem, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods far observing 00 perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, U.S. Patent Nos. 5,382,510 issued 7 September 1999, and 5,952,170 Issued 17 January 1995).

I Additionally, one can examine the methylation status of a 273P4B7 gene in a biological sample. Aberrant 0 demethylation andlor hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and Stransformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-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 et Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers 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 lymphoblastold 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 cleave 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 will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation Interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification Is an additional method for assessing the status of 273P487. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alteratively, antibodies are employed that recognize specific duplexes, including 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 273P4B7 expression. The presence of RT-PCR amplifiable 273P4B7 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 include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687- 1688).

A further aspect of the Invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 273P4B7 mRNA or 273P487 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 273P4B7 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 273P4B7 in prostate or other tissue is examined, with the presence of 273P4B7 In the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 273P4B7 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The C presence of one or more perturbations in 273P4B7 gene products in the sample is an indication of cancer susceptibility (or the 00 emergence or existence of a tumor).

I

N The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging CK aggressiveness of a tumor comprises determining the level of 273P487 mRNA or 273P4B7 protein expressed by tumor cells, comparing the level so determined to the level of 273P4B7 mRNA or 273P487 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 273P4B7 mRNA or 273P4B7 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 273P4B7 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the Integrity of 273P4B7 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 273P487 mRNA or 273P487 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 273P4B7 mRNA or 273P4B7 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 273P4B7 mRNA or 273P4B7 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 273P4B7 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 273P4B7 nucleotide and amino acid sequences In a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one 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 and diagnostic protocols known in the art. For example, another embodiment of the invention Is directed to methods for observing a coincidence between the expression of 273P4B7 gene and 273P4B7 gene products (or perturbations in 273P4B7 gene and 273P4B7 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the' status of a tissue sample. 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 etc.) as well as gross cytological observations (see, e.g., Bocking etal., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson etal., 1998, Mod.

Pathol. 11(6):543-51; Baisden et 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 273P4B7 gene and 273P487 gene products (or perturbations in 273P4B7 gene and 273P4B7 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment methods for observing a coincidence between the expression of 273P4B7 gene and 273P4B7 gene products (or perturbations in 273P4B7 gene and 273P4B7 gene products) and another factor associated with malignancy 0 entails detecting the overexpresslon of 273P487 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA C or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 273P4B7 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 273P4B7 and PSA.

mRNA in prostate tissue is examined, where the coincidence of 273P4B7 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.

Methods for detecting and quantifying the expression of 273P487 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art Standard methods for the detection and quantification of 273P4B7 mRNA include in situ hybridization using labeled 273P4B7 riboprobes, Northern blot and related 00 techniques using 273P4B7 polynucleotide probes, RT-PCR analysis using primers specific for 273P4B7, and other amplification N type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi- C quantitative RT-PCR Is used to detect and quantify 273P4B7 mRNA expression. Any number of primers capable of amplifying 273P4B7 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 273P4B7 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 273P4B7 The 273P487 protein and nucleic acd sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 273P4B7, as well as pathways activated by 273P487 via any 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 273P4B7 protein sequences.

In such methods, peptides that bind to 273P4B7 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 273P4B7 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 273P4B7 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.

Alternatively, cell lines that express 273P487 are used to Identify protein-protein interactions mediated by 273P4B7. Such interactions can be examined using immunoprecipitation techniques (see, Hamilton et al.

Biochem. Biophys. Res. Commun. 1999, 261:646-51). 273P4B7 protein can be immunoprecipitated from 273P4B7expressing cell lines using anti-273P4B7 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 273P487 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 3 S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 273P4B7 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 273P4B7's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA 0 molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 273P4B7-related ion channel, protein pump, or cell communication functions are identified and 0 used to treat patients that have a cancer that expresses 273P4B7 (see, Hille, Ionic Channels of Excitable Membranes 20d Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate 273P4B7 function can be C< identified based on their ability to bind 273P487 and activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligandsin which at least C one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 273P4B7 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library 00 O transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the C proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target S sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or O the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 273P487.

CAn embodiment of this invention comprises a method of screening for a molecule that Interacts with a 273P4B7 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 273P4B7 amino add sequence, allowing the population of molecules and the 273P4B7 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 273P4B7 amino acid sequence, and then separating molecules that do not interact with the 273P4B7 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 273P4B7 amino acid sequence. The identified molecule can be used to modulate a function performed by 273P4B7. In a preferred embodiment, the 273P4B7 amino acid sequence is contacted with a library of peptides.

X Therapeutic Methods and Compositions The identification of 273P4B7 as a protein that is normally expressed in a restricted set of tissues, but which Is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is-still able to survive. Examples of non-vital organs are ovary, breast, and prostate.

For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu Is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.

Despite the fact.that HER2neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, "cardiotoxicity," has merely S been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very C< low percentage of patients.

Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.

N Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth 00 factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.

Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.

SAccordingly, therapeutic approaches that inhibit the activity of a 273P4B7 protein are useful for patients suffering from a cancer that expresses 273P4B7. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 273P4B7 protein with its binding partner or with other proteins.

Another class comprises a variety of methods for inhibiting the transcription of a 273P4B7 gene or translation of 273P4B7 mRNA.

Anti-Cancer Vaccines The invention provides cancer vaccines comprising a 273P4B7-related protein or 273P4B7-related nucleic acid. In view of the expression of 273P4B7, cancer vaccines prevent and/or treat 273P4B7-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int J. Cancer 63:231-237; Fong et 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 273P4B7-related protein, or a 273P4B7-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 273P4B7 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, Heryin at Ann Med 1999 Feb 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response (e.g.

humoral andlor cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope an epltope present in a 273P4B7 protein shown in Figure 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope generates antibodies that specifically recognize that epitope). In a preferred method, a 273P4B7 immunogen contains a biological motif, see Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 273P4B7 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.

The entire 273P4B7 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g.,Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) microspheres (see, Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, Takahashi et al., Nature 344:873- 875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413,1988; Tam, J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et In: Concepts in vaccine development, Kaufmann, S. H. ed., p. 379, 1996; Chakrabarti, S. et a., 0 Nature 320:535, 1986; Hu, S. L. et Nature 320:537,1986; Kieny, et al, AIDS Bio/Technology 4:790, 1986; Top, F.

H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al, Virology 175:535, 1990), particles of viral or synthetic origin Kofier, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16,1993; Falo, L. Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. Vogel, F. and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; CGupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. etal., J. Immunol. 148:1585, 1992; Rock, K. Immunol.

Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et Science 259:1745, 1993; Robinson, H. L., CHunt, L. and Webster, R. Vaccine 11:957,1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. ed., p. 423, 1996; Cease, K. and Berzofsky, J. Annu. Rev. Immunol. 12:923, 1994 and Eldrldge, J. H. et al., O Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as Sthose of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

In patients with 273P4B7-associated cancer, the vaccine compositions of the invention can also be used in O conjunction with other treatments used for cancer, surgery, chemotherapy, drug therapies, radiation therapies, etc.

including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines: CTL epitopes can be determined using specific algorithms to identify peptides within 273P4B7 protein that bind corresponding HLA alleles (see Table IV; Epimer T M and Epimatrix

T

Brown University (URL brown.edu/Research/TB- HIV.Lablepimatrixepimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithl.bmi-heidelberg.com/).

In a preferred embodiment, a 273P4B7 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 XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif Table IV Table IV or Table IV andlor a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif Table IV or Table IV As is appreciated In the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., 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 acids can be 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 acids long, or longer than 25 amino acids.

Antibody-based Vaccines A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein a 273P4B7 protein) so that an Immune response is generated. A typical embodiment consists of a method for generating an immune response to 273P4B7 in a host, by contacting the host with a sufficient amount of at least one 273P4B7 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 273P4B7 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 273P4B7related protein or a man-made multiepitopic peptide comprising: administering 273P4B7 immunogen a 273P4B7 protein or a peptide fragment thereof, a 273P4B7 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 M peptide (Epimmune Inc., San Diego, CA; see, Alexander 0 et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 751-761 and Alexander et al., C Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an Individual against a 273P4B7 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 273P4B7 immunogen, the DNA sequence operatively linked to regulatory 0sequences 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 lipids; saponins; lectins; estrogenic compounds; Shydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be 00 administered that mimics 273P4B7, in order to generate a response to the target antigen.

_Nucleic Acid Vaccines:

C

Vaccine compositions of the Invention include nuclelc acid-mediated modalities. DNA or RNA that encode 0protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate Sprophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 273P487.

Constructs comprising DNA encoding a 273P4B7-related protein/immunogen 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 273P4B7 protein/immunogen. Alternatively, a vaccine comprises a 273P4B7-related protein.

Expression of the 273P4B7-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 273P4B7 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 et. 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 98104720. Examples of DNAbased delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, U.S. Patent No. 5,922,687).

For 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 practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang etal. J. Natl. Cancer Inst 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 273P4B7-related protein into the patient intramuscularly or Intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified 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 273P4B7-related nucleic acid molecule. In one embodiment, the fulllength human 273P4B7 cDNA is employed. In another embodiment, 273P4B7 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody 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 cells (DC) to present 273P4B7 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and 11-12, and are thus highly specialized antigen presenting cells.

0 In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et 1996, Prostate 28:65- O 69; Murphy et 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 273P4B7 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 273P4B7 peptides C capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 273P4B7 protein. Yet another embodiment involves engineering the overexpression of a 273P4B7 gene in Sdendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et at., 1997, Cancer Gene 0 Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA 00 Stransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp.

C Med. 186:1177-1182). Cells that express 273P4B7 can also be engineered to express immune modulators, such as GM- CSF, and used as immunizing agents.

XB.) 273P4B7 as a Target for Antibody-based Therapy 273P4B7 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 273P4B7 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 273P4B7-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 specifically reactive with domains of 273P4B7 are useful to treat 273P4B7-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

273P487 antibodies can be introduced into a patient such that the antibody binds to 273P4B7 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or 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 273P4B7, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or 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 273P4B7 sequence shown in Figure 2 or Figure 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, Slevers et al. Blood 93:11 3678- 3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell 273P4B7), the cytotoxic agent will exert its known biological effect cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known In the 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- 273P4B7 antibody) that binds to a marker 273P487) expressed, accessible to binding or localized on the cell surfaces.

A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 273P4B7, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 273P4B7 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 parenterally to said individual a S pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic andlor CN therapeutic agent.

Cancer immunotherapy using anti-273P4B7 antibodies can be done in accordance with various approaches that y) have been successfully employed In the treatment of other types of cancer, including but not limited to colon cancer (Aden et Sal., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et 1997, Blood 90:3179-3186, Tsunenarl et a., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi etal., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et 1996, Leuk. Res. 20:581-589), C colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et 1995, Cancer Res. 55:4398-4403), and 00 breast cancer (Shepard et 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of

\O

naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or 1131 to anti-CD20 antibodies Zevalin

T

IDEC

SPharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other Stherapeutic agents, such as Herceptinm (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 273P4B7 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamlcin Mylotarg", Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see US Patent 5,416,064).

Although 273P4B7 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 tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al.

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

Although 273P4B7 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 tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 273P4B7 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 273P4B7 imaging, or other techniques that reliably indicate the presence and degree of 273P4B7 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

Anti-273P4B7 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-273P4B7 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (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-273P4B7 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 273P4B7. 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-273P4B7 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

C

known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs Scan induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation 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 monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind 01 specifically to the target 273P4B7 antigen with high affinity but exhibit low or no antigenicity in the patient 00 Therapeutic methods of the invention contemplate the administration of single anti-273P4B7 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain S mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that C rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti- C N 273P4B7 mAbs can be administered 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- 273P4B7 mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-273P4B7 antibody formulations are administered via any route capable of delivering the ahtibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-273P4B7 antibody preparation, via an acceptable route of administration such as intravenous injection typically at a dose In the range of about 0.1, .2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin T M mAb in the treatment of metastatic breast cancer, 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- 273P4B7 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as 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 regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 273P4B7 expression in the patient, the extent of circulating shed 273P4B7 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.

Optionally, patients should be evaluated for the levels of 273P4B7 in a given sample the levels of circulating 273P4B7 antigen and/or 273P4B7 expressing cells) in order to assist in the determination of the most effective dosing regimen, 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).

Anti-ldlotypic anti-273P4B7 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 273P487-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-273P4B7 antibodies that mimic an epitope on a 273P4B7-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J.

Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can S be used In cancer vaccine strategies.

273P4B7 as a Target for Cellular Immune Responses C) 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 claimed peptides. A peptide can be present In a vaccine 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 00 peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with

NO

different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, recombinantly or by chemical synthesis.

SCarriers that 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, polyamlno acids such as poly L-lysine, poly L-glutamic 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 Freund's 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 tripalmitoyl-S-glycerylcysteinlyseryl- serine (P 3 CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-contalning (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000)) 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 cells that express or overexpress 273P4B7 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, It 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 embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, CA) molecule (described in U.S. Patent Number 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization 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. Vaccine 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 polyepltopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles 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.

S1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 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 Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one CN or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

1 Epitopes are selected that have the requisite binding affinity established to be correlated with 0 immunogenicity: for HLA Class I an ICso of 500 nM or less, often 200 nM or less; and for Class II an ICso of 1000 nM or less.

00 Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are l 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, Spopulation 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 providing 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 multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

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 acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concem is a junctional epitope that is a "dominant epitope." A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

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 class I binding peptide or the entire 9-mer core of a class II binding peptide'be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

X.C.1. Minigene Vaccines A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids 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.

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

67:348,1993; Hanke, R. et al., Vaccine 16:426,1998. For example, a multi-epitope DNA plasmid encoding supermotifandlor motif-bearing epitopes derived 273P4B7, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 0273P4B7 (see Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The Immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of c CTL induction responses against the epitopes tested. Further, the Immunogenicity of DNA-encoded epitopes in viv can be 00 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 ri recognized cells expressing the encoded epitopes.

SFor example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid 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 acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic 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.

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

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 cloning site for minigene Insertion; a polyadenylation signal for efficient transcription termination; an E. co/i origin of replication; and an E.

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

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 inclusion 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. co/l strain, and DNA is prepared using standard techniques.

The orientation and DNA sequence of the minigene, as well as all other elements included 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 immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

0In 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 C) polypeptides that could beneficially enhance the immune response if co-expressed include cytokines IL-2, IL-12, GM- SCSF), cytokineinducing molecules LelF), costimulatory molecules, or for HTL responses, pan-DR binding proteins C- (PADRE

T

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 Cthan that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune 00 Sresponse by co-expression of immunosuppressive molecules TGF-P) may be beneficial in certain diseases.

C- Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E coi, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker 0 flasks or a bloreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercolled 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 clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an altemative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, as described by WO 93/24640; Mannino Gould-Fogerite, BioTechniques 6(7): 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.

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 formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (5sCr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 5 Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity 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, s5Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs.

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

O Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S.

C- 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 epitopes of the Invention can be incorporated into a viral vector such as vaccinia.

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

00 For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence S which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be <1 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 acids 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.

SIn certain embodiments, the T helper peptide Is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions.830-843 (QYIKANSKFIGITE; SEQ ID NO: 26), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 27), and Streptococcus 18kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 28). 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 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 PADRE T M Epimmune, Inc., San Diego, CA) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAa (SEQ ID NO: 29), where is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either o-alanine or L-alanlne, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, 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 palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides 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. Lipids have been identified as agents capable of priming 1 CTL in vivo. For example, palmitic acid residues can be attached to the e-and a- amino groups of a lysine residue and then linked, via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide.

The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to s- and a- amino groups of Lys, which is attached via linkage, CK Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S- C glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate 00 peptide (see, Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to PaCSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, C because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor HTL Peptides An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail 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 Progenipoietin T M (Pharmacia-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 peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 273P4B7.

Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be Included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 273P4B7.

X.D. Adoptive Immunotherapy Antigenic 273P4B7-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 not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL 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 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 cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 273P4B7. 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 will 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 O the patient, and the judgment of the prescribing physician.

SFor pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 273P4B7. The peptides or DNA encoding Sthem 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 273P4B7-associated cancer.

This Is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The CK1 embodiment of the vaccine composition Including, but not limited to embodiments such as peptide cocktails, 00 polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses CK 273P4B7, a vaccine comprising 273P4B7-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient o 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 Wg to about 50,000 pg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, 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.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease 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 felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for 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 from about 500 pg to about 50,000 pg per 70 kilogram patient This is followed by boosting dosages of between about jg to about 50,000 pg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's 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 carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, water, buffered water, 0.8% saline, 0.3% glycine, 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 lyophilized preparation being Scombined with a sterile solution prior to administration.

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

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, from less Sthan 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.

00 \O A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a Shuman unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, Remington's SPharmaceutical Sciences, 17" t 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 jig, generally 100-5,000 pig, for a 70 kg patient.

For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic 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 fowlpox virus administered at a dose of 5-107 to 5x109 pfu.

For antibodies, a treatment generally involves repeated administration of the anti-273P4B7 antibody preparation, via an acceptable route of administration such as intravenous injection typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated.

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- 273P4B7 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill 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 immunogenicity of a substance, the degree of 273P487 expression in the patient, the extent of circulating shed 273P4B7 antigen, the desired steady-state 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 Non-limiting preferred human unit doses are, for example, 500pg Img, 1mg 50mg, 50mg 100mg, 100mg 200mg, 200mg 300mg, 400mg 500mg, 500mg 600mg, 600mg 700mg, 700mg 800800mg00mg 900mg, 900mg 1g, or 1mg 700mg. In certain embodiments, the dose is in a range of 2-5 mglkg body weight, with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3,4, 5, 6, 7, 8, 9, 10mg/kg body weight followed, in 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 polynucleotides 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 polynucleotide, molecular weight of the polynucleoide 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 polynucleotide 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 S independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, S 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: C 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 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 administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides 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 00 parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104

NO

cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5 x 1010 cells.

A dose may also about 106 cells/m 2 to about 1010 cells/m 2 or about 106 cells/m 2 to about 108 cells/m 2 SProteins(s) of the Invention, and/or nucleic 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 lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Uposomes include 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 monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids Is generally guided by consideration liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S.

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

For targeting cells of the immune system, a ligand 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 suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according 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 carriers may be used which include, for example, pharmaceutical 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 formed by incorporating any of the normally employed excipients, 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 peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% 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, linolelc, 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 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, lecithln for intranasal delivery.

0 XI.) Diagnostic and Prognostic Embodiments of 273P4B7.

As disclosed herein, 273P4B7 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, C both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled "Expression analysis of 273P4B7 in normal tissues, and patient specimens").

S273P4B7 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by S emedical practitioners for years to identify and monitor the presence of prostate cancer (see, Merrill et al., J. Urol. 163(2): 00 O 503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat Cancer Inst. 91(19): 1635- CK 1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., F; Tulchinsky et Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, Sthis disclosure of 273P4B7 polynucleotides and polypeptides (as well as 273P4B7 polynucleotide probes and anti-273P487 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 assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 273P4B7 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, Sharief et 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 level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 273P4B7 polynucleotides described herein can be utilized in the same way to detect 273P4B7 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies 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 Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, Alanen et al., Pathol. Res. Pract 192(3):233-7 (1996)), the 273P4B7 polypeptides described herein can be utilized to generate antibodies for use in detecting 273P487 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 or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 273P4B7 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 273P4B7-expresslng cells (lymph node) is found to contain 273P4B7-expressing cells such as the 273P4B7 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph hode and bone metastasis, respectively, this finding is. indicative of metastasis.

Alternatively 273P4B7 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 273P4B7 or express 273P4B7 at a different level are found to express 273P4B7 or have an increased expression of 273P487 (see, the 273P4B7 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-273P4B7) such as PSA, PSCA etc. (see, Alanen et al., Pathol. Res. Pract. 192(3): 233- 237(1996)).

O The use of immunohlstochemistry to identify the presence of a 273P4B7 polypeptide within a tissue section can C= indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.

The 273P4B7 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular r morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane 00 proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.

C The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et The Breast Journal, 7; 40-45 (2001); Zhang at al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemlstry, 45:1547-1557 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, etal, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).

Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information conceming the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 273P4B7, the 273P4B7 protein and immune responses related thereto are very useful.

Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 273P487 protein and immune responses related thereto very useful. Use of the 273P4B7 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.

Immunohistochemical reagents specific to 273P4B7 are also useful to detect metastases of tumors expressing 273P4B7 when the polypeptide appears in tissues where 273P4B7 is not normally produced.

Thus, 273P4B7 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.

Just as PSA polynudeotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 273P4B7 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides 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 polynucleotide 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 polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide 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 273P4B7 In normal tissues, and patient specimens," where a 273P4B7 polynucleotide fragment is used as a probe to show the expression of 273P4B7 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for 0 the corresponding mRNAs in PCR and Northern analyses (see, Sawai et al, Fetal Diagn. 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)).

Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleoide sequence a 273P4B7 polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.

SFurthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 273P4B7 polypeptide fragments and polypeptide Sanalogs 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 00 \O such as fusion proteins being used by practitioners (see, Current Protocols In Molecular Biology, Volume 2, Unit 16, SFrederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in Sorder to generate immune responses specific for different portions of a polypeptide of interest (see, U.S. Patent No.

5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 273P4B7 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill 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 273P4B7 polypeptide shown in Figure 3).

As shown herein, the 273P4B7 polynucleotides and polypeptides (as well as the 273P4B7 polynucleotide probes and anti-273P4B7 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 273P4B7 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 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 Pathol. Res. Pract 192(3): 233-237 (1996)), and consequently, materials such as 273P4B7 polynucleotides and polypeptides (as well as the 273P4B7 polynucleotide probes and anti- 273P4B7 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 273P4B7 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 273P4B7 gene maps (see the Example entitled "Chromosomal Mapping of 273P4B7" below). Moreover, in addition to their use in diagnostic assays, the 273P4B7-related proteins and polynucleotides 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, 273P4B7-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 273P4B7. For example, the amino acid or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to a 273P4B7 antigen. Antibodies or other molecules that react with 273P4B7 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 273P4B7 Protein Function O The invention includes various methods and compositions for inhibiting the binding of 273P4B7 to Its binding C partner or its association with other protein(s) as well as methods for inhibiting 273P487 function.

)XII.A.) Inhibition of 273P4B7 With Intracellular Antibodies In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 273P4B7 are introduced into 273P487 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti- 273P4B7 antibody is expressed intracelluarly, binds to 273P4B7 protein, and thereby inhibits its function. Methods for ri engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as 00 "'ntrabodles", are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory N activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and ri Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant 0cell surface receptors (see, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J.

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

Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, 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 polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to 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 include a nuclear localization signal. Lipid moieties are joined 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, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 273P4B7 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 273P4B7 intrabodies in order to achieve the desired targeting. Such 273P4B7 intrabodies are designed to bind specifically to a particular 273P4B7 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 273P4B7 protein are used to prevent 273P4B7 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus preventing 273P4B7 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 273P4B7 with Recombinant Proteins In another approach, recombinant molecules bind to 273P4B7 and thereby inhibit 273P4B7 function. For example, these recombinant molecules prevent or inhibit 273P4B7 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 273P4B7 specific antibody molecule. In a particular embodiment, the 273P4B7 binding domain of a 273P4B7 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 273P4B7 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the CH 2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 273P4B7, whereby the dimeric fusion protein specifically binds to 273P4B7 and blocks 273P4B7 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

SXII.C.) Inhibition of 273P4B7 Transcription or Translation C The present invention also comprises various methods and compositions for inhibiting the transcription of the 273P4B7 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 273P4B7 C mRNA into protein.

00 In one approach, a method of inhibiting the transcription of the 273P4B7 gene comprises contacting the 273P4B7 gene with a 273P4B7 antisense polynucleotide. In another approach, a method of inhibiting 273P4B7 mRNA translation CK comprises contacting a 273P4B7 mRNA with an antisense polynucleotide. In another approach, a 273P4B7 specific ribozyme is used to cleave a 273P4B7 message, thereby inhibiting translation. Such antisense and ribozyme based O methods can also be directed to the regulatory regions of the 273P4B7 gene, such as 273P4B7 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 273P4B7 gene transcription factor are used to inhibit 273P4B7 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above, The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 273P4B7 by interfering with 273P4B7 transcriptional activation are also useful to treat cancers expressing 273P4B7. Similarly, factors that interfere with 273P4B7 processing are useful to treat cancers that express 273P4B7. Cancer treatment methods utilizing such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 273P4B7 antisense, ribozyme, polynucleotides encoding intrabodies and other 273P4B7 inhibitory molecules).

A number of gene therapy approaches are known in the art. Recombinant vectors encoding 273P4B7 antisense polynucleotides, ribozymes, factors capable of interfering with 273P4B7 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 vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 273P4B7 to a binding partner, etc.

In vivo, the effect of a 273P4B7 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic 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 (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 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.

SIn vivo 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 Sthe 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 C compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally CK non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard 00 pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16 1 Edition, A. Osal., Ed., 1980).

CTherapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic Scomposition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, andlor 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 vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

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

XIII.) Identification, Characterization and Use of Modulators of 273P4B7 Methods to Identify and Use Modulator 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 identify 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 differentially expressed gene.

Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind andlor 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 pattem 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 cells containing these sequences are used in screening assays, Ssuch 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

L

C N Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Held, Genome Res 6:986- 94,1996).

LC The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer 00 proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for N compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the Invention. This is cl done on a gene itself or by evaluating the effect of drug candidates on a "gene expression profile" or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow 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 are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. "Modulation" in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original 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 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 is 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 acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself Is monitored, through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow 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 acid probes are attached to biochips 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 polynucleotide 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 subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds," as compounds for screening, or as therapeutics.

O In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a i cancer polypeptide 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.

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

00 If 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 and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nudeotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or SThe target sequence can be labeled with, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, 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 binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin 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 acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid 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 include 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, nuclease 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.

SBiological Activity-related Assays SThe Invention provides methods identify or screen for a compound that modulates the activity of a cancer-related Sgene or protein of the Invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention.

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

In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of C1 physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, 00 pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells cell-cell contacts). In IND another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that C1 modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify Scritical structural features of the compound.

In one embodiment, a method of modulating inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating inhibiting) cancer is 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.

In 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, senescence, 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, o( random or directed digests of protelnaceous 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 especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, substrates for enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators Normal cells require a solid substrate to attach and grow. When cells 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.

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

Evaluation of Contact Inhibition and Growth Density Limitation 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 foci. Alternatively, labeling index with 3 H)-thymidine at saturation density is used to measure density limitation of growth, 00 similarly an MJT or Alamar blue.assay will reveal proliferation capacity of cells and the the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.

SIn this assay, labeling Index with 3 H)-thymidine at saturation density is a preferred method of measuring density C1 limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with 3 H)-thymidine is determined by Incorporated cpm.

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

Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators Transformed cells have lower serum dependence than their normal counterparts (see, Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle etal., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of 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, Angiogenesis 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. Biol. Chem. 249:4295-4305 (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 Matrigel to Identify and Characterize Modulators The degree of Invasiveness into Matrigel 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.

SBriefly, the level of invasion of host cells 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 1251 and counting the radioactivity on Sthe distal side of the filter or bottom of the dish. See, Freshney (1984), supra.

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

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

To prepare transgenic chimeric animals, mice, a DNA construct is introduced into the nuclei of embryonic 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 containing the introduced genetic lesion (see, Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas 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, Giovanella 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 10 6 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., Student's 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 Western 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 nucleic 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 ri gene such as luciferase, 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. Curr.

SOpin. Blotechnol. 1998: 9:624).

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

00 In one embodiment, screening for modulators of expression of specific gene(s) Is performed. Typically, the 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 Sexpressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better C=K evaluate structure activity relationships.

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

Thus, 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 T 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 sterically block either the ligand 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 ligand/binding 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.

Alternatively, 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 S assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding Qassays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, Setc.) and the like.

A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the ri invention 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 Cr a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various 00 blocking and washing steps can be utilized as appropriate.

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

CK Alternatively, more than one component is labeled with different labels, 1125, for the proteins and a fluorophor for the compound. Proximity reagents, quenching or energy transfer reagents are also useful.

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

Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the 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 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 alternative 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 capable 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 0 drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins.

C 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 the activity of such proteins.

7) Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow 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 samples can be counted in a scintillation counter to determine the amount of bound compound.

00 A variety 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 Sor background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, Snuclease Inhibitors, anti-microblal agents, etc., can be used. The mixture of components is added in an order that provides N for the requisite binding.

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

Polynucleotide 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 91/04753. Suitable ligand-binding 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 oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be Introduced into a cell containing the target nucleic acid sequence, by formation of a polynucleotide-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 Nucleotides In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.

In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties 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 nucleotides of the invention. See, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Blosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill In the art Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, Stein Q &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

Ribozymes In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer- CN associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different 00 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 rC properties of different ribozymes).

The general features of hairpin ribozymes are described, in Hampel et al., Nuc. Acids 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 those of skill in the art (see, WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699- 703 (1995); Leavitt et al., Human Gene 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 acid 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 accomplished, PCT US97/01019. 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, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.

SMethods of Identifying Characterizing Cancer-associated Sequences Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for Identifying cells containing variant cancer genes, determining the presence of, all or part, the sequence of at least one endogenous cancer gene in 00 00 a cell. 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 Individual. This is generally done in at least one tissue of the individual, a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the C1 sequence of the sequenced gene to a known cancer gene, a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, 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 abnormalities such as translocations, and the like are Identified in the cancer gene locus.

XIV.) KitslArtlcles of Manufacture For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are 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, along with a label or insert comprising instructions for use, such as a use described herein. 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 protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotinbinding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable 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 or with the container to indicate that the composition is used for a specific therapy or nontherapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for 0 either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an Sinsert(s) or label(s) which is included with oron the kit The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a Slabel can be associated with a container when it is present within a receptacle or carrier that also holds the container, as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a Scondition, such as a neoplasia of a tissue set forth in Table I.

The terms "kit" and "article of manufacture" can be used as synonyms.

SIn another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid 00 sequence(s), small molecule(s), nucleic acid 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 Smanufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) andlor antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of282P1G3 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that 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 a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 282P1 G3 and modulating the function of 282P1 G3.

The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including 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 is intended to limit the scope of the invention.

Example 1: SSH-Generated Isolation of cDNA Fragment of the 273P4B7 Gene To isolate genes that are over-expressed in lung cancer the Suppression Subtractive Hybridization (SSH) procedure was used using cDNA derived from lung cancer tissues. The 273P487 SSH cDNA sequence was derived from lung tumor minus cDNAs derived from normal lung. The 273P4B7 cDNA was identified as highly expressed in cancer.

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

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

SRNA Isolation: u Tissues were homogenized in Trizol reagent (Life 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 Sspectrophotometric analysis 2601280 nm) and analyzed by gel electrophoresis.

Oliqonucleotides: The following HPLC purified oligonucleotides were used.

0 0 DPNCDN (cDNA synthesis primer):

NO

5'TTTTGATCAAGCTT3o3' (SEQ ID NO: Adaptor 1: 0 5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 31) C 3'GGCCCGTCCTAG5' (SEQ ID NO: 32) Adaptor 2: 5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 33) (SEQ ID NO: 34) PCR primer 1: 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: Nested primer (NP)1: 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 36) Nested primer (NP)2: 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 37) Suppression Subtractive Hybridization: Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in cancer. The SSH reaction utilized cDNA from lung cancer and normal tissues.

The gene 273P4B7 sequence was derived from lung cancer minus normal lung and a mix of 9 normal tissues cDNA subtraction. The SSH DNA sequence (Figure 1) was Identified.

The cDNA derived from normal lung mixed with a pool of 9 normal tissues was used as the source of the "driver" cDNA, while the cDNA from lung cancer was used as the source of the "tester" cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 pg of poly(A)* RNA isolated from the relevant tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804- The resulting cDNA was digested with Dpn II for 3 hrs at 370C. Digested cDNA was extracted with phenollchloroform and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from normal lung with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 pl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 pi of water. The diluted cDNA (2 pi, 160 ng) was then ligated to 2 pi of Adaptor 1 and Adaptor 2 (10 pM), in separate ligation reactions, in a total volume of 10 pi at 160C overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 pl of 0.2 M EDTA and heating at 72oC for 5 min.

O The first hybridization was performed by adding 1.5 pi (600 ng) of driver cDNA to each of two tubes containing 1.5 pi rN ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 pi, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98oC for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68oC. The two hybridizations were then mixed together with an additional 1 pl of fresh denatured driver cDNA and were allowed to hybridize O overnight at 680C. The second hybridization was then diluted in 200 pi of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 700C for 7 min. and stored at -200C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH: 0To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR 00 c reaction 1 pi of the diluted final hybridization mix was added to 1 il of PCR primer 1 (10 pM), 0.5 pl dNTP mix (10 pM), 2.5 pl F x reaction buffer (CLONTECH) and 0.5 pl 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 pl. PCR 1 was conducted using the following conditions: 75oC for 5 min., 940C for 25 sec., then 27 cycles of 94oC for 10 sec, 66oC for 30 sec, S72oC for 1.5 min. Five separate primary PCR reactions were performed for each experiment. .The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 pi 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 performed using 10-12 cycles of 940C for 10 sec, 6800C for 30 sec, and 72oC for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture ovemight To identify inserts, PCR amplification was performed on 1 pl 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 nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis: First strand cDNAs can be generated from 1 pg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturers protocol was used which included an incubation for 50 min at 42oC with reverse transcriptase followed by RNAse H treatment at 370C 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 cDNAs from multiple tissues was performed by using the primers 5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 38) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 39) to amplify P-actin. First strand cDNA (5 lp) were amplified in a total volume of 50 pl containing 0.4 pM primers, 0.2 pM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCI, pH8.3) and 1X Klentaq DNA polymerase (Clontech). Five pi 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 94C0 for 15, 65oC for 2 min, 72oC for 5 sec. A final extension at 72oC 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 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 273P4B7 gene,.5 pl of normalized first strand cDNA were analyzed by PCR using S 26, and 30 cycles 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 are listed below: 273P4B7.1 GCTAGTGCTCAGAATACCAGACTATGG 3' (SEQ ID NO: 273P4B7.2 CGCTTGACATAAAAAGTGCAGATCC 3' (SEQ ID NO: 41) A typical RT-PCR expression analysis is shown in Figure 14(A) and 14(B). First strand cDNA was prepared from vital pool 1 00 S (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas rC- cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles of amplification. Expression of 273P4B7 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas.

Example 2: Full Length Cloning of 273P4B7 The 273P4B7 SSH cDNA sequence was derived from a subtraction consisting of lung cancer minus a normal tissues.

The SSH cDNA sequence of 170 bp (Figure 1) was designated 273P4B7.

273P4B7 v.1 of 4194 bp was cloned from lung cancer, revealing an ORF of 1250 amino acids (Figure 2 and Figure 3).

Other variants of 273P4B7 were also identified and these are listed in Figure 2 and Figure 3.

273P4B7 v.1, v.3, v.7, and v.8 code for identical proteins of 1250 amino acids in length. 273P4B7 v.4, v.5 and v.6 differ from 273P4B7 v.1 by one amino acid as shown in Figure 2. 273P4B7 v.2 is a splice variant of 273P4B7 v.1 and code for a protein of 1127 amino acids.

273P4B7 v.1 shows 99% over only 1106 amino acids to the unnamed protein AK074719. The nucleic acid sequence of 273P4B7 v.1 aligns with 99% identity to the nucleotide position 159-4194, to cDNA FLJ31932 fis, clone NT2RP7006296, weakly similar to EXCISION REPAIR PROTEIN ERCC-6.

273P4B7 v.1 shows 72% identity to the mouse protein BC004701 shown to be a member of the family of DEAD-like helicase superfamily. Members of this family include the DEAD and DEAH box helicases. Helicases are involved in unwinding nucleic acids. The DEAD box helicases are involved in various aspects of RNA metabolism, including nuclear transcription, pre mRNA splicing, ribosome biogenesis, nuceocytoplasmic transport, translation, RNA decay and organellar gene expression.

Example 3: Chromosomal Mapping of 273P4B7 Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent In situ hybridization (FISH), human/hamster 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 Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).

273P4B7 maps to chromosome Xq13.1 using 273P4B7 sequence and the NCBI BLAST tool located on the World Wide Web at (.ncbi.nlm.nih.gov/genome/seqlpage.cgi?F=HsBlast.html&&ORG=Hs).

Example 4: Expression Analysis of 273P4B7 in Normal Tissues and Patient Specimens Expression analysis by RT-PCR demonstrated that 273P4B7 is strongly expressed in patient cancer specimens (Figure 14). First strand cDNA was prepared from normal tissues (bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon and stomach), and from pools of patient cancer specimens (pancreas cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, pancreas cancer pool, prostate cancer xenograft pool, prostate metastasis to lymph node, bone and melanoma cancer pool, cervical cancer pool, lymphoma cancer pool, stomach cancer pool, uterus cancer pool, and multixenograft pool). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 22, 26 and 30 cycles of amplification. In Figure 14(A) picture of the RT-PCR agarose gel is shown. In Figure 14(B) PCR products were quantitated using the Alphalmager software. Results show strong of expression N of 273P4B7 in prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary 00 cancer pool, breast cancer pool, cancer metastasis pool, pancreas cancer pool, prostate cancer xenograft pool, prostate metastasis to lymph node, bone and melanoma cancer pool, cervical cancer pool, lymphoma cancer pool, stomach cancer C pool, uterus cancer pool and multi-xenograft pool (prostate cancer, kidney cancer and bladder cancer xenograft pool). In Snormal tissues, 273P4B7 is predominantly expressed in testis and not in any other normal tissue tested.

SExtensive expression of 273P4B7 in normal tissues is shown in Figure 15. Two multiple tissue northern blots (Clontech) both with 2 pg of mRNANane were probed with the 273P4B7 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 7kb 273P4B7 transcript in normal testis but not in the other normal tissues tested.

Expression of 273P4B7 in pancreas, ovary and testis cancer patient specimens is shown in Figure 16. RNA was extracted from normal pancreas (NPa), normal ovary normal testis (NTe), pancreas cancer patient specimen (P1), ovary cancer patient specimen (P2,P3,P4), and testis cancer patient specimen (P5,P6,P7). Northern blot with 10 pg of total RNAlane was probed with 273P4B7 SSH sequence. Size standards in kilobases (kb) are indicated on the side. 273P4B7 transcript was detected in the patient specimens, but not in the normal tissues.

Figure 17 shows 273P4B7 expression in cervical cancer patient specimens. In Figure 17(A), total RNA was extracted from cervical dancer patient specimens (T1-T7), and HeLa cell line. Northem blot with 10 pg of total RNA/lane was probed with 273P4B7 SSH sequence. Size standards in kilobases (kb) are indicated on the side. 273P4B7 transcript was detected in all patient specimens tested as well as in the Hela cell line. In Figure 17(B), first strand cDNA was prepared from a panel of cervical cancer patient specimens, normal cervix and HeLa cervical cell line. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles 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 273P4B7 in most of the cervical cancer tissues tested.

Expression of 273P4B7 in bladder cancer patient specimens is shown in figure 18. First strand cDNA was prepared from a panel of bladder cancer patient specimens, normal bladder and bladder cancer cell lines (UM-UC-3, TCCSUP, J82). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel Figure 18(A), and PCR products were quantitated using the Alphalmager software Figure 18(B). Expression was recorded as absent, low, medium or high. Results show expression of 273P4B7 in most of the bladder cancer tissues tested, but not in the normal bladder tissues.

Expression of 273P4B7 in colon cancer patient specimens is shown in figure 19. First strand cDNA was prepared from a panel of colon cancer patient specimens, normal colon, and colon cancer cell lines (LoVo, CaCo-2, SK-CO1, Colo205, and T284). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles 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 high. Results show S expression of 273P4B7 in the majority of the colon cancer tissues tested, but not in the normal colon tissues. Expression Cl was also detected in the cell lines LoVo, CaCo-2, SK-CO1, Colo205, but not in the T284 cell line.

Figure 20 shows 273P4B7 expression in ovary cancer patient specimens. First strand cDNA was prepared from a panel of ovarian cancer patient specimens, normal ovary and ovarian cancer cell lines (OV-1063, PA-1, SW626).

0 Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 273P4B7, was performed at 26 and 30 cycles 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 high. Results show expression of 273P4B7 in the majority of ovary cancer tissues tested as well as in the cell lines, but not in normal ovary.

00 The restricted expression of 273P4B7 in normal tissues and the expression detected in cancer patient specimens

\O

suggest that 273P4B7 is a therapeutic target and a diagnostic marker for human cancers.

SExample 5: Transcript Variants of 273P4B7 As used herein, the term variant comprises Transcript variants and Single Nucleotide Polymorphisms (SNPs).

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. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and Is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding or 3' end) portions, from the original transcript. Transcript variants can code for the same, similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the 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 times. Proteins encoded by transcript variants can have similar or different subcellular 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 cloning experiments, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or Indirect identity with each other. Second, ESTs in the same cluster 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 yet a full-length clone, that portion of the variant Is very useful as a research tool, for antigen generation and for further cloning of the full-length splice variant, using techniques known to those skilled In the art.

Moreover, computer programs are available to those skilled in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH Salamov and V.

Solovyev, "Ab initio gene finding in Drosophila genomic DNA," Genome Research. 2000 April;10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrallForm) and GenScan (URL genes.mit.edu/GENSCAN.html). 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 Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad. Sci 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 cloning, 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 al, Differential splicing of pre-messenger RNA produces Smultiple forms of mature caprine alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR-based Validation: Wellmann S, et Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, et al., Discovery of new human beta- C defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation: Brigle, et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem C Biophys Acta. 1997 Aug 7; 1353(2): 191-8).

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

CDisclosed herein is that 273P4B7 has a particular expression profile related to cancer (See, Table Alternative transcripts and splice variants of 273P4B7 are also be involved in cancers in the same or different tissues, thus serving as Stumor-associated markers/antigens.

Using the full-length gene and EST sequences, four additional transcript variants were identified, designated as 273P4B7 v.2, v.9 and v.10. The boundaries of exons in the original transcript, 273P4B7 v.1 are shown in Table LI. The structures of the transcript variants are shown in Figure 10. Variant 273P487 v.2 added 22 bases to the 5' end of exoni and an additional exon in the first intron of variant v.1. Variants v.9 and v.10 were shorter and matched part of the last exon of v.1, with a few different base pairs.

Tables Lll(a)-(d) through are set forth on a variant-by-variant bases. Tables Lll(a)-(d) show nucleotide sequence of the transcript variants. Tables LIll(a)-(d) show the alignment of the transcript variant with nucleic acid sequence of 273P4B7 v.1. Table LIV(a)-(d) lay out amino acid translation of the transcript variant for the identified reading frame orientation. Table display alignments of the amino acid sequence encoded by the splice variant with that of 273P4B7 v.1.

Example 6: Single Nucleotide Polymorphisms of 273P4B7 A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C 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. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called 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 Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, "The use of single nucleotide 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(1):15-26).

SNP are identified by a variety of art-accepted methods Bean, "The promising voyage of SNP target C discovery," Am. Clin. 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 pharmacogenetic studies by high-throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNP can be 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 Ssequences from different DNA samples. With the rapid accumulation.of sequence data in public and private 00 databases, one can discover SNP by comparing sequences using computer programs Gu, L. Hillier and P. Y.

Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998; 12(4):221-225). SNP can C be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays Y. Kwok, "Methods for genotyping single nucleotide Spolymorphisms," Annul. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, 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, six SNP were identified in the transcript, 273P4B7 v.1, as shown in Table LVI. The transcripts or proteins with alternative allele were designated as variant 273P4B7 v.3 through v.8, as shown in Table LVI and Figure 12. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 273P4B7 v.2, as listed in table LVI) that contains the site of the SNP, as laid out in Figures 11 and 12.

Example 7: Production of Recombinant 273P4B7 in Prokarvotic Systems To express recombinant 273P4B7 and 273P4B7 variants in prokaryotic cells, the full or partial length 273P4B7 and 273P4B7 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 273P4B7 variants are expressed: the full length sequence presented in Figures 2 and 3, 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 273P4B7, variants, or analogs thereof.

A. In vitro transcription and translation constructs: pCRI: To generate 273P4B7 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 273P4B7 cDNA The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 273P4B7 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 273P4B7 at the RNA level. Transcribed 273P4B7 RNA representing the cDNA amino acid coding region of the 273P4B7 gene is used in in vitro translation systems such as the TnT" Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize 273P4B7 protein.

B. Bacterial Constructs: pGEX Constructs: To generate recombinant 273P4B7 proteins in bacteria that are fused to the Glutathione Stransferase (GST) protein, all or parts of the 273P4B7 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 273P4B7 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. 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 cleavage site, such as the PreScissionT M recognition site in pGEX-6P-1, may be employed such that it permits S cleavage of the GST tag from 273P4B7-related protein. The ampicillin resistance gene and pBR322 origin permits selection C= and maintenance of the pGEX plasmids in E coli.

CpMAL Constructs: To generate, in bacteria, recombinant 273P4B7 proteins that are fused to maltose-binding protein (MBP), all or parts of the 273P4B7 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant 273P4B7 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxylterminus. The MBP and 6X His tags permit purification of the recombinant protein from Induced bacteria with the appropriate

C

affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6X His epitope tag is 00 generated by adding.6 histidine codons to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 273P4B7. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the

C

cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs: To express 273P4B7 in bacterial cells, all or parts of the 273P4B7 cDNA protein coding Ssequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 273P4B7 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag T that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 273P4B7 protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs: pESC Constructs: To express 273P4B7 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 273P4B7 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either FlagT or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein Interactions of 273P4B7. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations that are found when expressed in eukaryotic cells.

pESP Constructs: To express 273P4B7 in the yeast species Saccharomyces pombe, all or parts of the 273P4B7 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 273P4B7 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 FlagTM epitope tag allows detection of the recombinant protein with anti- FlagTM antibody.

Example 8: Production of Recombinant 273P4B7 in Higher Eukarvotic Systems A. Mammalian Constructs: To express recombinant 273P4B7 in eukaryotic cells, the full or partial length 273P4B7 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 273P4B7 were expressed in these constructs, amino acids 1 to 1250 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 273P4B7 v.1, v.4, v.5, and v.6; amino acids 1 to 1127 of v.2 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 273P4B7 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-273P4B7 polyclonal serum, described herein.

SpcDNA4/HisMax Constructs: To express 273P4B7in mammalian cells, a 273P4B7 ORF, or portions thereof, of N 273P4B7 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovlrus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six /histidlne (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth Shormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

c0 pcDNA3.11MycHis Constructs: To express 273P4B7 in mammalian cells, 273P4B7 ORF, or portions thereof, of 273P4B7 with a consensus Kozak translation initiation site was cloned 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 carboxyl-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 origin for eplsomal 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 resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.11CT-GFP-TOPO Construct: To express 273P4B7 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 273P4B7 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-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 ColEI origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an aminoterminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 273P4B7 protein.

PAPtaL A 273P4B7 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 273P4B7 protein while fusing the IgGic 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 273P4B7 protein. The resulting recombinant 273P4B7 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 273P4B7 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-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. coll.

A 273P4B7 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 273P4B7 protein with an amino-terminal IgGic signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 273P4B7 protein is 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 273P4B7 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. coll.

O PsecFc: A 273P4B7 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by K cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, Califomia). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 273P4B7 proteins, while fusing the IgGK signal sequence to N-terminus. 273P4B7 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 273P4B7 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as

L

C immunogens or to identify proteins such as ligands or receptors that interact with 273P4B7 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian

L

C cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E coli.

00 pSRa Constructs: To generate mammalian cell lines that express 273P4B7 constitutively, 273P4B7 ORF, or portions thereof, of 273P4B7 were cloned into pSRa constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRa constructs into the 293T-10A1 packaging line or co-transfection of pSRa and a helper plasmid S(containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of CK1 mammalian cell lines, resulting in the integration of the cloned gene, 273P4B7, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRa constructs were made that fuse an epitope tag such as the FLAG T tag to the carboxyl-terminus of 273P4B7 sequences to allow detection using anti-Flag antibodies. For example, the FLAGTM sequence 5' gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 42) 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 273P4B7 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 273P4B7.

High virus titer leading to high level expression of 273P4B7 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 273P4B7 coding sequences or fragments thereof are amplified by PCR and subconed into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 273P4B7 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 273P4B7 in mammalian cells, coding sequences of 273P4B7, or portions thereof, are cloned 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 273P4B7. These vectors are thereafter used to control expression of 273P4B7 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems To generate recombinant 273P487 proteins in a baculovirus expression system, 273P4B7 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-273P4B7 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 273P4B7 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified S baculovirus. Recombinant 273P4B7 protein can be detected using anti-273P4B7 or anti-His-tag antibody. 273P4B7 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 273P4B7.

Example 9: Antigenicity Profiles and Secondary Structure Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9 depict graphically five amino acid profiles of 273P4B7 variant 1, each assessment available by accessing the ProtScale website located on the World Wide Web at (www.expasy.ch/cgibin/protscale.pl) on the ExPasy molecular biology server.

These profiles: Figure 5, Hydrophilicity, (Hopp Woods 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 00 3828); Figure 6, Hydropathicity, (Kyte Doolittle 1982. J. Mol. Biol. 157:105-132); Figure 7, Percentage Accessible SResidues (Janln 1979 Nature 277:491-492); Figure 8, Average Flexibility, (Bhaskaran and Ponnuswamy 1988.

Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-tur (Deleage, Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the c 273P4B7 variant proteins. Each of the above amino acid profiles of 273P4B7 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (Figure Hydropathicity (Figure 6) and Percentage Accessible Residues (Figure 7) profiles were used to determine stretches of hydrophilic amino acids values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity 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-turn (Figure 9) profiles determine stretches of amino acids 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 273P4B7 variant proteins indicated, by the profiles set forth in Figure 5, Figure 6, Figure 7, Figure 8, andlor Figure 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-273P4B7 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10,11, 12,13,14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 273P4B7 protein variants listed in Figures 2 and 3. In particular, peptide immunogens of the invention can comprise, 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 Hydrophilicity profiles of Figure 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 Hydropathicity profile of Figures 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 profiles of Figure 7; a peptide region of at least 5 amino acids of Figures 2 and 3in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles 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 acid position having a value greater than 0.5 in the Beta-turn profile of Figures 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.

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

SThe secondary structure of 273P4B7 protein variant 1, namely the predicted presence and location of alpha Ci helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147- C 150 Combet Blanchet Geourjon C. and Del6age http:llpbil.ibcp.fr/cgi-bin/npsa.automat.pl?page=npsann.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (www.expasy.ch/tools/). The C l analysis indicates that 273P4B7 variant 1 is composed of 41.60% alpha helix, 11.12% extended strand, and 47.28% random coil (Fgure 13A).

C Analysis for the potential presence of transmembrane domains in the 273P4B7 variant protein 1 was carred out 00 using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the S World Wide Web at (www.expasy.ch/toolsl). Shown graphically in Figure 13B and Figure 13C are the results of analysis of variant 1 using the TMpred program (Figure 13B) and TMHMM program (Figure 13C). The TMpred program predicts the 0presence of 2 transmembrane domains, whereas the TMHMM program does not predict transmembrane domains. Taken together with analysis using other programs summarized in Table VI and Table L, the data suggest that 273P4B7 is most likely a soluble protein.

Example 10: Generation of 273P4B7 Polvclonal Antibodies Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 273P4B7 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles and Secondary Structure"). Such regions would be predicted to be hydrophilic, 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 273P4B7 protein variant 1).

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 273P4B7 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described (see the Example entitled "Generation of 273P4B7 Monoclonal Antibodies (mAbs)").

For example, in 273P4B7 variant 1, such regions include, but are not limited to, amino acids 1-16, amino acids 23-43, amino acids 170-194, amino acids 324-368, amino acids 430-461, amino acids 735-753, amino acids 774-792, amino acids 1002- 1043, and amino acids 1105-1158. 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 1-16 of 273P4B7 variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 273P4B7 variant proteins, analogs or fusion proteins thereof. For example, the 273P4B7 variant 1 amino acid 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. In another embodiment, amino acids.1000-1250 of 273P4B7 variant 1 is fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed Include maltose binding protein, LacZ, 1- thioredoxin, NusA, or an Immunoglobulin constant region (see the Example entitled "Production of 273P4B7 in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Unsley, r Brady, Urnes, Grosmalre, 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 273P4B7 in Eukaryotic Systems"), and retain post-translational modifications such as glycosylatlons found In native protein. In one embodiment, the complete cDNA of 273P4B7 variant 1 is cloned into the mammalian secretion vector, and expressed in 293T cells. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified 00 273P4B7 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 17 response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and SMPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

CKl In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 pg, typically 100-200 jig, 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 pig, typically 100-200 pg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of Immune serum, such as the rabbit serum derived from immunization with the GST-fusion of 273P4B7 variant 1 protein, the full-length 273P4B7 variant 1 cDNA is cloned into pCDNA 3.1 myc-hls expression vector (Invitrogen, see the Example entitled "Production of Recombinant 273P4B7 in Eukaryotic Systems"). After transfection of the constructs Into 293T cells, cell lysates are probed with the anti-273P4B7 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 273P4B7 protein using the Westem blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 273P4B7-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 273P4B7 are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 273P4B7 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 irrelevant fusion protein. For example, antiserum derived from a GST- 273P4B7 variant 1 fusion protein 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- 273P4B7 fusion protein covalently 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 273P4B7 Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 273P4B7 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 273P4B7 variants, for example those that would disrupt the interaction with ligands and binding partners.

Immunogens for generation of such mAbs include those designed to encode or contain the entire 273P4B7 protein variant sequence, regions predicted to contain functional motifs, and regions of the 273P4B7 protein variants predicted to be S antigenic from computer analysis of the amino acid sequence (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, c and the Example entitled "Antigenicity Profiles"). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 273P4B7 variant, such as 293T-273P4B7 variant 1 or 300.19-273P4B7 variant 1murine Pre-B cells, are used to immunize mice.

To generate mAbs to a 273P4B7 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 lpg of protein immunogen or 107 273P4B7-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently S immunized IP every 2-4 weeks with, typically, 10-50 jig of protein immunogen or 107 cells mixed in incomplete Freund's 00 adjuvant. Alternatively, MPL-TDM adjuvant is used In'immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 273P4B7 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, the complete cDNA of 273P4B7 of variant 1 is cloned into the Tag5 mammalian secretion vector and the recombinant vector will CN then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 273P4B7 variant 2 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxylterminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 273P4B7 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, immunoprecipitation, fluorescence microscopy, 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 273P4B7 monoclonal antibodies, a GST-fusion of variant 1 antigen encoding amino acids 1000-1250 is expressed and purified from bacteria. Balb C mice are initially immunized intraperitoneally with pg of the GST-273P4B7 variant 1 protein 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 GST-fusion antigen and a cleavage product from which the GST portion is removed determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 273P4B7 variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 273P4B7 variant 1 cDNA (see the Example entitled "Production of Recombinant 273P4B7 in Eukaryotic Systems").

Other recombinant 273P4B7 variant 1-expressing cells or cells endogenously expressing 273P4B7 variant 1 are also used.

Mice showing the strongest reactivity 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 273P4B7 specific antibody-producing clones.

The binding affinity of a 273P4B7 variant monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 273P4B7 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated 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 monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, 1- dissociation rate constants, equilibrium dissociation constants, and affinity constants.

O

SExample 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 protocols PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 S(1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 1 25 1-radiolabeled probe 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 experiments, each MHC preparation is titered in the 0 0 presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

SSince under these conditions Pabel]<[HLA] and ICso>[HLA], the measured ICso values are reasonable CKl approximations of the true Ko values. Peptide inhibitors are typically tested at concentrations ranging from 120 pg/ml to 1.2 ng/ml, 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 ICso 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 and/or HLA motif-bearing peptides (see Table IV).

Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes 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 confirmation 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 epitopes The searches performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles" and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 273P4B7 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.

All translated 273P4B7 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/supermotif 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 specific 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: AG"= a aixa a3 xan where ai is a coefficient which represents the effect of the presence of a given amino acid at a given position (i) S along the sequence of a peptide 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 position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide rirrespective of the sequence of the rest of the peptide.

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

C 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363- 3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding

C

(ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate ofji. For Class 00 II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure.

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

Selection of HLA-A2 supertype cross-reactive peptides Protein sequences from 273P4B7 are scanned utilizing motif identification software, to identify 9- 10- and 11mer sequences containing the HLA-A2-supermotif 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 vitro (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-bearing epitopes The 273P4B7 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 supermotlf bearing epitopes The 273P4B7 protein(s) scanned above is also analyzed for the presence of 9- 10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele the prototype 87 supertype allele). Peptides binding B*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, 8*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7supertype alleles tested are thereby identified.

Selection of Al and A24 motif-bearing epitopes To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 273P4B7 protein can also be performed to identify HLA-A1- and A24-motif-containing Cr sequences.

High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 14: Confirmation of Immunogenicitv Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology: 00 Target Cell Lines for Cellular Screening: The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -C null mutant human Blymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line Is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and S(v/v) 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 Dendritic Cells PBMCs are thawed in RPMI with 30 pg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, Lglutamine and penicillin/streptomycin). The monocytes are purified by plating 10 x 106 PBMC/well in a 6-well plate. After 2 hours at 37"C, the non-adherent cells are removed by gently shaking the plates and aspirating the supematants. 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 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFa is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250x106 PBMC are processed to obtain 24x10 6 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30pg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20x10 6 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140pl beads/20x10 6 cells) and incubated for 1 hour at 4 0 C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at 100x106 cells/ml (based on the original cell number) in PBS/AB serum containing 100pl/ml detacha-bead® reagent and 30 pg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ 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 40pg/ml of peptide at a cell concentration of 1-2xl0 6 /ml in the presence of 3pg/ml I2- microglobulin for 4 hours at 20 0 C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at ix10 s cells/ml) are co-cultured with 0.25ml of CD8+ T-cells (at 2x10 6 cell/mi) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human Is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 Is added 48 hours later at 10 IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5x106 cells/ml and irradiated at -4200 rads. The PBMCs are plated at 2x10 s 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 0 tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 0lpgiml of peptide in the S presence of 3 pg/ml 112 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37°C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the Induction cultures (CD8+ cells) 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 C human IL2 is added the next day and again 2-3 days later at 501U/ml (Tsai et al., Critical Reviews In Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 5'Cr release assay. In some r N experiments the cultures are assayed for peptide-specific recognition in the in situ IFNy ELISA at the time of the second 00 restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays S for a side-by-side comparison.

C Measurement of CTL lvtic activity by 5 1 Cr release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51 Cr release assay by S assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10pglml peptide overnight at 370C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200pCi of slCr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 370C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3x106/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°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 slCr release sample)/(cpm of the maximal 5 sCr release samplecpm of the spontaneous 5'Cr 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 pg/ml 0.1M NaHCO3, pH8.2) overnight at 4*C. The plates are washed with Ca 2 Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 il/well) and targets (100 pI/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 1x106 cells/mi. The plates are incubated for 48 hours at 37*C with 5% CO 2 Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200pg1100 microliterwell and the plate incubated for two hours at 37°C. The plates are washed and 100 p.l of biotinylated mouse anti-human IFNgamma monoclonal antibody (2 microgramlml in PBSI3%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 microliter/well 1M 4 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.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5x104 CD8+ cells are added to a T25 flask containing the following: 1x106 irradiated (4,200 rad) PBMC (autologous or allogenec) per ml, 2x105 irradiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (anti-CD3) at 30ng per ml In RPMI-1640 containing 10% human AB serum, non-essential amino acids, C) sodium pyruvate, 25pM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 Shours later at a final concentration of 2001U/ml and every three days thereafter with fresh media at 501U/ml. The cells are split if the cell concentration exceeds xl10 6 /ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 3 and 1:1 in the s 5 Cr release assay or at Ix106/ml in the in situ IFNy assay using the same targets as before the expansion.

O Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic 0 activity against peptide and endogenous targets are selected and 5x10 4 CD8+ cells are added to a T25 flask containing the following: Ix10 6 autologous PBMC per ml which have been peptide-pulsed with 10 pg/ml peptide for two hours at 37°C and S irradiated (4,200 rad); 2x10 5 Irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 25mM 2-ME, L-glutamine and gentamicin.

C1 Immunogenicity of A2 supermotif-bearing peptides A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptidespecific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptidespecific CTLs In at least individuals, and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 273P4B7. Briefly, PBMCs 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*03/A11 Immunogenicity HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 immunoqenicity Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2-and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, HLA-A1, HLA-A24 etc. are also confirmed using similar methodology Example 15: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules.

Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

AnalomQin at Primary Anchor Residues Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-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.

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.

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

SThe selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the 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 SA2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient S quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and crossreactivity by T cells specific for the parent epitope (see, Parkhurst et J. Immunol. 157:2539, 1996; and Pogue et al., CNL Proc. Natl. Acad. Sci. USA 92:8166,1995).

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

Analoging of HLA-A3 and B7-supermotif-bearinc peptides SAnalogs 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. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue I, L, or F) at the C-terminal primary anchor position, 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.

Analoging 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 273P4B7expressing tumors.

Other analoging strategies Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with aamino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of a-amino butyric acid for cysteine not only alleviates this C 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 1. Chen, John Wiley Sons, England, 1999).

SThus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands Sfor HLA supertype molecules can be modulated.

Example 16: Identification and confirmation of 273P4B7-derived sequences with HLA-DR binding motifs Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using 00 methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-supermotif-bearing epitopes: To identify 273P4B7-derived, HLA class II HTL epitopes, a 273P4B7 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR- C= supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood et 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 273P4B7-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 02, DR6wi9, 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, DR5w11, 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. 273P4B7-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 specificity 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 273P4B7 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 IIM or better, less than 1 pM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes Identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

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

Example 17: Immunogenicity of 273P4B7-derived HTL epitopes rThis example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

c Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models.

Immunogenicity is determined by screening for: in vitro primary induction using normal PBMC or recall responses from 00 patients who have 273P4B7-expressing tumors.

NO

SExample 18: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine Sbreadth of population coverage This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or 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-(1-Cgf) 2 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-loci 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-like supertype may also include A34, A66, and A7401, these alleles were not included 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, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, 8*5301, B*5401, B*5501-2, B*5601, 8*6701, and B7801 (potentially also B*1401, B*3504-06, B*4201, 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 ethnicities when Al and A24 are combined with the coverage of the A2-, A3- and B7-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.

Immunogenicity studies in humans Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et 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 inclusion in a vaccine that is immunogenic in a diverse population.

With 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, which is known In the art (see Osborne, M.J. and Rubinstein, A. "A course in game theory" MIT Press, 1994), can be r used to estimate what percentage of the Individuals in a population comprised of the Caucasian, North American Black, r Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is Example 19: CTL Recognition Of Endogenously Processed Antigens After Priming This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, native antigens.

O0 Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 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 s5Cr labeled Jurkat-A2.1/Kb target cells in the Sabsence 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 273P4B7 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 273P4B7 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenlc mouse models including mice with human All, which may also be used to evaluate A3 epitopes, and B7 alleles 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 Conjugated Epitopes In Transgenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 273P4B7-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 273P4B7-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 immunogenicity 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 peptides may be lipidated, if desired.

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

Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic 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 lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPSactivated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene Vitiello et J. Exp. Med. 173:1007,1991) In vitro CTL activation: One week after priming, spleen cells (30x106 cells/flask) are co-cultured at 37°C with syngeneic, 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.

SAssay for cytotoxic activity: Target cells (1.0 to 1.5x106) are incubated at 37°C in the presence of 200 pl of 5 1 Cr.

After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 pg/ml. For the assay, 104 5 Cr-labeled 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 supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamria counter. The percent specific lysis is determined by the formula: percent specific release 100 x (experimental release spontaneous Cr release)l(maximum release spontaneous release). To facilitate comparison between separate CTL assays run under the 00 same conditions, 5 sCr release data is expressed as lytic units/10 6 cells. One lytic unit is arbitrarily defined as the number S of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 5 1Cr release assay. To obtain specific lytic Sunits/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence 0of peptide. For example, if 30% 61 Cr release is obtained at thp effector target ratio of 50:1 5x10 5 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 specific lytic units would be: [(1/50,000)-(1/500,000)] x 106 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 Immunogenicity." Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes andlor 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 273P4B7-specific vaccine.

This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences minigene) that encodes peptide(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 composition.

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 273P4B7 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 273P4B7. For example, if it has been observed that patients who spontaneously clear 273P4B7-expressing cells generate an immune response to at least three epitopes from 273P4B7 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 class 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.dcrt.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.

When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as

O

Sthose employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine C 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, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each O 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 cleavage from the native source. Altematively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepltopic peptide. Such a vaccine composition is administered for therapeutic or 00 prophylactic purposes. This embodiment provides for the possibility 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 that is presently unknown. Furthermore, this embodiment (absent the creating of any N analogs) directs the immune response to multiple peptide sequences that are actually present in 273P4B7, 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 epitopes 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 clears cells that bear or overexpress 273P4B7.

Example 22: Construction of "Minigene" Multi-Epltope DNA Plasmlds 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 plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -87 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 273P4B7, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 273P4B7 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 epitopes as described in the art, wherein the CLIP sequence of the II protein Is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. 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 murine kappa Ig-lght chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed 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.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as S appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by S extending the overlapping oligonucleotides in 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 0 C for 15 sec, annealing temperature below the lowest 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 annealed and extended: In an example using eight oligonucleotides, four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 pl reactions containing Pfu polymerase buffer (Ix= 10 mM KCL, 10 mM (NH4)2S04, mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 pg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu 00 polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. 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- Slength product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

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

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 deterrriining 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 epitope-HLA class I complexes on the cell surface.

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

Immunol. 156:683-692,1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class 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).

Alternatively, 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 capacity 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 cDNA 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 51 Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the In vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. 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.

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for 7- those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized Intramuscularly with 100 gg of plasmid DNA. As a means of comparing the level of HTLs Induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.

CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3 H-thymidine incorporation proliferation assay, (see, Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo Immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein 0 Barnett et al., Aids Res. and Human Retmviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, Hanke et at., Vaccine 16:439- 445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177- S181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

CN 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/Kb transgenic mice are immunized IM with 100 i.g of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3- 9 weeks), the mice are boosted IP with 10 7 pfulmouse of a recombinant vaccinia virus expressing the samesequence encoded by the DNA minigene. Control mice are immunized with 100 jig 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 ELISPOT assay.

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

It is found that the minigene 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 supermotif 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: Peptide Compositions for Prophylactic Uses Vaccine compositions of the present invention can be used to prevent 273P4B7 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid 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 273P4B7-associated 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 lpg, generally 100-5,000 jg, 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 CTLpopulations 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 273P4B7-associated disease.

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

Example 25: Polyepitopic Vaccine Compositions Derived from Native 273P4B7 Sequences A native 273P4B7 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I Sandlor 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 0 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 C- generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is C selected because it has maximal number of epitopes contained within the sequence, it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping frame shifted relative to one another). For 00 0 example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present In a 10 amino acid peptide.

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

The vaccine composition will include, for example, multiple CTL epitopes from 273P4B7 antigen and at least one SHTL 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 and/or binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possibility 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 273P4B7, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid 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 273P4B7 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 273P4B7 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 273P487 as well as tumor-associated antigens that are often expressed with a target cancer associated with 273P4B7 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Altematively, 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 slecific antibodies, CTL or HTL directed to 273P4B7. 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.

In this example highly sensitive human leukocyte antigen tetrameric complexes ("tetramers") are used for a cross- S sectional analysis of, for example, 273P4B7 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 273P4B7 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain S (A*0201 in this example) and p2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy 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 (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Streptavidin-phycoerythrin conjugate Is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer- 00 0 phycoerythrin.

SFor the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended in 50 pl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CDB-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min r and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include 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 readily indicating the extent of immune response to the 273P4B7 epitope, and thus the status of exposure to 273P4B7, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28: Use of Peptide Epitopes to Evaluate Recall Responses The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acut or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 273P4B7-associated disease or who have been vaccinated with a 273P4B7 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 273P4B7 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 (50U/ml), streptomycin (50 pglml), 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 pg/ml to each well and HBV core 128-140 epitope is added at 1 pg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4 x 10 s PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 .l/well of complete RPMI. On days 3 and 10, 100pl of complete RPMI and 20 U/ml final concentration of rlL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rlL-2 and 105 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 siCr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med.

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

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 al. J. Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 pM, and labeled with 100 pCi of 51 Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.

cl Cytolytic activity is determined in a standard 4-h, split well 51 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target ratios of 20-50:1 on day 14. Percent C cytotoxicity is determined from the formula: 100 x [(experimental release-spontaneous release)/maximum release- 0O spontaneous release)]. Maximum release is determined by lysis of targets by detergent Triton X-100; Sigma Chemical S Co., St. Louis, MO). Spontaneous release is <25% of maximum release for all experiments.

CK The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 273P4B7 or a 273P487 vaccine.

SSimilarly, 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 pig/ml synthetic peptide of the invention, whole 273P4B7 antigen, or PHA. Cells 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 10U/ml IL-2. Two days later, 1 jpCi 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-

thymidine incorporation in the presence of antigen divided by the 3 H-thymidine incorporation in the absence of antigen.

Example 29: Induction Of Specific CTL Response In Humans A human clinical 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 jg of peptide composition; Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 pg peptide composition; Group II1: 3 subjects are injected with placebo and 6 subjects are injected with 500 [ig 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 immunogenicity. 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 mononuclear 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.

The vaccine is found to be both safe and efficacious.

Example 30: Phase II Trials In Patients Expressing 273P4B7 1. Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 273P4B7. The main objectives of the trial are to determine an effective dose and regimen for C inducing CTLs in cancer patients that express 273P4B7, to establish the safety of Inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows: 0The studies are performed In multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

0 0 There are three patient groupings. The first group is Injected with 50 micrograms of the peptide composition and

NO

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 273P4B7: C- 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 273P4B7associated 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 Immunogenicity," 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 vaccine, 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 nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 Ipg) 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 fowlpox virus administered at a dose of 5-107 to 5x10 9 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 mondnuclear 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 273P4B7 is generated.

Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC) Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professional" 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 elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 273P4B7 protein from which the epitopes in the vaccine are derived.

SFor 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 M (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 Speptides.

SAs 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).

Although 2-50 x 106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided.

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

00 In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin

T

are injected into patients without 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 S immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 106 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 Progenipoietin T M is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex vivo activation of CTL/HTL responses Alternatively, ex vivo CTL or HTL responses to 273P4B7 antigens can be induced by incubating, in tissue culture, the patient's, 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 cells express only a single type of HLA molecule.

These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 273P4B7. 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 acid conditions and their amino acid sequence determined, by mass spectral analysis Kubo et al., 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 273P4B7 to isolate peptides corresponding to 273P4B7 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

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 Sthat means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 34: Complementary Polvnucleotldes 0 Sequences complementary to the 273P4B7-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 273P4B7. Although use of oligonucleotides comprising from about to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.

Appropriate oligonucleotides are designed using, OLIGO 4.06 software (National Biosciences) and the coding sequence 00 of 273P487. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 273P4B7-encoding transcript.

C Example 35: Purification of Naturally-occurring or Recombinant 273P4B7 Using 273P4B7-Specific Antibodies Naturally occurring or recombinant 273P4B7 Is substantially purified by immunoaffinity chromatography using antibodies specific for 273P4B7. An immunoaffinity column is constructed by covalently coupling anti-273P4B7 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 273P4B7 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 273P4B7 high ionic strength buffers in the presence of detergent).

The column is eluted under conditions that disrupt antibody/273P4B7 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 273P4B7 273P4B7, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 273P4B7, washed, and any wells with labeled 273P4B7 complex are assayed. Data obtained using different concentrations of 273P4B7 are used to calculate values for the number, affinity, and association of 273P4B7 with the candidate molecules.

Example 37: In Vivo Assay for 273P4B7 Tumor Growth Promotion The effect of the 273P4B7 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 273P4B7. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of either 3T3, cancer cell lines expressing 273P4B7 (Table or cancer cell lines containing tkNeo empty vector. At least two strategies may be used: Constitutive 273P4B7 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 heterologous 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, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and followed over time to determine if 273P4B7-expressing cells grow at a faster rate and whether tumors produced by 273P4B7-expressing cells demonstrate characteristics of altered aggressiveness enhanced metastasis, vascularization, Sreduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1 x 10 5 of the same cells orthotopically to determine if 273P4B7 has an effect on local growth, and whether 273P4B7 affects the ability of the cells to metastasize, specifically to lymph nodes, and bone (Azuma H et al, J Urol. 2003,169:2372; Fu X et al, Int J Cancer. 1991,49:938). The effect of 273P4B7 on bone tumor formation and growth may be assessed by injecting tumor cells intratibially.

C1 The assay is also useful to determine the 273P4B7 inhibitory effect of candidate therapeutic compositions, such as for example, 273P4B7 intrabodies, 273P4B7 antisense molecules and ribozymes.

Q Example 38: 273P4B7 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo SThe significant expression of 273P4B7 in cancer tissues, together with its restricted expression In normal tissues, C1 makes 273P4B7 an excellent target for antibody therapy. In cases where the monoclonal antibody target is a cell surface protein, antibodies have been shown to be efficacious at inhibiting tumor growth (See, (Saffran, et al., PNAS 0 10:1073-1078 or on the World Wide Web at (.pnas.org/cgildol/10.1073/pnas.05162 4 698). In cases where the target is not on the cell surface, such as for 273P4B7, and including PSA and PAP in prostate cancer, antibodies have still been shown to recognize and inhibit growth of cells expressing those proteins (Saffran, et al., Cancer and Metastasis Reviews, 1999.

18: p. 437-449). As with any cellular protein with a restricted expression profile, 273P4B7 is a target for T cell-based immunotherapy.

Accordingly, the therapeutic efficacy of anti-273P4B7 mAbs in human xenograft mouse models, including bladder, pancreas, cervix, lung and the other cancers set forth in Table I, is modeled in 273P4B7-expressing cancer xenografts or cancer cell lines, such as those described in the Example entitled "In Vivo Assay for 273P4B7 Tumor Growth Promotion", that endogenously express 273P4B7 or that have been engineered to express 273P4B7.

Antibody efficacy on tumor growth and metastasis formation is confirmed, in a mouse orthotopic 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. It is confirmed that anti-273P4B7 mAbs inhibit formation of 273P4B7-expressing tumors.

Anti-273P4B7 mAbs also retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice.

These results indicate the utility of anti-273P4B7 mAbs in the treatment of local and advanced stages of cancer. (See, e.g., Saffran, et al., PNAS 10:1073-1078 or on the World Wide Web at (.pnas.orglcgildoV/10.1073/pnas.051624698).

Administration of anti-273P4B7 mAbs retard established orthotopic tumor growth and inhibit metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 273P4B7 Is an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-273P4B7 mAbs for the treatment of local and metastatic cancer.

This example demonstrates that unconjugated 273P4B7 monoclonal antibodies effectively to inhibit the growth of human bladder tumors grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor inhibition using multiple unconjugated 273P4B7 mAbs Materials and Methods 273P4B7 Monoclonal Antibodies: Monoclonal antibodies are raised against 273P4B7 as described in the Example entitled "Generation of 273P4B7 Monoclonal Antibodies (mAbs)." The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation, in accordance with techniques known in the art, for their capacity to bind 273P4B7. Epitope mapping data for the anti- 273P4B7 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 273P4B7 protein.

Immunohistochemical analysis of cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G

C

l Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -200C. Protein determinations are S performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a Smixture of Individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of bladder tumor xenografts.

Cancer Cell Lines Cancer cell lines expressing 273P4B7 are generated by retroviral gene transfer as described in Hubert, et 0al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Nati Acad Sci U S A, 0 1999. 96(25):14523-8. Cancer cell lines endogenously expressing 273P4B7, including prostate, bladder, kidney, and the other tissues set forth in Table I are also used for in vivo and in vitro models. Anti-273P4B7 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL f low cytometer.

CN In Vivo Mouse Models.

Subcutaneous tumors are generated by injection of 1 x 10 6 273P4B7-expressing cancer cells, mixed at a 1:1 dilution with Matrigel (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 monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth.

Tumor sizes are determined by vernier 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. Circulating levels of anti-273P4B7 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, (Saffran, et al., PNAS 10:1073- 1078) Orthotopic injections are performed, for example, in two alternative embodiments, under anesthesia by, for example, use of ketamine/xylazine. In a first embodiment, an intravesicular injection of cancer cells is administered directly (Peralta, E. et al., J. Urol., 1999. 162:1806-1811). In a second embodiment, an incision is made through the abdominal wall, the tissue is exposed, and tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are surgically glued onto the exterior wall, termed "onplantation" (Fu, et Int J. Cancer, 1991. 49: 938-939; Chang, et al., Anticancer Res., 1997.

17: p. 3239-3242). Antibodies can be administered to groups of mice at the time of tumor injection or onplantation, or after 1- 2 weeks to allow tumor establishment.

Anti-273P4B7 mAbs Inhibit Growth of 273P4B7-Expresslng Tumors In one embodiment, the effect of anti-273P4B7 mAbs on tumor formation is investigated in subcutaneous models of the cancers listed in Table I, by Inoculating the right flank of SCID mice with the appropriate 273P4B7-expressing cell line, and comparing its growth in the presence or absence of anti-273P4B7 mAb, as described below, In another embodiment, the effect of anti-273P4B7 mAbs on tumor formation is tested by using the orthotopic model. As compared with the s.c. tumor model, the orthotopic model, which requires surgical attachment of tumor tissue directly, results in a local tumor growth, development of metastasis in distal sites, and subsequent death (Fu, et al., Int. J.

Cancer, 1991. 49: p. 938-939; Chang, et al., Anticancer Res., 1997. 17: p. 3239-3242). This feature make the orthotopic model more representative of human disease progression and allows one to follow the therapeutic effect of mAbs, as well as other therapeutic modalities, on clinically relevant end points.

Accordingly, 273P4B7-expressing tumor cells are onplanted orthotopically, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-2000pg, usually 20 0 -500pg, of anti-273P4B7 Ab, or b) PBS, three times per week for two to five weeks. Mice are monitored weekly for indications of tumor growth.

O As noted, a major advantage of the orthotopic cancer model is the ability to study the development of metastases.

Formation of metastasis in mice bearing established orthotopic tumors is studied by histological analysis of tissue sections, dL including lung and lymph nodes (Fu, et al., Int. J. Cancer, 1991. 49:938-939; Chang, et al., Anticancer Res., 1997.

C/3 17:3239-3242). Additionally, IHC analysis using anti-273P4B7 antibodies can be performed on the tissue sections.

Cr Mice bearing established orthotopic 273P4B7-expressing tumors are administered 1000pg injections of either anti- 273P4B7 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (1-2 weeks Cgrowth), to ensure a high frequency of metastasis formation in mouse lungs and lymph nodes. Mice are then sacrificed and their local tumor and lung and lymph node tissue are analyzed for the presence of tumor cells by histology and IHC analysis.

00 O These studies demonstrate a broad anti-tumor efficacy of anti-273P4B7 antibodies on initiation and progression of cancers in mouse models. Anti-273P4B7 antibodies inhibit tumor formation and retard the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-273P4B7 mAbs demonstrate a dramatic inhibitory effect on 0the spread of local tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-273P4B7 mAbs are efficacious on major clinically relevant end points including lessened tumor growth, lessened metastasis, and prolongation of survival.

Example 39: Therapeutic and Diagnostic use of Anti-273P4B7 Antibodies in Humans.

Anti-273P4B7 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-273P4B7 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 273P4B7 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-273P4B7 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-273P4B7 mAb specifically binds to carcinoma cells. Thus, anti-273P4B7 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy 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 273P4B7. Shedding or release of an extracellular domain of 273P4B7 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. Hepatology 27:563-568 (1998)), allows diagnostic detection of 273P4B7 by anti-273P4B7 antibodies in serum and/or urine samples from suspect patients.

Anti-273P4B7 antibodies that specifically bind 273P4B7 are used in therapeutic applications for the treatment of cancers that express 273P4B7. Anti-273P4B7 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 radioisotopes. In preclinical studies, unconjugated and conjugated anti-273P4B7 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 "273P4B7 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo". Either conjugated and unconjugated anti-273P4B7 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-273P4B7 Antibodies In vivo 0 Antibodies are used in accordance with the present invention which recognize an'epitope on 273P4B7, and are c- used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, Including 273P4B7 Sexpression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these Sindications, three clinical approaches are successfully pursued.

Adjunctive therapy: In adjunctive therapy, patients are treated with anti-273P4B7 antibodies in CK combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-273P4B7 antibodies to standard first and CK second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to 00 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-273P4B7 antibodies are utilized in several adjunctive C clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

SII.) Monotherapy: In connection with the use of the anti-273P4B7 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 disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

III.) Imaging Agent: Through binding a radionucide iodine or yttrium (1131, Y 9 0) to anti-273P4B7 antibodies, 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 273P4B7. In connection with the use of the anti-273P4B7 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 postzoperative follow-up to determine what tumor remains and/or returns.

In one embodiment, a (11 ln)-273P4B7 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 273P4B7 (by analogy see, Divgl et al. 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 skill In the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-273P4B7 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-273P4B7 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-273P4B7 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-273P4B7 antibodies can be lower, perhaps in the range of 50 to 300 mg/m2, and still remain efficacious. Dosing in mg/mz, 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 all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-273P4B7 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 perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

O Clinical Development Plan (CDP) SOverview: The CDP follows and develops treatments of anti-273P4B7 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-273P4B7 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 273P4B7 expression levels in

C

their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to cytokine Srelease syndrome, hypotension, fever, shaking, chills; (il) the development of an immunogenic response to the material 00 development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to S normal cells that express 273P4B7. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anticr 273P4B7 antibodies are found to be safe upon human administration.

SExample 41: Human Clinical Trial Adiunctive Therapy with Human Anti-273P4B7 Antibody and Chemotherapeutic Agent A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-273P4B7 antibody in connection with the treatment of a solid tumor, a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-273P4B7 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-273P4B7 antibody with dosage of antibody escalating from approximately about 25 mg/m2 to about 275 mg/m 2 over the course of the treatment in accordance with the following schedule: Day Day 7 Day14 Day21 'Day28 mAb Dose 25 75 125 175 225 275 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 Chemotherapy (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: 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, (iii) toxicity to normal cells that express 273P4B7. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-273P4B7 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42: Human Clinical Trial: Monotherapy with Human Anti-273P4B7 Antibody Anti-273P4B7 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human 0 clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same S safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-273P4B7 antibodies.

O. Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-273P4B7 Antibody COnce again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-273P4B7 antibodies as a diagnostic imaging agent. The protocol Is Sdesigned in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

NO

Example 44: Homology Comparison of 273P4B7 to Known Sequences: The 273P4B7 protein of Figure 3 has 1250 amino acids with calculated molecular weight of 141.1 kDa, and pl of 0 5.19. 273P4B7 is predicted to be a nuclear protein (65% by PSORT) with a possibility of being a cytoplasmic protein PSORT). Although some prediction programs indicate that 273P4B7 may have a transmembrane domain, it is equally likely that the 273P4B7 protein is a soluble intracellular protein.

By use of the PubMed website of the N.C.B.I. available on the World Wide Web at(.ncbi.nlm.nih.gov/entrez), it was found at the protein level that 273P4B7 shows best homology to an un-named protein (gi|22760345) of unknown function, with 99% identity and 99% homology over the entire length of the protein (Figure 4A). The 273P4B7 protein demonstrates similarity to a hypothetical human protein named BJ-HCC-15 tumor antigen (gij22002580) with 99% identity and 100% homology over the last 419aa of the 273P4B7 protein (Figure 48). The mouse ortholog of 273P487 has been identified showing 72% identity and 81% homology to 273P4B7 (Figure 4C). Bioinformatic analysis revealed the presence of a SNF2 motif at aa 99-417 and a helicase motif at aa 490-574 of the 273P4B7 protein. These motifs are also found in the mouse SNF2/RAD54 family protein (gil27414501) which carries 72% identity to 273P4B7.

The SNF2 domain is often found in proteins involved in transcription regulation, DNA repair, DNA recombination, and chromatin unwinding (Alexeev A, Mazin A, Kowalczykowski SC. Nat Struct Biol. 2003, 10:182; Solinger JA, Kiianitsa K, Heyer WD. Mol Cell. 2002, 10:1175; Martens JA, Winston Genes Dev. 2002, 16:2231). By remodeling DNA complexes, SNF2 makes nucleosomal DNA accessible to regulatory factors, thereby regulating gene expression (Fan HY et al, Mol Cell.

2003, 11:1311). Evidence in Saccharomyces cerevisiae indicates that SNF2 regulates transcription in these organisms. It has been shown that SNF complexes with SWI and the SWI/SNF is recruited to the promoter of specific genes inducing their transcriptional activation (Kingston, R.E. and Narikar, G.J. Genes Dev. 1999, 13: 2339-2352). A similar chromatin remodeling complex has been Identified in mammalian cells, known as Brm/ Brgl. This complex was found to regulate gene expression as well as cell cycle (Muchardt C and Yaniv M, Oncogene 2001, 20:3067). Finally, a "proliferation-associated SNF2-like gene" which contains SNF2 motifs has been associated with AML (Lee D et al, Cancer Res. 2000, 60:3612).

Our findings that 273P4B7 is highly expressed in several cancers while showing a restricted expression pattern in normal tissues suggests that the 273P4B7 gene may play an important role in various cancers, including the cancers set forth in Table It is provided by the present invention that 273P4B7 controls tumor growth and progression by regulating proliferation, cell cycle, gene expression as well as cell survival. Accordingly, when 273P4B7 functions as a regulator of proliferation, cell cycle, gene expression, and cell survival, 273P4B7 is used for therapeutic, diagnostic, prognostic or preventative purposes.

Example 45: Identification and Confirmation of Signal 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, transcription factors have been shown to regulate O mitogenic and survival pathways (Neeley K, Biochim Biophys Acta. 2002,1603:19). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 273P4B7 and mediate signaling events. Several pathways 0 known to play a role in cancer biology can be regulated by 273P4B7, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, CN p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.). Biolnformatic analysis revealed that 273P4B7 can become phosphorylated by serine/threonine as well as tyrosine C kinases. Thus, the phosphorylation of 273P4B7 is provided by the present invention to lead to activation of the above listed C0 pathways.

IO Using, Western blotting techniques the ability of 273P4B7 to regulate these pathways is confirmed. Cells C expressing or lacking 273P4B7 are left untreated or stimulated with cytokines, hormones and anti-integrin antibodies. Cell Slysates are analyzed using anti-phospho-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect 0phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and other signaling molecules.

To confirm that 273P4B7 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional 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 factors, signal transduction pathways, and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress 2. SRE-luc, SRFITCF/ELK1; MAPK/SAPK; growth/differentiation 3. AP-1-luc, FOS/JUN; MAPK/SAPKIPKC; growth/apoptosis/stress 4. ARE-luc, androgen receptor; sterolds/MAPK; growth/differentiation/apoptosis p53-luc, p53; SAPK; growth/differentiation/apoptosis 6. CRE-luc, CREB/ATF2; PKA/p38; growthlapoptosis/stress Gene-mediated effects can be assayed in cells showing mRNA 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 luciferin substrate and luminescence of the reaction is monitored in a luminometer.

Signaling pathways activated by 273P4B7 are mapped and used for the identification and validation of therapeutic targets. When 273P4B7 plays a role in the regulation of signaling pathways, mitogenic and survival pathways, phospholipid pathways and adhesion and migration pathways whether individually or communally, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes. Additionally, when 273P4B7 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 46: Involvement in Tumor Progression The 273P4B7 gene can contribute to the growth of cancer cells. The role of 273P4B7 in tumor growth is confirmed in a variety of primary and transfected cell lines including pancreas, cervix, bladder, lung, prostate, kidney, colon, ovary, breast, bone, skin, lymph node, stomach, and uterus cell lines as well as NIH 3T3 cells engineered to stably express 273P4B7. Parental cells lacking 273P4B7 and cells expressing 273P4B7 are evaluated for cell growth using a welldocumented proliferation assay (Fraser SP, Grimes JA, Djamgoz MB. Prostate. 2000;44:61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7:288).

C To confirm the role of 273P4B7 in the transformation process, its effect in colony forming assays is investigated.

Parental NIH3T3 cells lacking 273P4B7 are compared to NHI-3T3 cells expressing 273P4B7, using a soft agar assay under stringent and more permissive conditions (Song Z, et al. Cancer Res. 2000, 60:6730).

0 To confirm the role of 273P4B7 in invasion and metastasis of cancer cells, a well-established assay is used, a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999, 59:6010). Control cells, including, but not limited to prostate, colon, bladder and kidney cell lines lacking 273P4B7 are compared to cells expressing 273P4B7. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane 00 analog. Invasion is determined by fluorescence of cells In the lower chamber relative to the fluorescence of the entire cell

NO

population.

273P4B7 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 273P4B7 are Scompared for differences In cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988, S136:247). 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 G1, S, and G2M phases of the cell cycle. Altematively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 273P4B7.

Engineered and parental cells are treated with various chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 273P4B7 can play a critical role in regulating tumor progression and tumor load.

When 273P4B7 plays a role in cell growth, transformation, invasion and metastasis,, and cell cycle and apoptosis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

i Example 47: Involvement in Angiogenesis Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell.

1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays, endothelial cell tube formation, and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the effect of 273P4B7 on angiogenesis is confirmed.

For example, endothelial cells engineered to express 273P4B7 are evaluated using tube formation and proliferation assays. The effect of 273P4B7 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 273P4B7 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemlstry techniques.

When 273P4B7 affects angiogenesis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 48: Regulation of Transcription The localization of 273P4B7 to the nucleus and its similarity to SNF2 containing proteins known to regulate gene expression and chromatin structure, support the present invention use of 273P4B7 based on its role in the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, by studying gene expression in cells expressing or lacking 273P4B7. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 273P4B7-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 cells treated with FBS or androgen 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 K et al., Thyroid. 2001,11:41.).

SIn the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs Including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc.

CN These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of wellcharacterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for CN positive and negative modulators of pathway activation.

0S0 Thus, when 273P4B7 plays a role in gene regulation, it is used as a target for diagnostic, prognostic, preventative O and therapeutic purposes.

Example 49: Protein-Protein Association SNF2 containing proteins have been shown to Interact with other proteins, thereby forming protein complexes that can regulate protein localization, chromatin structure, gene transcription, and cell transformation (Papoulas et al, Development, 1998,125:3955; Cao et al, Mol. Cell. Biol. 1997,17:3323). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 273P4B7. Immunoprecipitates from cells expressing 273P4B7 and cells lacking 273P4B7 are compared for specific protein-protein associations.

Studies are performed to determine the extent of the association of 273P4B7 with receptors, such as the EGF and IGF receptors, and with intracellular proteins, such as IGF-BP, cytoskeletal proteins etc. Studies comparing 273P4B7 positive and 273P4B7 negative cells, as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors and anti-integrin Ab reveal unique interactions.

In addition, protein-protein Interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem Biol.

1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 273P4B7-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with surface receptors and effector molecules directs one of skill to the mode of action of 273P4B7, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 273P4B7.

When 273P4B7 associates with proteins to regulate protein localization, chromatin structure, gene transcription, and cell transformation or associates with small molecules it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.

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 thescope 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.

TABLES:

TABLE 1: Tissues that Express 273P4B7: Maligqnant Tissues a. Prostate b. Bladder c. Kidney d. Colon e. Lung f. Ovary g. Breast h. Pancreas i. Bone j. Skin k. Cervix 1. Lymph Node m. Stomach n. Uterus TABLE It: Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cystelne W Trp, tryptophan P Pro proline H His histidine 0 Gin glutamine R Arg arginine Ilie Isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G -Gly glycine TABLE III: A~mino 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 CK1 ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V, WY.

4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A 9 -3-4 -2 -3-3 -1-3 -1I- 1-3-3 -3 -3 1- 1-2-2 C C16 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -3 -4 -3 D -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 .1 3 F 6 6-2 -4 -2-4 -3 0-2-2 -2 0-2-3-2 -3 G 8-3 -1 -3-2 1-20 0-1 -2 -3-2 2 H 00 4 -3 213-3-3-3 -3-2-1 3-3 -11 -2-1 0 -1 1 2 0-1 -2 -3-2 K 4 2 -3-3 -2 -2-2-1 1 -2-1 L -2 -2 0 1 11 -I IM 6 -2 0 0 1 0 -3-4 -2 N 7 -1-2 -1-1 -2-4 -3 P 1 0 -1 -2 -2-1I Q -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: KiA Class 1111 Motifs!Supermotifs TABLE IV HLA Class I SupermotlfslMotlis SUPERMOTIF POSITION POSITION POSITION 2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor) Al TIL VMS FWY A2 LIVMATQ

IVMATL

A3 VSMATLI RK A24 YFWIVLMT ______FIYWLM 137 p VILFMWYA B27 RHK FYLWMIVA B4ED

FWYLIMVA

B58 ATS FWYLIVMA B62 QLIVMP

FWYMIVLA

Al TSM Al

Y

A2.1 LMVQIAT A3 LMVISATFCGD All VTMLISAGNCDF A24 YFWM A*3l01 MVTALIS RK A*3301 MVALFJST RK A*6801 AVTMSLI RK B*0702 P B*3501 P 651 P 8*5301 P B*5401 P __________ATIVLMFWY Bolded residues are preferred, italicized residues are less preferred: A pepUde is considered motif-bearing .if it has primary anchors at each primary anchor position for a motif or supermotif as specified In the above table.

TABLE IV HLA Class It Supermotif 1 6 9 W F, L A, V,1, L, PC,S, T A V, 1,LCS, T, MY TABLE IV HLA Class 11 Motifs MOTIFS 1 *anchor 1 2 3 4 5 1 0anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WOE DR1 preferred MFLIVWY PAMQ VMVATSPLIC M AVM deleterious C CH- FD OWD GDE D DR7 preferred MFUIVWY M W A IVMSACTPL M IV deleterious C G GIRD N G DR3 MOTIFS 1 *anchorl1 2 3 1 *anchor 4 5 1 aanchor 6 Motif a preferred LIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR Supermoif MFUIVWY

VMSTACPLI

Italicized residues indicate less preferred or "tolerated" residues TABLE IV HLA Class I Supermotifs POSITION: 1 2 3 4 5 6 7 8 C-terminus

SUPER-

MOTIFS

Al 1' Anchor I O Anchor TIL VMS

FWY

A2 l" Anchor 1 *Anchor LIVMATQ

LIVMAT

A3 Preferred 10 Anchor YFW YFW YFW P 1* Anchor VSMAThI (415) RK deleterious DE

DE

P A24 10 Anchor Anchor YE WI VLMT

FIYWLM

B7 Preferred FWY 1" Anchor FWY EWY 1 "Anchor LIVM P

VILFMWYA

deleterious DE DE G QN DE P(515); QN(315) B27 1 Anchor 1 "Anchor RHK

FYLWMIVA

844 I -Anchor 1 0 Anchor ED

FWYLIMVA

B58 I' Anchor 1 Anchor ATS ,FWYLIVMA 862 1* Anchor 1 0 Anchor QUVMP

FWYMIVLA

Italicized residues indicate less preferred or "tolerated" residues TABLE IV HLA Class I Motifs POSITION I 3 4 5 6 7 8 9 termilnus or C-terminus Al preferred GFYW l *Anchor DEA YFW P DEQN YFW I 0 Anchor 9-mer STM y deleterious DE RHKLIVMVP A G A Al preferred GRHI ASTCUIVMV 1 Anchor GSTC ASTC LIVM DE 1 0 Anchor 9-mer 5DEAS

Y

deleterious A RHKDEPYFW DE PQN RHK PG GP Al preferred YFW 1 Anchor DEAQN A YFWQN PASTC GDE P 1 0 Anchor STM

Y

mer deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A Al preferred YFW STCLIVM I 0 Anchor A YFW PG G YFW I 0 Anchor DEAS

Y

mer deleterious RHK RHKDEPYFW P G PRHK QN A2.1 preferred YFW 1 *Anchor YFW STC YFW A P 1 0 Anchor 9-mer LMIVQAT

VUIMAT

deleterious DEP RERKH RKH DERKH POSITION:l1 2 3 4 5 6 7 8 9 0- Tenminus A2.1 preferred AYFW I 0 Anchor LVIM G G FYWL I 8 Anchor LMVQAT vim VLIMAT mer deleterious DEP DE RKHA P RKH DERKHRKH A3 preferred RHI l1 0 Anchor YFW PRHKYF A YFW P 1 0 Anchor deleterious DEP LVSTCDDE wKRF All preferred A 1 Anchor YFW YFW A YFW YFW P 1 Anchor WTLMISAGNCD

KRYH

F

deleterious DEP A G A24 preferred YFWRHK I 0 Anchor STC YFW YFW I Anchor 9mrdeleterious DEG YFM DE G QNP DERHKG AQN FI A24 Preferred 1 *Anchor P YFWP P 1 0 Anchor YFWM

FLIW

mer Deleterious GDE QN RHK DE A QN DEA A31011Preferred RHK I 9 Anchor YFW P YFW YFW AP V Anchor Deleterious DEP MVAS DE ADE DE DE DE R A3301 Preferred I1 *Anchor YFW AYFW 1 Anchor MVALFIST

RK

Deleterious GP DE A6801 Preferred YFWSTC 1 *Anchor YFWLIV YFW P 1 Anchor ATMalI M

RK

deleterious GP DEG RHK A 80702 Preferred RHKFWYP 1 0 Anchor RHK RHK RHK RHK P A 1I-Anchor p

LMFWYAI

deleterious DEQNP DEP DE DE GDE QN DE B3501 Preferred FWYLIVMV I 0 Anchor FWY FWY V Anechor

P

A

2 3 4 5 6 7 8 9 C- POSITION I 234 5 6 7 8 9 C terminus or C-terminus Al preferred GFYW 1 0 Anchor DEA YFW P IDEQN YFW I 0 Anchor 9-mer STM

Y

deleterious DE RHKLIVMP A G A Al preferred GRHI( ASTCLIVM I 0 Anchor GSTC ASTC LIVM DE I 0 Anchor 9-mer IDEAS

Y

deleterious A RHKDEPYFW DE PQN RHK PG GP deleterious AGP G G 851 Preferred LIVMFWY l 0 Anchor FWY STC FWY G FWY l 0 Anchor P

LIVFWYA

M

deleterious AGPDER DE G DEQN GOFE

HKSTC

85301 preferred LIVMFWY 1 *Anchor FWY STO FWY LI VMFWYF WY I 0 Anchor P

IMFWYAL

V

deleterious AGPQN G RHKQN DE 85401 preferred FWY I 0 Anchor FWYLIVM LIVM ALIVM FWYA I 0 Anchor P P ATIVLMF

WY

deleterious GPQNDE GDESTC RHKDE DE QNDGE DE 00 TABLE IV Summary of HLA-supertypes Overall phenotypic frequencies of HLA-supertypes in different ethnic populations Specificity Phenotypic frequency SupertypePosition 2 C-Terminu CaucasianN.A. Black JapaneseChinese Hspanic verage B7 AILMVFW 3.2 55.1 57.1 43.0 9.3 49.5 A3 AILMVST RK 37.5 42.1 45.8 52.7 3.1 44.2 2 AILMVT AILMVT 45.8 39.0 2.4 5.9 3.0 42.2 24 YF (WIVLMT)F (YWLM) 3.9 38.9 58.6 40.1 8.3 40.0 B44 E FWYLIMV 3.0 21.2 42.9 39.1 39.0 1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 27 RHK FYL (WMI) 8.4 6.1 13.3 13.9 35.3 23.4 B62 QL (IVMP) FWY (MIV) 2.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY(LIV) 10.0 25.1 1.6 9.0 5.9 10.3 TABLE IV Calculated population coverage afforded by different HLA-supertype combinations HLA-supertypes Phenotypic frequency Caucasian N.A Blacks Japanese Chinese Hisanic verage 36.1 87.5 88.4 86.3 86.2 2, A3 and B7 99.5 98.1 100.0 99.5 99.4 99.3 A2, A3, B7, A24, B44 99.9 99.6 100.0 99.8 99.9 99.8 and Al A2, A3, B7, A24, B44, Al, B27, 862, and B 58 Motifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

Table V: Frequently Occurring Motifs Name avrg. Description Potential Function Nucleic acid-binding protein functions as transcription factor, nuclear location zf-C2H2 34% Znc finger, C2H2 type robable Cytochrome b(N- membrane bound oxidase, generate cytochrome_b_N 68% terminal)/b6/petB uperoxide domains are one hundred amino acids long and Include a conserved g 19% Immunoglobulin domain intradomain disulfide bond.

tandem repeats of about 40 residues, each containing a Trp-Asp motif.

Function in signal transduction and 18% WD domain, G-beta repea protein interaction may function in targeting signaling PDZ 23% PDZ domain molecules to sub-membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions conserved catalytic core common to both serinelthreonlne and tyroslne protein kinases containing an ATP Pkinase 23% Protein kinase domain binding site and a catalytic site leckstrin homology involved in ntracellular signaling or as constituents PH 16% PH domain of the cytoskeleton 30-40 amino-acid long found in the extracellular domain of membrane- EGF 34% EGF-like domain bound proteins or in secreted proteins Reverse transcriptase (RNA-dependent DNA Rvt 49% polymerase) Cytoplasmic protein, associates integral Ank 25% Ank repeat membrane proteins to the cytoskeleton NADH- membrane associated. Involved in Ubiquinone/plastoquinone proton translocation across the Oxidoredql 32% (complex various chains membrane calcium-binding domain, consists of a12 residue loop flanked on both sides by a Efhand 24% EF hand 12 residue alpha-helical domain Retroviral aspartyl Aspartyl or acid proteases, centered on Rvp 79% protease a catalytic aspartyl residue extracellular structural proteins involved n formation of connective tissue. The Collagen triple helix repeat sequence consists of the G-X-Y and the Collagen 42% (20 copies) polypeptde chains forms a triple helix.

Located in the extracellular ligandbinding region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide Fn3 20% Fibronectin type III domain bonds seven hydrophobic transmembrane eglons, with the N-terminus located 7 transmembrane receptor extracellulaly while the C-terminus is 7tm_1 19% (rhodopsin family) cytoplasmic. Signal through G proteins Table VI: Post-translational modifications of 273P4B7 N-glycosylation site 795- 798 NVTT (SEQ ID NO: 43) 827 830 NSSL (SEQ ID NO: 44) 903 906 NESQ (SEQ ID NO: 911 914 NVSl (SEQ ID NO: 46) 966 969 NFSS (SEQ ID NO: 47) 1047 1050 NTSL (SEQ ID NO: 48) 1084-1087 NKSM (SEQ ID NO: 49) 1155-1158 NKSS (SEQ ID NO: Tyroslne sulfation site 1119-1133 EAKGPEDYPEEGVEE (SEQ ID NO: 51) 1134- 1148 SSGEASKYTEEDPSG (SEQ ID NO: 52) 1193 -1207 AAEATNDYETLVKRG (SEQ ID NO: 53) cAMP- and cGMP-dependent protein kinase phosphorylation site 335 338 KKKS (SEQ ID NO: 54) 336 339 KKSS (SEQ ID NO: Protein kinase C phosphorylation site 4-6 SRR 214-216 SFR 237- 239 STK 250- 252 SNR 282- 284 TLK 285-287 TFK 362-364 SRK 418-420 SAR 459-461 SGK 503-505 TLR 683-685 SVK S739-741 STK S740-742 TKK r 769-771 SSK S791-793 SIK CN 872-874 STK 1004-1006 SEK 1036-1038 SFK C 1055-1057 SVK 1063-1065 TPK 00 1089-1091 SRR O 1095-1097 SRR 1162-1164 TSK Casein kinase II phosphorylation site 180-183 SKDE (SEQ ID NO: 56) S269- 272 SLFD (SEQ ID NO: 57) 303- 306 TPGE (SEQ ID NO: 58) 329- 332 TKED (SEQ ID NO: 59) 339- 342 SNPE (SEQ ID NO: 431 -434 SAQD (SEQ ID NO: 61) 454-457 TLME (SEQ ID NO: 62) 589- 592 TVEE (SEQ ID NO: 63) 608 611 TTGE (SEQ ID NO: 64) 620- 623 SKQE (SEQ ID NO: 629 632 TIED (SEQ ID NO: 66) 672-675 SDHD (SEQ ID NO: 67) 683 686 SVKE (SEQ ID NO: 68) 724- 727 TRNE (SEQ ID NO: 69) 763- 766 TQEE (SEQ ID NO: 798-801 TLQD (SEQ ID NO: 71) 820 823 SVEE (SEQ ID NO: 72) 838-841 TKNE (SEQ ID NO: 73) 847- 850 TLQE (SEQ ID NO: 74) 873- 876 TKAD (SEQ ID NO: 913- 916 SIE (SEQ ID NO: 76) 1004 -1007 SEKD (SEQ ID NO: 77) 1028- 1031 SDGE (SEQ ID NO: 78) 1036 -1039 SFKD (SEQ ID NO: 79) 1134-1137 SSGE (SEQ ID NO: 1142 -1145 TEED (SEQ ID NO: 81) 1188-1191 SPQD (SEQID NO: 82) Tyrosine kinase phosphorylation site 655 662 KLDEHIAY (SEQ ID NO: 83) N-myristoylaion site 117-122 GGILAD (SEQ ID NO: 84) 125-130 GLGKTV (SEQ ID NO: 138 -143 GMFDAS (SEQ ID NO: 86) 196 201 GVIITT (SEQ ID NO: 87) 277- 282 GSLLGT (SEQ ID NO: 88) 281 286 GTLKTF (SEQ ID NO: 89) 428-433 GTFSAQ (SEQ ID NO: 540- 545 GVGLTL (SEQ ID NO: 91) 542-547 GLTLTA (SEQ ID NO: 92) 574- 579 GQKENV (SEQ ID NO: 93) 804-809 GTGSAD (SEQ ID NO: 94) 806 811 GSADSI (SEQ ID No: 831 836 GMEKSF (SEQ ID NO: 96) 983 988 GSAPNS (SEQ ID NO: 97) 1130-1135 GVEESS (SEQ ID NO: 98) Amidation 113 -116 DGRK (SEQ ID NO: 99) Table VII: Search Peptides 273P4B7 vadant 1 for 9-mers, l0mers and 15-mers (SEQ ID NO: 100)

MEASRRFPEA

QKIQEALEELi AflDMGLGKTV

KDERTRNLNR

AICARAIPAS

DATPGEKPJJG

LSRKNDLIIW

ACCLI

2

NLGTF

VFSQSRQILN

EAILSPEQAAH

AEQGDDEFTD

QIIAFLSGMF

IQORNGVI IT NRLzLLTGTPI

FKISENLMAI

IRLVPLQEEI

SAQDGNEGED

IIERLLKNRH

YLRYVKEAKE

VCNSGLLLYR

DASLVNHVLL

TYQMLINN1WQ

QNNLQELWSL

IKPYFLRRTK

YRKFVSLDHI

SPDVDHIDQV

FKTLRIDGTV

ATDAQAVDRV

KQELRELFTI

IDLSVKEELDV

ATKNGDLEEA FKLFNLAKDI

ELHNQLFEHQ

IMPTNLINTW

QLSSFRGQEF

FDFACQGSLL

EDVQKKKSSN

KELLMETRSP

TDDTLMEESG

THLLEREKRI

KEGIAFLYSIJ

VKEFIKWTPG

VWD'LVILDEA

GTLKTFKMEY

PEARLNEKNP

LAELGVLKKL

KMIFLMDLLK

N7LFQQNXDYS VGLTLTAATR VVIFDPSWNP KDSLIRQTTG EKKNPFRY5FS AYLQSLGIAG ISDHDLMYTC QQRTRNEGAW LREPVFPSST KKKCPKLNKP PKEGEKQDLS SIKVNVTTLQ DGKGTGSADS EAVQ1XETLQE GPICQEALQED ITNESQNAES NVSI IEIADD ADNRQNFSSQ SLEHVEKENS SKARRIVSDG EDEDDSFKDT SSVNKSMNSR RSLASRRSLI YTEEDPSGET LSSENKSSWL ETLVKRGKEL KECGKIQEAL 273P487 v.4 9-mers, aa 164-180 aa 163-181 aa 158-186 273P4B7 9-mers, aa 356-372 lO-mers, aa 355-373 aa 350-378 273P487 Y.6 9-mere, aa 881-897 aa 880-898 aa 875-903

PLESFNYVLS

LSASHSALQD

LCGSAPNSRA

SSII'PFNTSL

NMVLDMVDM

MTSKPSALsAQ

NCLVKALDIK

YRIGQKENVV VY1RLITCGTV EDLQNSVTQL QLQSLHAAQR VEESHYIQQR VQKAQFLVEF QPQPSPLLST HHTQEEDISS IATLPKGFGS VEELCTNSSL KSTKAflIGPN LDOLKDDEIL AQASEAKLEE EPSASSPQYA GFVIISKTCLS WEFSEKDDEP FQFSSVKQFD ASTPKNDISP EERLDDSSEA KGPEDYPEEG ETSLGAPEPL SGEQLVGSPQ SADPEVMLLT LSLYKQLNNN

FPNEKVLSRI

YRDGRKGGIL

MRVKTFHGPS

HKIKTSSTYKS

ENPITRAREK

DVDAICEMPS

CDHPRLLSAR

RLRDEGHQTL

VFLLTTQVGG

EEKIYRRQVF

KSDIKTJDEHI

ESQNKEFLME

KMASVVIDDL

GMEKSFATKN

RHCNPWPIIS

CDFNLFLEDS

EEVVVKAKIR

PGRFFS SQl P

VEESSGEASK

DKAAEATNDY

120 180 240 300 360 420 480 '540 600 660 720 780 900 ,960 1020 1080 1140 1200 1250 FIKWTPGMGVKTFHGPS (SEQ ID NO: 101) EFIKWTPGMGVKTFHGPSK (SEQ ID NO: 102) NTWVKEFIKWTPGMGVKTFHGPSKDERTR (SEQ ID NO:.

CEMPSLSRRNDLIIWIR (SEQ ID NO: 104) ICEMPSLSRRNDLIIWIRL (SEQ ID NO: 105) PDVDAICEMPSLSRRNDLIIWIRLVPLQE (SEQ ED NO: LDQLKDDEVLRHCNPWP (SEQ ID NO: 107) NLDQLKDDEVLRHCNPWPI (SEQ ID NO- 1.08) ADIGPNILDQLKDDEVLRHCNPWPIISITN (SEQ ID NO: 103) 106) 109) Tables Vill XXI: Table VI II-VI -HLA-AI -9mers- 273P4B37 1 Each peptlde 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 peptidle Is the start I position plus eight, Start Subseqauence Iscore] [Fill LALGLK 001 0 00 L~il]ICEMPLSR 45,00~ ADAQA DR 25.0 [1231 SAD PEVMLL][ 2500J TF7 TLI 12.0 58D9EA 18.0010 1 IDEAH [1 111.00 FLEDSADNRTLV 9000 F[11LAEQDDEK 109.00 [226 D1EAKL .00 FROF]I NGDEF 5.000 1671 SHLY I~ 763 TQEIS 2.0 F31 ELMII2.0 j81F][VNGLY250 K NDSPG 12.0 F410[LDHRS2.0 IiFi[ ISHLM 1.50 F46]75 DLRL ~P0 IF~] DG EEDDSFJ250 456][MEGMF220 EL SE 225 Table Vill-VI-HILA-All-9mers-1 273P4B7 J 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.

LStart ISubse uencej IScore 12 FVKEAKEATK 1.800 F8_3_1FGMKSFATK 1.80 F9 EAEALSPEQ1.0 6 9_0 ESYQ 1.8001 I1152]LNK WL fj I 376] QEEIYRF 1.35 85=7 [LQ EDPLEF 1.50 36 KDI IWIR 1.250 81_2 ]LLPKGFGSJ 1.25 FTVNSL1120 i1 GTVEEKIYRl 250I L73EI1 DPMLLTL 125l [4 iMWMLLtOO [6177 ADLAS .00 1655 lDEH IY iOO] Fbz2z71I MLLTLSLYK FOl.OO 774 JISVD DLPK FT1 VDEEJ 0.90 73-1-1 EVPS .0 1 971 I HEE .0 SVE82N 090 5-60-7DD] EWV0.0 F5 QKE5VY 0.0 1329 II TKED=VQKKK 090 [2?jFI.IELQNSV =0.900 Table ViII-V1-HL-A-A1 -9mers- L 273P4B7 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 L position plus eight.

j[ Subsequence I Sor L_9ZiI7 F NAESNVSII 1I 0.00 F130GVEESSGEA 0.900 1 394 1 M-ETRsPLA 090 1651~IE KSDI K LD EH E. 750] F1 PGFF 0.750 F1133][LESSGEASK 0.750 F44I][ SDD I~I.625] 450 I[ VDTLMEE I0.625~ [177][SQ7PSSVNKJ 0600 [iii ALYSLY jlEgo [iF l LIMPTNLI 0.5001 [892] ONW PIISJ .0 453 DTL M EE SGKj[E.O 563]LAQ2YDRVY 19.POj [320j[ IIKPYFL RR 001~~so L[DNLFQQN-KDY. [0.5001 I 38RRQN1 SLKL .500] IiF37 EDWE3 0.5001 1F-56A75SYR 0.5001 F4510] VTLEE]0.5001 316 NLAIKP 0.500 99 EKAP E .0 747 ADIPE .0 1.IK92 EATNDY] 0.5001 1100691 EPEVVK EiIM 70,TRNJ 0=.450 Table VIII-V1-HLA-AI-9mers- 273P4B37 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 I SubsequenceScr 455 1LMEESGKMI 1j50 8911YEHQFI.5 709 EFSQNKEF L45-0 Table VIII-V4-HLA-A1- 9mers-273P4B7 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.

7Start I ubsequen~c ore] G: GV KP. 1-0 0I F=4 WPGGK 0.0501 TPGVKTF 0.25 [L-V.IMrFHGI1~ LY]q y EO 90 F6 PGMGVKTFH 0~ WID MG@VEFHG a00 Table VIII-V-L-1 9mers-7PB I 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.

,[Kii~ ubsequence or F9[ RRN=DLIIWIR 0.20 F[1- CPSLSURL 02 [II LSRRN DLI .00011 Table VIII-V5-HLA-AI-1 9mers-273P487 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 !S ubsqunc LScore T 1 LSRRNDLII0.0 F-7 ]I[RNDLI 000 F7i -m PSLSRRND .00 Table ViII-V6-HLA-AI- 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 I position plus eight.

trSa cr EE 0.020 W VRHCNWP 0.000 ELIDQKDEL0.0 Table IX-V1-1-LA-Al-1 Omers- 273P4B37 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Isl10 amino acids, and the end position for each peptide is the start position I plus nine.

S ubsqunc 180 ITnDSATLPK 50000 Table IX-Vi -HLA-A1 -1 Omers-1 273P4B7 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.

Star!Sbeq 76I1 ELDWVEESHY[ 2-5.00 1o f .ilE5 EN5T 1[ 22.500 110ES0IF[225 0 00D7] VVK~ K5 ICMSSR 80001 121ASEAHY1.0 264 LEW D 372RVPQEY 14 PE QA.AHYLRI450 DGEDDDSF 4.500_ 114 SG-ETLSSENK .0 7 100EEVAK ,7914][IEIADDL=SA I450 69L0 E ESHYIQQ .0 9 83 A NSRAGFII300 960SDNNFSI250 386 [L DHIKELLMI250 [226 IDAKK .0 *874 AIPLQf.0 35[oNSGE1 2.500 1141L7TEEDPSGETI250 {Table IX-V1-HLA-AI -1 Omers.

273P4B37 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 pepilde Is the start position -plus nine.

1171APEPLSGEQL 2.250 473 RDGQL FI 2.5 18761 OIGPNLDqLK ]2.000 [11] VEESSGEASK 1.0 ~1[KEESSS .0 9g44 ASSQYACDF150 7173AWD DLPK1.0 928ff~q 1.DQAE~j500 671JSDHDLMYTC-j1.0 2r SSENKSSWLm 1.5 E8i FTDVCNGLL 1 250 450 VTE)DTL-MEES1.2501

KNDSGRFJ

561 FDQADvl.2UOI LOJATPGEKALqGFII1.250j 1191 LADOMGLG1[ 1.000 655 flKDHAL 100 104

SINPFNTSLFEJ

"i RLDDSSEAKG 1.000 488 ILNIIERLLK q00o~

SDYETLVKRGK

401 LAELLK 0.900 589]I TVEEKIYRRQj 0.900] 97~ EAEAILSPEQA 10.900 844 QKETLQEGPK 0.900 7E!'gooE 971] SLEHVEKENS 0.900 6291 TIEDLQNSVT] o.9__ 820j1SVEECTS 0.900 Table IX-Vi -HLA-A1-1 Omers- 273P4B37 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 L plus nine.

StrSubsequen~ce[Scr F956 FLEDSADNRQ 0.9002 E l .LAEQGDDEFT 0. 900 P102I vSDGEDDSI .5 [106 I.SPPGRFFSS[070 6 5 1 K SDI KL D EHI I 0. 750 FN G -NDL E EAFKL Jj0625 2 81 GTLKTF KMEYJO625] F9(- J AISOOj0.625 F9Ji8 SSQISLEHVEKj[0.0 qj SQI1PSSVNKJ0.0 K6 FJ M DPPF S L f 0 7~.500 7W ILPEE 0.500 58-6] 7CTVEKY 2P.

150 PPIS .0 M1 DLK 0.500 103 GALSY .0 679 DSKE .0 ]12 TTRW5 0.00 762 HTQEEDISSK 0.50 E7 _IADDLSASHS10.5007 446]F HIDQVTDqTL Jj0.500 Table IX-VI-HLA-A1-l10mers 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 pepfide Is the start position plus nine.

iStar] Subsque c e;] )588FIGTVEEKIYRR]o0.500 F57 IFDPWNPAT F0-7 50o5 FR-DGTvTH-LL 0.500 1 373 LPLEEY 0.500 1 Table IX-V4-HLA-A1 -1 Omers- 1 273P4137 Each peptide Is a portfion o7f 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.

[sart Susequence IScore WLY]V PGMGV KTFI L iOjf GVKTFHGDsKj 04 I. .4LKI/TPGM V FO0010 =7l FPGMGVKTFHG I 0.00 I(WTPGMGVK 0.,00 1 F~W 8MGVTFHGP 10_0011 LI]7 MGVK-TFH-GPS 0.00Qj fIKWPGMGEo oo Table IV5HAl-10Diers-j 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__tart position plus nine.

FStart Subsequence Score] LiJ~~ IEPSR[9.000 [EI RRNDLIIWIR L.-6i1 ISLSRR0.00I [ill CEMPS-SIRRN] PSLSRN La03 Table IX-V5-HLA-A1 -1 Omers-1 273P4B7J 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.

TI!PSLSRNDLI J0002] 7IISFODII 10 01] 31MPLRN IKo Table IX-V6-HLA-A1-1 Omers- 273P34B7I Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino ac 'ids, and the end position for each peptide is the start position plus nine.

[Stat Susequnce Scorel NLDQLKDDEV 050 WI2~P~YLRCN_10.025] W IJ DDVRCP 0.005I LFTLDLKDVLI 0.003 R ~i 0.003i FT-O][_EVLRHCNPWP ffO1j W-1 LDQLKDDEVL[aoF-0J 9= ELRCPW- =.000 Table X-VI-HLA-A0201-9mers-1 273P4B7 Each peptide is a portion of SEQ1 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 II Susequence Scr [651KLDEHIAYL If 1 5 1 1 4 F3973 I LLMETRSPL 5I .1 67-6 I MTDSJ2322 [~KLNAKDI 113.105 Table X-V1 -HLA-A0201-9mers- 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.

S tartl Subsequene 148 VLLIMPTNL7 I34-.3691 F99 YACDFNLFL16.5 F12151 KIQEALNCL 19.4 F368 IIWIRL-VP 9-5.325 [1159 _WLMTSKPSAF8II.557 FT011 KLCDHPRLL I.I6-2.310i1 461] 1MFML i60.857] F291ALNCLKA 493] [42741 LLNLGTFSA !14a8.984] [i62l[ KIQEALEEL j47.50-4] F32]KISENLMAI I7 [2521 RLLLTGTPI 0 ,VILDEAHKI II 36.995]1 [F76 LLSARACCL i 3.16 533[ FLTVG I35.3531 I-5230 F N DYS 9-3248] Fj9ISLGMEKSFA FI27.324]1 I~o] LMTSKPSALI 26.28 ti~[ SVNHLL2-3.9951 VFLLT~yJ22.5171 [i12]2 TLVKRGKEL F21.362 [i1i51[ IMPNINJ2-1.044] F17 1W'KEFIKW 20.6-90 K5~] RLLSARAC 18.382 F10 99 LINMVLDHV 18.323, [F 47 LLIMPTNLI 17.736 F81l3 TLPKGFGSV 1FI7.1791 F266]I ELWSLFDFA l 15.8361 F1278[ KTVQIIAFL 1-3-.136 I!121] MVLDHVEDM ][12.634] 1325] [FLRRTEDV[1.5 1042 SPTL 11.162] 118 GIADML I j-.868 I Table X-VI-HLA.A0201-9mers- 273P4B37 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 foreach peptide is the start position plus egt i[Starene] 11score] 1091 SRL 110.433] 622)j QELRELFTI ]1-67613] 7876 FSIV]95-773] 262] NNQL FL[8.9700 8671 YVLSKSTKA 7.T399 640fl LQLQSLH 7. F287 830l L EK F T72767 4868 ILNIIERLL 7.6 696] YIQQRVQKA ]F7.227 543:] LTLTAATRV 6.6 1311 QIIFGM 5.970 913 jSEADLI .901 TLMTR F5.7 03 43 6U G F 545 F512 FLKIj5-.3817 F706] FLVEFESQN 31 788 LSSIVNV 5.21-6 F372 RLVPLQEEI F5-.112 F3670 SLSRKNDLI J.F 5112 iF63[ LQQSHA9 4.9-68 F213 SSR EF 4.2 [26][SLFDFACQ 4.296] LF38J6 SLOHIKELL JF4.187 F-663]1 LQSLGIAGI I F4.136 1196][ ATNDYETLV Ij 3-.961 F57721 RIGQKENW 3.3921 [iF63l DLQNSVTQL ]F 3.685 SMNSRRSLA F3.588 F4-0721 AELGVLKKL 11F3.535 F4551[ LMEESGKMI].113.361 Table X-VI-HLA-A0201-9mers 273P4137___ Each pepide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amlno acids, and the end position for each peptide is the start position plus eight.

Start SuI ec cr 17048 LT F FSSV I .92 1239 LThSLYKQL 2. 17 99 318 IAIPF .7 F505-51 RI D GT VT H L 2.7 02 F351 GDLEEAFKL 2.T611] F49 5 LLKNRHFKT 12.572 r62 9 [I TD LQNSJ 2.509 17 QMAHYLRYV I.2.4441 L!LJ1IIDXYLi .2.439 483 SQRI FI2.433 36 DLIIWIRLV 2 .400 F967 FSSQSLEHV JF2.3 5 4 F471 RLDEHQ I2.-322 95641 RQNFSSQSL 2.6 [1075] FSSQIPSSV F2.0-88 Li 7] GQFWDV2.068 816 KGGSVEL J2.035] 1l230o Ls A-DEVK.PIL 2.001 537 QGVGT 1-.-9 625l RELFTIEOL F 172? Table X-V4-HLA-A0201-9mers-1 273P4B37 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.

[sta1 Subsequence I Scor 2]j IKWTPGMGVII1 1.3631 411 WTPGMGVKT Ij 0.476 LU7 iMG ITFJ 0312 I [IZIP5VKF7 .0 WH 1 VTHG 0.000 F 7 PGMGVKTFH .F0.000 F i 1 FIKWTPGMGI.F 0.-000 F GVKTFHGPS ]10. 0 E3]KTPGMGVK F- 000 TbleX-V-Hk-A0201-9mes 2734B7 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 positon plus eight.

Subsequence j Soe SLSRRNDI 5.11-21 [jRRNDLIIWI T0.7075 PSLSRRNDL ]0.011 W- LSRRNDLII0.4 2~j EMPSLSRRN ]0.001 [ZIILjY!PSLSRRND I000 Table X-V6-HLAA21 9es 273F47 SDN:3ectart poSubsquenc Is~jj seiidQthe etho epid i W 1 LDQLKDDEV IF0.0801 W9 1 LHNW 0.011 F3jj QLKDDEVLR F002 L4I1 LKDDEVLRH 0.001 W 1 DEVLRHCNP =0000 WE ODEVLRHCN] 0-.000 6 [able XI-V1-HLA-A0201-1 Omers-1 (273P34B7 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.

FStartj Subsequence I Scorel FQ5]FLFDASL73J 4 1 WLTKPA 3635] Table XI-Vl-HLA-A0201-1 Omers-] 273P4837] Each peptide is a portion of SEQ ID NO: 3; each start posiion is specified, the length of peptide is amino acids, and the end position for each peptide Is the start position plus nine.

Start f Subsequence Soe 4161 LLSARACOLL 3631] 552 1VIFDPSWNPA]2994 26JILDEAHKIKT 2067 84771 TL-QEGPKQEA 21 936 31 ENM2] L%2 751DLMYTCDLSVI F'1 3 0 1 [628] FTIEDLQNSV_~ 18. 921] L868 17.ST~i73 [F~LQEIRF 37617.30 [iuKEGIAFLYSL F-77 [E524 JQNYSVFL l [367 V IWIRLVPL F1.4 [639] QLQLQSLHAAF174 F26]RGQEFVWDYV 8.528 VQIIAFLSGM I7.48-4j [471 11 RLRDEGHQTL 6.65 [156 LlN~yl~ F6.7572 [1160 lj LMTSKPSL 6.0 [ble XI-V1-HLA-A0201-1 Omers- 273P4B37 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.

jF LSPE37HY 5.346 1151 LSSENKSW 5.34 681 DL-SKEELDVg521 717 FLMEQQRTRN- 5.200 L1671 ALAQETSLGA F.:§68 r288 MEYENPITRA 4.956 480 LVSSQL 4.821 62 KIQEALEELA 4.803 Fli43-l1 SLVNHVLLIM 4.685 F1-23-0] KSADPEVMLL 4.63 [152 j__MPTNLINTWV4.24 5 F22411 YVILDE-AHKI I4.199 [Ii2][ EQGDDEFTV 4.120 tF1L-1j MDHVEDM 14F4I L. I RIGQKENVW 3.921 633 LF36TLQ 8-821 L 8:0 ]I VVYRLITCGT 3.547 F621-] KQELRELFTI3.71611 F1 1 FPNEKVLSRI2-.9541 667 GIAGISOHOL .2.7937 F2461 AIPASNRLLL 72937] E34] KN y 12.8811 l-196-I1 GVITYML 2.8041 710 FESQNKEFLM 2.7577 F486 Il RQiLL 2.7441 Iii197I1 VIITTQ 2.4 39 I-11841 QLVGSPQD-DKA 2.434 [564j AQAVDRVYR:1 2.433 F495-] LLKNRHFKTL 2.415 F261-] QNNLQELWSL 2.40 f5131 LLEREKRINL I2-.324 4871 QILNIIEL 2.7 [E36]QVGG h 2.166 Table XI-VI-HLA-A0201 -1 omers-] 273P4B37 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 L start position plus nine.

s ta rti Subse uence liscore] L. iL9yvf2.TT E08LP8] 558 j[WNPATDAQAV 2088 F47W9ILTYVFSQSRQI F27087l [-212 1 SFGEV 2.080f 386 ISDIKLM 1.987 =3L38I GMFDASLVNH1.878 F6381 TQLQLQSLHAJ 1.864 50fl~P LNSFFS IF835 F-2651FOF 1.830 '/85TSLDHIKELL 1.7621 L METRSPLAEL .2 KTPGMVRV 150 Table XI-V4 A-A0201 -1 Omers-] 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.

r Startij Subsequence Scor 1211FIK~rrPMGV11.5401 1J GMGVKTFHGP 0.20 ]IwrPGMGVIKfF 0.011 7T]~ TPGMGVKTF 0.0034 =6j _PMGVKTFH 0.00 [TJJ GMGV~FHG 0.000 10 GVKTFHGPSK 0.00 1] IKWTPGMGVK 10000 TableA .IV HAA201-10;mr-]I 2 137 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 I start position plus nine.

~a~Sbseqence W1 SLSRRNDLIIl 4.77 1 MPSLSRRNDLJg023 RNDLW!RL 0.95 Li L SSRDLI 1 000 V IEMPSLSRRND 1000 [ZIL LRRNDLIIW J 000 [l1-iP]ICEMSLR 0.0001 ITable XI-V6 HLA-A0201 -1 Omers-] 273P4B37J 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 osiltion pus nine.

=Start 1_iubil Soej 1 LLDQL~qjfl. 3632 1 VLRHCPP -5.109 F47 QLKDDEVLRHI05 DQLKDDVL o01 M~I LKDDE-VLR-HC 000 Wa EVLRHCNPWP0.2 6II7 KDDEVLR CNI 00 0 1 LEI. DQLKDDEVLR] 0.000 LZDDEVLRH~ 0.000 TbeXiI-VI-HLA-A39mers- =273PQ67 Each peplide 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 pepide Is the start Position plus eight.

Start _Subsequence Scre OD4 SLFQFSSVK 300.0 00 Table Xii-V1-HLA-A3-9mers- 273P467 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.

831 1 GMEKSIFATK 18O00 Table XII-VI -HLA-A3-9mers- 273P34B7 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 Lstart position plus eight.

[Stagj Subsequence Scr [5725 GLGK1VQII 5400 [42 I[ KLFNLAKDI 4.50 F585-11 ITCGTVEEK 14.50 [-328 RTKEOVQK 4.500 F655 [I 46IKMIFLMDLL 1-956I FLEDSADNR 400 58j QVFKDSLIR 400 2-7~i] fl LGLT 13: .375]I [FT] F31-7- LMAIIKPYF 3.000 L 263jNLQELWSLF 3.000 _2 GLLRELH 2.70 F-867 ESFNYVLSK [2.700 F589 Jf TVEEKIYRR GTVLTHLi~ LER 1L2.700 [-1L !9 !IIPYFLR7j 2.700 5E881 Gi !VEKIYRJ .700 FTT9]0I GISDI-DMY 2.40 LT95J][ Nv1TLQDGK 12.0001 F707- LVEFESQNK] 2.000 F6761 LMYTCDLSV 2.00 [1203 LVI(RiKEL-J 2.000 [FT1j 0-81SLINMVLDH 180 F574] GQKEVY F734 PVFPSSTKK7 1.5001 F149 -LLIMPTNLI F36] DLEE AFKLF F-931 LLERP 1.350 F-7631 TQEEDISSK 1.350 372[ RLVPLQ '1077 j SQIPSSVNK 1.350 401 LAELGVLKK 120 ]14 IAFLYSLYR 120 1031 GIAFLYSLY 1.2001 lF7TK!(EAVQW]I1.ffl0I F.2 GLTLTAR 12.00 130 10DAT .0 [1]NIIKPY 6750 59 RNLFQQNK 6.000 77 SWIDDLPK 6.000 14I SLVN HVLU7[5-400 Table XX-V4-HLA-A3-9mers- 273P4B37j 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 FStartQ Subsequen c -e] [IL]j GMGVKTFHGj~ 0.18 FT-Il KWTPGMGVKT71.0411 Table XX-V6-HLA-A3-9mers- 273P4B37 Each pepfide 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 [S-tartI Subsequence score Lii 2 QKDEL .0 LIKODEV-RT]J0.01 LK 148 I VLLIMPTNL J[0.9001 L JI..VK JUUJ I13.j1.>u n ju.uuuI 717J[ FL EQTFO90-~LY!F G S I00111 1~ LDQLKDDEV 0.001 [1 F156 fl LINTWVKEF [0.90 0 WTPGMG-vKT- 0o.0057 1 DEVILRHN .0 [-478h] QTLVFSQSR] 0.900 jF~WTGG 2 0.003 l[a] DDVRC Emil~ ELWSLFDFA =0.9001i] Fl-219][LCVA 1F 960 8 7THPIoo Table Al-1-HLA-A3-1 Omers- ][52 RL.LGTP PGMEGVKTFH.Ef 1 273P4B37 0O.00001 Each peptide' is a portion of SEQ I..Z2.I[ WREPVPS 0.101ID NO: 3; each start position Is F3-.111KSNAI .10 Table XX-V5-HLA-A3-9mers- I specified, the length of peptide Is E ach p e p tid e is a p o rtio n o f S E Q po sitio n fo r e a ch p e p tid e is th e [BK I HLEEKR 067 IID NO: 3; each start position Is start position plus nine. F1082 1 SVNKSMNSR 0O.60-0J specified, the length of peptide Is So Dpositio for each peptide is the 41 J~RCO .60 9io d, an d the0en j~GFSV tr osition plus eight.- 18FML K 00 I ii0:1[ TSKP-sAL 0.600 [SWa~I Subsequqen coe Y L 1_ SRRNDLJ [676] LMYTCDLSVK 0 4092 0.600RSL 00:~ V i RNDLIIWIR VMLSLI 9 69 1. 0.600P~ L~tMPRLS Z3 DGR1TSLYK 0 E04L]INLGFS 0.600 I [7_1 RRDLIW 1LP 4 24 LLLFAHYLRVK [600 E I L -RMoL!.. 0.001 106 =8 1 AAHILADDM E60]G L I o.W [278]_ SLLTLrFK]i [FJ RQ 8 ILNIIERF 0.5401 [I2711EMSLSRRN I F8Y-6RRNL] 0.000]00.0 F7-71 LSFYL 0.5401 II88 MPSLSRRNDK J .0 1 F-63j2 DLQNSVTQL7 060 Table XX-V6-HLA-A3-9mers- LDMGK 00 KSSWLMTSK 1 0.4501 273P4137 F11 F-01 TVRKL10.450 I Each peptide is a portion of SEQ 42 LF NLKD 0 I .!-fll][QThVKKL 045 ID NO: 3; each start position isLJ L J I ilFQSSVQFspecdfied, the length of peptide is 00 1Ai DTLMEESGK E.450 9 amino acids, and the end[120 LKGE 0 E~iIILMESKMII .401position for each peptide is the 368 start _position plus eight.F8] MYNIR F IWRLPL 0450 FStart] Subsequence]oe 180 jss KVLR I 005 LiI 3 QLKDDEVLR 4.00 401 PALVK 62 KIQEALEEL J0.45 VLRH NPWP1 0.020

N

M 1.~1 KIQEALNCL 0.4050201 ISCNP7 0.00 Table XIii-VI -HLA-A3-1 0mers.' 273P4137 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.

[tr] Subsequence Scar 9 AIIKPYFLRR 10 %80 f37]I RLVPLQEEIY 9.00 SLR]TE 9.000 31 GIAFLYSLYR: F7200 ffI ALQEDPLESF 6.50 F58]8 GTEK ER i 60 H4]J LIOGVE K K600 (9870 SLGANRiZ 6.0001 flj LMEESGKMI7F 6.000 FE62 E iMFMLK16000 F12 LPQMY 600 [9I1l FTKFMYJ5.7400, 138 JGM ASNH 4.5001 E6]1 NLMAIIKPYF 4.500 QFEH QKEGI 450 L~ 4jf QLQ L ]AQ 4.000 406I _[VLKKLCDHPR7F7000l 266 fl WLFA 2.7001 5741[ GQKENVVVYR 2.430j 17621I HTQEEDISSK_ 2.7250, lj21J[ QL JR E 12.0001 ff1J RVKTFHGPSKJ 2.000 117i1 ATND ETL K 0 18251 CN G E 2.0001 [-604]1 _LIRQTTGEKK .0 1251!j GLGKTVQIIA711.8001 F317]1 LMVAIIKPYFL 1.80 [8879 IL-RH-CNIPWP 1.800 F34]IEMPSLSR 11.800 [379 1 EAFKLIFNL-AK 1Ei [_7LEHLLIMlt~ [Table XIII-VI-HLA-A3-1 Omejrs- I273P4137 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.

Pat SubseunePcr 148 11VLLIMVPTNLI 1.350 F374 VPEEYR 13 5-13f ILLEREKIRINL 1.2001 12 1 6 IQANLK1.20 [687 i1 ELDVVEESHY 1-.2001 [32][1RTKEDVQKKKj M111251 LEFYVS 1.0601 [i1 NMVLDHVEODM Eo900 [2031 QMINWQ 0.7900f 69 I[ ELAEQGDDEF I0.00 Ei I YLQSLGIAGI 10.9001 9I8-]E NSAGVHK 9001 ]ILLKNRHFKTL .900q.2 I 38-911 HIKELLMETR 090 2 iroi AFIEL 09001 L99][ HQKEGIAFLY0.810 I 373 f LVPLQEEIYR 0.80-0 734]J1 PVFPSSTK 0.750 I 1098]1 SLINMVLDHV 0.675J I4 74t TLMEESGKMI ]j0.6751 1184J[ QLGP D EN075 j49-fl[ NIIRN 10,6751 F 642][ LQSMRK 0.00 44 FLDU ]L 0.600J 928 lQDQSA 060 880 I NLDQLKDbEI 0.600 723f RTNEALR 0.60 17] QMYLY 0-600 544 TLTAATRWI Tle 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. j [Statld Susqe c r [4 IGLTLTMATRV 0.6001 fl[ LLGTLKTFKM7 0.600O F8 ILY8 KSTKADI .60-0 [2751 CQGSLLGTLK 0.6001 F[i71I -Tlrrv'mLI 1.54 217 GQEVW:] 0.486 F847 TLQEGPKQEA70.45 1221 1_NCLVKALDIK 0.450 r393] LLMTRPL 045 1l 04-4f NE9SFQ [K44 J1 RLLKNRHFKT .0-.45-0! 10131I VWKAKIRSK- 0.74501 L able XIII-V4-HLA-A3-l0mes1 273P4137 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 ubeuec Score 10I GVKTFGS WTPGGKF 0.22-5 GMGVKTF] 0.1801 F2l[ FIKTPGGV .0760! 76 _TPGMGVKTFH 0.003! [II KWPMVT 000 ti9l GVKTFHF-G.00-0 TbeXIII-V5-HLA-A3-lomers- [Tbe 273P4B37 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. Fstr II Subsequence] Score0 F CMSLR 1I0601 [EJ RRNDLIIWIR q- 60.27- RNDLIIWIRL 00 4 F- 1LSRRNDLIIW _150031.

[T5j1 PSLSRRNDLI IO-.00o0] I~a~ e X iV-LA-A3-1 Omers- 23P4B7 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.

Start j~isqe [Soe LIIIF~~ TUDE-~I1200 DQ DEL j[ 0.054 [-9ii1 EVLRHCNPWP][ 001] L-8IJ DEVLRHCNPW].000-o F6- D IRHC j 0.000 ]0.0 Iii! DDEVLRHCNP fl 00 Table XIV-V1 -HLA-AI 1 01-9mers- 1 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.

FStart Subsequence 11Score F7741 SWIDDLPK 16.00 Fi57 TWVKEFIK J6000 1057 I KQDASTPK][0 Table XIV-V1-HLA-AI 101-Smers- 273P4B7 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of peptlde is 9 amino acids, and the end p~osition for each peptide is the start position plus eight.

FStart Subsequence cr -224-1 YVILDEAHK 13.00 I593 1[ KIYRQVK j 2.400! 12 03 1LVKRGKELK 2.00 L795] NV1TLQDGK ]EOO]O LZPL-7][ LVEFESQNK 12.0001 GTVE EKR1.801 E K] _TVHLERIII.800I F-462 II MIFLMDLLK ]V1600I f--81 QVFKDSLIR7 =1.600 F51 1 RINL-FQQN-K- 1.200! [1-113]1 RLDDSSEAK]1.00 F4-86-] ]LTSY 1.080 8-37 A KNEAvQK Ii~ F-585 CTVEK 1[ .]0 5- 89 I[ FO -TOKYR 0.80 LQSV 0.800! 66 NYVLSKT [0.60 0 ri12-22ii CLVKALDIK 0.6001 F-7-637 1 TQEEDISSK 0.60 196911 SQSLEHVEK 10.6001 F6 95-~ HYIQQVQ 0.600 1184] QLVGSPQDK 0.600 F617 RYFSKQELR 0.48 F-4537 DTLMEEG 0.45 1ii-34][ PVFPSSTKK 0.400! L-604] LIRQTT E .40 [i0 1 LADDMGLGI( 0.40 F9987 CLSWEF SEK 0.400 F 2-9-1 LLGTLKTFK 10.00 I-087 fI SVNKSMNSR 0.400 [1 ~AAH-YLRYVK 040 Table XIV-V1 -HLA-Al 101-9mers- 273P4B7 Each peptide is a portion of SEQ ID NO: 3; each start position is specifiedt the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

-Start Subsequence S.core -iLIAFKLFNLAK 0.40 21 ]LKA [0 04-00 613 319 L 542 11 GLTLTMATR _I 0.240I F10 4 -j WKAKIRSK 1 g i F226 1j ILDEAHKIK] 0.200 F237-11 STKSAICAR 0.200 F301NPEARLNEK =0.20 F6-47[ ]ARSDK 0.200 [s6i1 ATDAQAVDR 0.200 [16 GVIITTYQM_0.180 [701 VKQL 0.1801 10301!] DED 0.180! =148 ]~-GTSEK 0.180! 51L~LEP 0.180 IIPYLR .0.6 F1i0i1L IAFLYSLYR 0.1601 [i8-831] QLKDDEL 0.1601 F3 22][ KPYFLRRTK 0FO.1-201 F7411 KKKCPKLNK] 0.120 F15411I TNLINTWVK 0F-.1201 [Z9] LNIIERLLK. 0 120! 3=5 6 1 CEMPSLSRK 10.20 F463 ILDLK .2 121711 QEALNCV .2 314][ ENMIK 010 1564] AQAVORVYR 0.20 F-642[ LQSLHAAQR 0.120 374 VPLQEEIYR0.2 1 735 11 VFPSSTKKK ]0.100 F510 ]j VTHLLEREK igo I55] KVLSRIQKI 0.00 F405 1 GVLKKL-CDH 110.090 ~Table XIV-V1-H-LA-AI 101 -9mers- 273P4B37 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.

[F S ubsequence Scorel I02[EVVVKAKIR_0090 550o] RWIFDPWb 00 F-73-31 EPVFPSTJ099] FPEVS .080 F1 197 TDELV .8 F956 FLEDSADNR I0.080J (10711 LYLRGR 000 L ILE= KELMQRI0.7 327 T[LrPKt a .060f [1207 j( 0KLKCKIO060! F11[7 KSSWLMTSK06J L. GVGLTLTA :JI0.6 [ji~~ojj GVESGEA Jlo. p LiKJL KAKRSKARi .0601 364 1 KNDLIIWIRJ[ T8 28LMEYENPITR 0.048] =2 GE~99y 0.045 8 1L K1VQHIAFL I0.0451 [ijLC(SPSJ .4 I ale XIV-V4-HLA-AI 101-9mers- 273P4B37 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.

7Start]I Subsequence co 1 JFIKWTPGMG L oo [=6J PGMGVKTFH ]O] [Table XIV-VS-HLA-AI 101-9mer- 273P4137 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.

L -CEMPSLSR 0[024 W LSRRNDLI 0. 4 WL~No~ll.7 0.001 WLISRR DLIIJ0.000 LlISRRNDLIIW 0.0 [7 PSLSRRNDL II0.00 7 MPS LS R R ND j 0.000] Li lEMPSLS-R-RN I 0.0003 Table XIV-V6-HLA-AI 101-9mers1 273P4137 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 s tart position pius eight, Start~ Subsequence Scr W QLKDDEVLR0.8 W EVLRHCNPW 0.009 2 DQLKDDEVL 9j~~ VLRHCNPWPJ oo 4 IILKDDEVLRH 0.000] 1 LDQLKDDEV000 FT DEVLRHCNp 0000 _LKDDEVRHCI1 Table XIV-V6-HLA-AI 1 01-9mers-] 273P4B7 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 kstart position plus eight, [Star gdI Sbseq ue nce Sce Ta-ble XV-Vi -HLA-AI 101.

I Omers-273P34B7: 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.

FStartj Subsequence Scorej [172 (VKF HGP-SKJ600 F K4IFLMDLL 3.600 24 YVKEAKEATK 2 00 [115 [ArNOYE 1 2.00 [38B1RTKEDVQKKKI 15001 ~723] R

T

RNEGAWL1 .0 26J1yMLL T-LSLYK j0 Ii6]L EALN LVKJ 1.200j 57 RQVFKDSLIR 11.080 F721 HTQEEDISSK 488 ILNIIERLLK 110.800 F676 LYODSK .0 19 ILADOMGLGK 0.0 F373 LVPLQEEIY1 0.800 F W27][ SLLGTLKTFK[0.0 92 ILQDAQASEAK[[060 20] HYLRY EK 0.0 374 VLQEIR 060 F603 SLIRQTTGEK[[5 3 F25j CQGStIGTLK 0j.60 1I202 TLV0.600DK L_ 642 1 LQSLHAAQRKj0600 7061 FLVEFESQNK 0.600 287 KMEEPT 0.8 9103 IALYSLYR Table XV-V1 -HLA-AI 101 -1 0mers-273P4B37 j 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 pston for each peptide Is the satposition plus nine. liiia r t Score F SVNKSMNSR 84 LCGEE]0.400J F17 QAHLYVIAO -8057SA DsIALPK 0.400 F 107] =YLRGR .400 F60-4 ILRQTTGEKK j 0.400 F3 MIL9D9K-1 0.320 1 F 1-221-YK1 IS] 0.300 122511 VILEAHKIK]0.0 L-3l!.I-[AjKPYFLRR [L~9-24 LTL9][EAFKLNLAKIIa~o ]TVTHL LERE K 1 0.200] F- -][Pm~lNwvK 200 1 608 F]E-I(KN IPFR .200] E 66WLAAR-KSDK 0.200 F3-55]IEPL .0 F8 36- FAKEVK a0 F510o IIVHLRK .0 F467Jj LAKDIFPNEK[ F303] =IP.EAGKL.200 F734 PFSSK1 .0 F612 KNFRF] .8 F223 DVLDAK .8 F7 84 GEQLSI 0.8 F298] KAPEK010 F732 RPVFST]010 I~ IFLYLYDGII0.60 D32 KFVSLDIKU.120] Table XV-V1 -HLA-AI 101 1 Omers-273P4B7 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 11 Subseguencel Score 33 11KN GDLEP FKJ 0.120 [53 61LA IRS!SQK I 0.120 [86]DGPLDFQLK 0.120O [-53-]EFNVLSIKI.2 F82-]{TKIKEI 0.1001 F 196-I1TTQM I 0,090] L733 1EVPSK] .9 406- =LKCHP .080 F1 4F[1EQAAHYLR0.8 [7]QLQSLHQ]008 [jo I[PLAELGVu[ 0. 0 80 1 865 FN VLSST 0.080 I81IVCNSGLLLYRI0.8 F389-] IELMT 0.080 F773] F-SWI 6LP .060 206]I RGKELKECG 1~ 0.060] 32]RRTKEDVQK F.0]60 F951 FL[LbDSADNR_.6 F 1-1 ESGE3K 1006 4-905][NERKR[0.6 F-6[2 ELELF1]O.54 [i[F HGPK7E 5[ 0.4 1313 IELM]K .4 F24-3 CRAPNR 0.4 F15-8 N VEFKW 0.4 F 480 LFSSRI Li I 830 LGM EKSA K Table XV-V1-HLA-AI 101-1 lomers-273P4B37 J Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amlno acids, and the end position for each peptide is the start position plus nine. I start ]I Subsequence Score 339_ ISNPEARLNEK] 0.4 694 1H QRQ F0.040 92 JFvHSK C=LSw 0.4 [Table XV-V4-HLA1 -me-I 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.

Istrt Subseqec IScorel 10 o1 GVKTFHGPSK]600 [G-l DKTGMV 0O.0401 [2j[ FIWTGM 0.0081 LF6 .I7V F O. 002 [IlL G V KHPJ 0.001 F711- EFIKWTPGMG- 10.0990 W4i KWP VTJ 0.000 llf][ PGGKF~ 000 Tabl XVV5~LA-A1 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 I start positon plus nine.

F17 1 ICEMPSLSRR- 0F.040 9 -1 RR" III ]FL99?±J F6T] SLSRRNDLII_] I 8 F WI[ NL IIRL] FT SSRND 1FO)022 a SRNDIWI 0.0 I7 1 LSIRRND)LIIW -0.000 I IILPSL-SRRPL 0E.000: Tabl XVV5-LA-AI 1-1 Omers- 273487 Each peptide is a portion of SEQ ID NO: 3; each start position Is 1 specified, the length of peptide Is amino acids, and the end position for each peptide is thestart position plus nine. I =Start Susquence Score W3- EMPS=SRRND0.0 L~I1_CEMSLSR NJ0.000 Table XVI-VI-HI.A!A24-9mers- 273P4B 7= Istart iti luseneiht.

Table XVI-VI-HLA-A24-9mers- 273P4B7 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.

Sr-- t i Sbeune Scorel F723_]_TNGAL1oo L--0i1 RIDGTVTL [KO00] EL87i GLYE 7.920 38 VSLDHIKEL 7.920 9 FFHKG 7500 F-661 AYLQSLGIA 750 F28971 EYENPITRA ]7.5 001 F393-1 LLMETRSPL 7.200 L.637I1 vTQLQLQSL] 7.2001 1I235 ]1 EVMLLTLSL 7.200 120411l MLINNWQQL LL200I F10-42 il SINPFNTSL 7.2001 1l2391 LTLSLYKQL 7.200 F 110 6 F~HEME ]7-.200 =97i] VITYM E200] F1_ 2 [A-c97 7.2001 F581 1 W IVRLITCGT FTO0.50 F930 K.AA EAKL160 =0Z2J TLKGE] F .60 0 =166 IISAA ETSL 0001 F1 42 ALNVL_6. 00 F.2-6 ItNLQLSL26.0 1-48][1 VLLIMPTNL ]6.000 F102-1 EGIAFLYSL 16.000 11l521 SSENKSSWL 6.00 F8821 1~ I~ 118DMGj.00 I T531 DAICEMPSL ]6.0001 632 ]IDLQNSVTQL 6.00 F1-4 11SPEQMAHYL 6.00 1 17 2 TSGPEL F399 SLEGV .0 F1233] F318 MAIIKPYFL_ 6.00 F53 5 TTVGG .0 F246-] AIPASNRLL600 EI U KGLEF 1.60 273P417 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 Startd Subsequence Score 772 MASWIDDL 1212 ECGKIQEAL 5.600 [F J DASLVNHV5.600 [309]LGFKISENL 5.6001 [j5377 QVGGVGLTL ]5.001 ]86[SLDHIKELL 5.6001 [-376 LEEIYRK 5.5441 F57 WR5GQKE- 5.500 .28 DYSVFLLTT 5.000 .525. LNKDYSVFL 14.8001 275 QGSLLGTL IF.80 9 D1FI PNEKVL 99 HQKEGIAFL 40 1231 SADPEVMLL 4.80 F379 J EIYRKFVSL 4.800 Di6i II SADSAL 76 DIGPNLDL 4.8001 =9J1 DATPGEKAL [4.800 949] YACDFNLFLJ 4.800 372 [RLVPLQEEI 4.752 =739 STKKKCPKL J 4.400 12..IIISW~lALI4.400 648 jj AQRKSDIKL 4.4001 F-619 II FSKQELREL 114.4001 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.

[Kt Subsequence I 1 5 TPGMGVKTF 2.000 LE~] WTPGMGV777 0.165 [E GVKTFHGPS 0.100 Li]7 1 KWTPGMGV 0024 8 1 MGVKTFHGP 0.018 GMGVKTFHG 0.010 i IKWTPGMGV I0.010 LLL]L-±9MGVKTFH I0.002 Table XVII-V5-HLA-A24-I0mers- 273P4B7 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 Subsequence Score 5J SLSRRNDLI [T000 L l LSRRNDLII [4 PSLSRRNDLJ 0.720 L 1 RRNDLIIWI 0.432 Lii EMPSLSRRN F 9 EDLIWUFT 0028 [III1 SRRNDLIIW II0.0101 [jii CEMPSLSRR 1 0.02 Table XVI-V6-HLA-A24-10mers- 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 II Subsequence I score 1 DQLKDDEVL .000 LII] EVLRHCNPW 0.180 K6] KDDEVLRHC 0.034 76] DDEVLRHCN 0.018 1 1 LDQLKDDEV lZ01T Table XVII-V-HLAA24-0mers-] 273P47 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 Subsequence Score j1 QLKDDEVLR 10.012 111 VLRHCNPWP 10.010 S7 IJELHCNP 0.002 LKDDEVLRH 10.001 Table XVII-V1-HLA-A24- 10mers-273P4B7 J 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.

IStart j Subsequence II~o~ [948 QYACDFNLFL I .82 IYFSKEL EOL 1224.01 00 [46] 00 F709] 1115QNKE 30.00 0 24.00 0 2.00 F618j YFKQELEL1 F70] LFDFAQGS 0 11 .8 LF FSKQ F 1 0 2451 RAIPASNERLL F1-4 3 1 KQVEAQEPLI 5: 1 207 Table XVI-V4-HLA-A24-9mers- 273P487 Table XVII-V1-HLA-A24-I 1 Omers-273P4B37 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.

Startl I SbeunelScorel =1 COw 398] RSPLAELGVL [1270] KNDLIIWIRL 11 KMASWILD DGQL 11.2 112 DPEGVES9.00 1123011 F1 KSDPVM I0 [364] QLIIEIRL 84 ACQGSLLTL 1.20 1176 PEPLSEQ 11.200 13- LSPEQAAHYL 7.20 F230 KAETVGKL.L6001 647U11 MQSIIDIKL .600 [6fli TCDLSKEETKA .60 Table XVII-V1-HLA-A24- 10mers-273P4B7 Each peptide is a portion of SEQ I D 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.

[StiartISbsqec IEcRe F3921 ELLMETRSPL 6.000 F46ll SPQYACDFNL 16.000 Fj T5- WLMTSKPSAL I[OD 15241 QQNKDYSVFL 6.000 lD3GGLDDG 6.000 790~ SSIKVNVTTL6.0 246 LAPSN ]L 6.000 F26-1 QNNLQELWSL 600 [F1] SSINPF1TS 6.000j [367 I[ LII1WIRLVPL 6.00 [i8Zii]I LLYRELHNQL 15.7601 [3081 ALGFKISENL 560 FB-2-1 STKADIGPNL 5.60 NSa3]EL5.8 F570 I RGKN 5.000 [58111 VYRLITCGTyJ 5.00 112811LLTSLYQL 1800 F636fI SVTQLQLQSL 4.800 i17[5:ii LSSENKSSWL 4.80 I 6968 I QQRVQKAQFL jp F4-80-1 LVFSQSRQIL 4.80 F3558Q MPSLSRKNDL 1±.8001 [525QJ QNKDYSVFLL 4.80 F 4-l1LLKNRI-FKTL 4.800 F3841FVSLDHIKEL4.0 F781SSTKKKCPKL 4.40 F316-1 NLMAIIKPYF 4.20 IT 77l1 ETSLGAPEPL 400 I -0-51 KNDISPPGRF 4I0 11311 FLSGMFDASL 4.000 1 31-71 LMAIIKPYFL 4.000 781FTDVCNSGLL 4000 Table XVII-V1-HLA-A24- I O0mers-273P4B37 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.

[si~I Subsequence EooFej [i16i1 LLSARACCLL 4.000 [141 I DASLVNHVLL 14.00 I 42 I KLFNLAKDIF 14.000 4i1 SARACCLLNL 14.700 F990 IAGFVH-SKCL 4.00 459] SGKMIFLMDL 14.000 F8051 TGSADSIATL 4.000 667 ]I GIAGISOHO10L 400 I446-[ 1IQTDL 4.0001 j80-9][ ITPKF 360 [4-5I5 1MEGKJJ3.6001 [1-4-2]1 SINPFNTSLF 3.600 r 86-] ALQEDPLESF 3.600 [-2621 NNLQELWSLF 3.60 =155 1 NLINTWVKEF'r~ 277IL.LL KTF I~ Tabl XVI-V4HLA-A24-10mers- 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.

[sarti Subsequenc Scorel [I-Il[ WTPG M GVKT-F 3-.00-01 [41KWTPGMGVKT 0.2641 F-9]1_ MGVKrFHGPS LLJL IIW~TGMGV 0.100 [ZEJI E1KWPG0-.07-5] F6]TPG MGVKT F H7 0.0 141 GMVK FHGP .01 21 L!Pi GVTHGPSK 0.10 [74 PGMVK FHG .002 3 I[IWPMGK 001 Table XVII-V5-HLA-A24-l0mers- 273P4B7I 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.

FStart ISubsequence Score] 1101 RNDLIIWIRL 1 11.2001 =4 MPSLSRRNDL 4.800 FT]1LLRRo~ 1.000.

F PSSRNL 0150.

a SRNDIII 7I LSRRNDLIIW iFAo1 EMPSILSRRND F0.015 I-1 T L2EMSLSR I=.015] _L RRNLIIWIR 0=0] Tal V -V6-HLA-A24-1 Omers- 273P4B37 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.

S§tart IL §An 9 i oe- [io l~~RC~Pj1.000j EL~_LDQKDDEV 10.600 F 61 KDDEVLRHCN 002 [811] DEVLRHCNPW 008 KDDELRHC10.0171 [-T1I DQLKDDEVLR7 0.0151 iI EVLRHCNPWP 0.01 F 4] QLKDDEVLRH 002 i] DDEVLRHCNP 002 T[;able XVIII-VI-HLA-B7-9mers-1 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._ FStartj Subsequence Sor 247 IPASRLLL 1100 Table XVIll-V1-HLA-B7-9mers- 273P4B37I Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide Is the start position plus eight.

FS t 7aa[ SubsequenceF] [-6478 AQRKSDIKL l~o [7-491 KPQPQPSPL j o 1 396 TSPALL6l I 7I23 RTRNEGAWLLL 40.000 0=94_ ASRRSLINM ]00, F5Ii sPEQ IIYL 02000] T[yGVGT =[2000 F8-fl] DVCNSGLLL 120.00 [F1I IPSSVNKSM 120.000 139 31 1 LLMETRSPL_j18=000 1 0851 KSMNSRRSL 18.000 a l 1APEKL]12.0001 E_ DAKV4V if12.0001 19 5 EATDYE L12=.000 131811i MAIIKPYFL I112. 000] Fl166 SALAQETSL 1512. 000]f I 772f Ii MASWVIDDL 12.000] 124611l AIPASNRLL Ii F12.o0o0 F142] ALNL 12.000 F24.lI1 RAIPASNRL 1120I DAQASEAKL IFI-2.-ol0 F49 IYAC DF N LF L][12 0-][o [3531i DAICEMVPSL f1200 1219~~ F1 ALCVA 2. 000 1l2301 KSADPEV-ML 6.000] F1 0 9711 RSLASRRSL 1I600 F3-3i7] KSSNPEARL ]F 6.-0001 1106I [HVEDMER Fl6ao000 F 5-14- LERERIN 0 0- E411 LVHLIM ~5.0-00 1 Table XVIII-V1-HLA-B7-9mers- 273P4B37 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. I siar] Subsequence j[ Score] 1961 GVIITIYQM ~f~I )J11 HVD IF 5.000 74 84 QSRQILNII 4.FT000] F46711 KMIFLMDLL 1.4.000 F49 Il DIFPNEKVL ]j 4.000] 48711 QILNIIERL r525 IQNKDYSVFLI 4.000 F417n1 LSARACCLL .4.0 0 0 806 IGSADSIATL 14.000 51215]1 1IELOI 4.000 _920 1 DLSASHSAL _400I [F-j QNFSQSL 4.000 [619 IIFKERLl4.0001 F25611 TGTPIQNNL 1 =P.000 [LSiE IADD J 4.000 1 [1221 LvKR KEL II 4.000I 11121 TSGPP 4.000 fL 4LYQQ T[4] F45ELEEL 0-1 =81 i[ KGFGSVEEL I f -5 0 LGFKISENL iL.-455501 10421 F591 RIQKIQEAL -F4.000o 876 LDGN 1Q 4.000 N2]i1 EGIAFLYSL I LF-. 00 0 F2 46 iiLLAACL 4.000 I F3851 VSLDHIKEL 4.T0700 F-997.1 I-QKEGIAFL J[4.000 5 7]I VTQLLQS ][ooo l iF LTLSLYKQL [f 1 VLLIMPTNL J.F-.0] F368 Il IIWIRLVPL F4.000 [1-28f KTVQIIAFL GILADMGLFl4.000 Table XVIII-V-HLA-87-9mers-1 273P4B37 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 pius eight._ Start li usqene1S-core 409 Z KFTRL 0 40-0 11781ELS QL 4.0 262-8- 1: NNL L 4.000 1 1 0 18 4.0 1559I NP A1[ 4. 00 0 1TQVGGVGLIF L40 _0 [739 Il STKKKCPKL ]j 4.000 84 1 GLYE 4.000 [1160o LMTSKPSAL 1I 4.000] [-632 IOQSTL[400 [7911_S I KVN VTTL j.4.0 00 1 [361 LR~JIf40 F1-9-7] 'ML J[4.0 1i8I LYEHQ F 4.0001 _____8t20 7 4.000 136)[LSGFDS 4:00 041. MLINNYWQQl)J 4.0= j 45=4 1[ TSAOPE ML 3.00] [669 AGISDHDLM 1.1 3.000 I FL R K D I -2.00 184 NPWPIISIT:i 0] Table XVIII-V-HLA-7.9mer- 273P4B7 9 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.

[s~trt[ Subsequence FScor-e )5j1 TPGMGVKTFJ040 [jj7 GVKTFHGPS 0.10 E ll W(PGMGVKT J0. 1-0 0 -21- GMGY] 0. 0-30O Table XV1iI-V4-HLA-B37-9mers- 273P4B37 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 Susqec ii[ FScore] (F8-l MGVKTFHGP7F 0 i1 I6]1_PGMGVKTFH [2:0903 KWTPGMGVK I 0001- 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. [Starj Susqe c oe F 4 S-LSR-RNDL:][6 -PSLSRR7ND 0[.300 LYJLMPSLRRN 0,020 Tabe X'I.V4LA-B7"Mmer5- 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 jSubsequenc [72[ DQLKOOEV 4.000 [T]Z VL.RHCNPWP 0.100 j( EVLRNCNPW 0.100 17 iLDQLKDDEV 0.0201 F3jj QLKDDEVLR 0.01 EIIIL DDELRHC I0.0031 Table XVII-V6-HLA-B7-9mers- 273P4137 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 61 DDEVLRHCN 0.00 ET FLKDDEVLRH [0.000 Table XIX-V1-HLA-B7-1 Omers-] 273134137 Each peptidle 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 peptidle is the start position plus nine.

FStartil Subsequence] scoreI =Z[I F-W 413] HPRLLSARAC 20.00~ 381 FVSLDHIKEL oo ~246fl3~RLLL 1[.00J 2 4 ACQGSLLGTL 12 %0 F9 A0GFVHSKTCLJ~ ~IEJLN LV 12.00 245][ RAPMN E~I LMTSKSAL 12.00j S349 NMPDVDAICE! J 6.00 [T 37 ASRRFPEAE 4.0 [49I5 LLKNRHFKTL 4000 [11711 ETSILGAPEPLJ .0 QNKDYSVFLL 4.00 [F385 DHIEL ]R4000 [i2jI PTNLI N 4.000 [T47 SRRFPEAEAL 4.00 1872 JSTKADIGPNL 4.00 1 LSEQHL .00 F273F INNQQ 00 1 31-7-1 [LMvAIIKPYFL 4.000 [jj] "TGAS J 4.000 Table XIX-V1 -HLA-B37-1 Omers-] 273P4B37 J Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle is 10 amino acids, and the end position for each peptide is the start position plus nine.

Stai][ Subsequence lIScorel 486[ ]QK9IR 400 667[ GIAGISDHDL 14.000 7101ETLVKRGKEL 4.0001 [1095 'SRRSLINMVVL 4.0001 [257[ LTGTPIQNNL 14.000 [i1041 SSINPFNTSL 4.00-01 [91I2 VSIIDL14.0001 LTTQV-GGVGL 4.00 F48il7 QILNIIERLL 4.0001 iF41I6 LLSARA-CCLL1 4.0001 F1375 I FLSGMFDASL L4:0]P 73]8 ISSTKKKCPKL 4.00 57230~ rr 14.000~ F45791 SGMIFLMVDL Ii83]1 NSGLLLYREL 4.000 l88791 ILRHCNPWP71i ±O IiE LREHQ I 4.O70 11633[ LQSVQQ .00 15361 TQVGGVGLTL 4.O0j [3617 LIIWIRLVPL 4. 0-001 ILSSENKSW 4g00 668]jIGSHL .0 F 751 IQPQPSPLLST 3.00 F10~ KIR- AR 81'lF- 3.000j F[-18[SPQDKAEA .00 Table XIX-V1 -HLA-B37-1 Omers- 273P4B37 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[ Subsequence I cr 224 YVILDEAHKI 27.0001 94111l EPSASSPQYA I2.00 122J1 GPEDYPEEGVj 1.8001 [51371ILLREINL 11.00 L ]NGDLEEAFKL 1.20 F848 ILQEGPKQEALI 1.200 1875 IADIGPNLDQL 1.20 [364 KNDLIIWIRL 11.200 [454 ITLMEESGKMI1 =.200j F8573KQEALQEDPLl1J 1 [1-94[ AEATND)YETL]1.200 LZ1:j[ SPDVDHIDQV 1=.2001 Fl-272 ADPEVMVLLTL 1.00 142] ASV LI 1.20 679] =iP~KE .200 ARIASR 6=4.2001 Table i XI-4- -B7-1 Omers- 27P4B37 Each peptidle 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 peptidle is the start position plus nine.

Sstartj Subsequence Sc o re LF2I1 FlKMNPGMVGV 0.30 F-671 TPGMGVKTFH 0.00 GVKTFHGPSK 0.05 F9]1 MGVKTFHGPS 0.20 F~ 5]1 WTPGMGVKTF 0.20 F4T1[KWTPGMGVKT 0010 W87 GMGVKTFHGP 0.01 LI7Z1 PGMGVKTFHG 0.00 FI rrGMV NI-00 Table XIX-V4-HLA-B7-1 Omers- 272P4B7 Each peptide Is a portion of SEQ I D 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 pius nine.

Start Subsequence Score LFIKWTPGMG 0.0011 Table XIX-V5-HL-7loes 2724B 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.

MIStar Subsequence ]Score~ jEThi RNDLIIWIRL 111200 lII SSRD4j 0.400 91 RN n 10.4001 EElIL~SRR NDLtlIIW.11200 [1LLSE L 0.0401 E~iI EMSLSRRND 001-51 Trable XIX-V -HLA-710mers- 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. 1Start iis.~iu I F§:orel 110 VLRHCNPWP 4.00 FT1] QLDEVLRH 0.1 E1Lgq. DEIR 0.010 Table XIX-V6-HLA-B7-1Oes 272P4137 Each peptide is a porton 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 t plus nine.

FS ta rt I Susqec lScorel LKDDEVLRHC 003 DEVLRHCNPW]1.02 Tale X-V1-LA-B3501 -1 9mrs23P4B7 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 FStart Sbeqec Score 1 079 IPSSVN KS

I~~

KPQQPP 1140.00 301 TPGEKALGF140*0 If- 9 SPLAELGVLM H KAAATNY2400I 61FSKQELRELM [247 IPASNRLLL 2000 LSPEQAHY 20.001 F2]1KSADPEVML 200 ESGEAS~j]15.00 [TEVY 120 Table XX-VI-HLA-B3501- 9mers-273P4B37 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. Subsequence 1 Scorel Fi4ijDASLVNHVL 300 15871[CTEKIl.0 F93f I[ DAQASEAKL I[C.00 F56--JP IAGISHlj 3.00 F1 56 178 ADWR 2.400 [1 -11 KIRSKARRI IF409_ K169]l SPPGRFFSS1 12.0001 [31-6]i LAIP .0 F9-4-61 SPQYACDFN 2.0001 1.280] LGLKFK 2.00 [964 RQNFSSQSL 2.0001 1236 IVLTS 120IE 2.000 398 ~RSPLALGVII.0 0 [Table XX-V4-HLA-B3501- 9mers-272P4B7 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 posito orec peptide is the star t position plus eight.

tari I SubsequenceP or [211 G TFHGP 20.300 PGMGVKF 010 F 7 FIKWTPGG 0.03 F27i1 IKWTPGMGV 02 8]I MGVKTFHG 0.01 L1L7 GKTH 31 KWTPGMGVK 10.00 Table XX-V4-HLA-B3501- 9mers-272P4B7 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the lengthl of peptide is'9 amino acids, and the end position for each peptide is the start position plus eight FStart ISubsequence scr F Tabe XX-5-HLA-3501 -1 9es272P4B37 J 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. E~rf Suseue c oe F T]SRDIW]025 i MPS-SRRN] =0.100 -IL=MPSLSRR INii Table XXI-V1-HLA-B3501- 1 Omers-27334837 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of pptie Is10 mino acids, and the end position for each peptide Is the start position plus nine.

~~_ence 17-801 LPKEGE-KQDL 100 F8-6j DPLES FN Y-V [4 0. 0 0 7-491 FK PQPQPSPLI][40.000 L1 KSDEML00 2-58 TPIQNNLQEL 2.0 4 NPFNTSLFQF 2=0 F9461 SP _QYA C DFN L 20.000 3 58 MPLRKDI 20=000 F511 FNK VLSRI EI 6.0001 398SLAE LGVL 15.000 9941 HSTLWE 5000 iiiJI RL RDEGHQTLI 20j L .J HQEIAFLYJ100 491 NPVDIEM[ 2000] 3-651 SRNLI]11.2501 385] L -i650 IsDl~j ooo J LS7SEN.KSSWL 000 il3L=PEQAAHYL 0000 12 DIKADEIA F6-5I3 =AIDDL].000] 413 =PLSAA .0001 525I FAP L EQ VL~ .0 GSLLGTLKTF =DYS0F0 Table XXI-V1-HLA-B3501- 1 Omers-273P4B37 Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the length of pepide is 10 amino acids, and the end position for each peptide is the start position plus nine.

!St a en]r Score 944 FASS-PQYACDFl 5.000 Fl80 I DSIATLPKGF 5.0006- 7381 FsSTKKKCPKL 15.000] [83 NSGLLLYREL 5.-000 1 F7901 SSIKVNVTTL .00 983GSAPNSRAGF 5.000 4.000 F1521 MPTN INTWV [±4.000 372R LVPLE EY 4.000 4-59] S=MFLD .000 [6981 IIQQRj\QKAQFLI3.0 [i6] =Gr.iAjj00] L15SSENKSSLM1L[300:] 58 611TGVEI 3.0001 F6-4-7 QKSIK 300 I SKM3300 iTl ISSEAPEDY 30] 7 1 PSDRRL[301 8 ALCLVAL 3.000 E111 ll NVLf .0 1 l8 SKDIPL j3.0 RHKL GQFWI .0 Table XXI-11A-B3501-1 l0mers-273P3417 -j 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 posiion for each peptide is the start position plus nine.

J

!a=ubsqenceEE 4971KNRHFKTLRI F2.40 0 8961 WPIISITNES j 2. 000 57j I KEVVj2.000] 143 SVHLI .0 194 EPASPY 2.000 1861 lQDLS[ 2.000 17511 PPPLS] 2.000 Lib NMLD DM 2000] 12-4710 IPSNRLLLTI 2.000 IJL1.SSKMASVWi]II2.000] 27-91 LLGLKT K .0 7771 KMASWIDDL 1482 SSQLI 14-1 555 EPSWNPATDAI .0 142EALNHLI .0 I 80]J 30-9LFIEL .0 2-81 42 KADI F [8341 FTNA .0 TO.LYIAFSM 2.000 [Table XXI-V1-HLA-B3501- I Omers-273P4B7 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and te end position for each peptide is the start position pjlus nine.

[rubsequence L[Soj 1I951 NGVIITTYQM 11 2.000 FIjJ[iALSPEQAAHY I .O j 6 69 AFSHLY2. 00 0 894INWIST I200 1 -2 I KS EN LM A!J i.60 F682LVEL .0 KSTADIPN =.500 FTable XXI-V4-HLA-B35- 1 1 Omers-273P4B7I 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.

K art IK Susqun e DDPGGVr I E. O2I0 FIKWTGMG l0 0 F-6-1 TPDGMVKT1.20! Table )(XI-V4-HLA-B35- 10mers-27334B37 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 I Subsequence Soe LI]l GMGVKTFHGP 10010 F] IKTPMGKJ00-1 711 ElKWTPMIF001: Tal X-5-HLA-B35- 1 mr-7354B37 Each pepfide 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.

Table XXI-V5-HLA-B35- I 0mers-,273P4B37_ Each peptide is a portion of SEQ ID NO; 3; each start position Is spe cified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position I plus nine.

I E MPL 0,00 Table XXI-V6-HLA-B35- I Omers-273P4837 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide is, 10 amino acids, and te end position for each peptide is the start position plus nine.

tIt Subsequence

H

10LHC-NPJ 1.2006 Lij LDQLKDDEV 0o.1=50 FT]D VL~c IFP.o91 FBIEL HCNPW_~ F-3I1 DLKDDEVLHC O. 0006! F[iL DDEVLRHCN L o L .4j [MPSLSRRNDL 7jE L wJRNDIF12 1JI~2!!~LWIR I]0600 F61SLSRRNDLII 0,4001 L-51L tPsRR-NDLI =0-200l F81SRRNDLIIWI010 211CMSS]NI000 F-3 I EMPSLSRRND1 FO00 jGVKTFHGPSK -4 KWTPGMGVKT' Tables XXII XLIX: TableX)XJ-Vl-HLA-A1- 9merg-273P4137 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.

Pos123456789 Iscorel FjicO] FQ! E GIAFLY1 F28 F8]-1 VC NSG LLY][F26] 6 70][!SHLM 221 F I1L FEQA HY L-RY1 F2 FD E-LAHY!RY 21 F4 1[L RL 1L2 F2-2]flVWDYVIL-DE][ 12 31 SA1E 9 19 1563 1 DAQVDVY 1 F78 I EEKQ lS 1l F741 YIEEP3G 1181 F111 VML L 18 FQh1IGI3L L 1817 FT2]LADGIGf 17] F1-0I3] SLR] 17] 450 TDTEE17 F37431 GQEV 1 11 F4 R5AGA0R 17 F117 SEA5]E 171 119- 7 TNYFK17] 136 7 AK 16 I 21-6 1RQFVD] 1 227 LEH T 1 I[TEFKM-EY I-Li i~~F28 AIKP2] i TableXXl-V1-HLA-A1 Smers-273P4B7 Each peptide is a portion of SEQ ID NO: 3;'each start position Is specified, theti length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Po~ 1234567897 scorel F786'-K-]Ls s-voHD 16 110271 VSDGEDEDD [i1s] 111521 SSENKSSWLI 161 1J210J FLK-EC-GK-QEf 161 [3-211 AKG1E 1151 F 1 688]LWEH 6891 893 [-LSF 91-7 SLVKE 1100371 D PE V 15 I SFIT0 S!P 1R 11 26 YEG ES 11143 EF1-5TL [i] 11 76 FPL~E 11 92 K ANY F313 I IENLMAlI 1 1 13201 I!KPYFLRR7[ 14 F3291[TEDVQKK141 330 1 [KEDVK1SF4 TableXXII-V4-HLA-AI- 9mers-273P487 I Eac petid isa =portion I Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is39 amino acids, and the end position for each peptide is the start position plus eight Posl 123456789 scr 65-5TKUJE-HIAYL 29 1I21 9 A-L NC LV KAL 2 F62 QE EL 2 368]IWRVL 2 464 FLD!KL27 149]LIPNI 2 533] LLTTQVLGGV 2 1042 SNFN 26 12-15 IE~NL26] 143]SVHLI 2 3441RNKPV 2 36-6 DLI!L 3-9-31L TSL 2 79 1]SKNTL 2 31-2 KSNI 24 487]QLIEL 2 505]RDTTL 2 6 7-6] LMTD24 813]TPGFS 24 913 EADL 2 1 25 G TQI2 148]VLMTL 2 2-25 EAK 23 372]RVLEI 2 4 2-4]LN FA 2 74881 ii.NliERLL1 23 6-3-2]DQS L 2 876 DGNL 2 Fl11 F-22-]L 204 22NN~QQ f3-86]SDIKL 4 0-9]KCHRLL 76 7 DSKMS 806]GAS!T 10O92SARSL

I

1231 SADPEL Ei272; TableXXlI-V6-HLA-A1-1 9mers-273P4B7 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.

Iposi 2468 soe F-4] DEVRH 1 I 9els77P4B7

TableXXIII-VI-HLA-A0201.

9mers-273P34B Each peptide is a portion ol SEQ ID NO: 3; each start position Is specified, the length of peptide is 9 ain acids, and the end position for each peptide Is the start position plus eight.

FPos I13579Iscore F 6 FIA LE EL AEQ G 114 [Fi1 LLY~H [14 135 FLSGMFDAS 1 4 [141] F14SL7H LJ[A [1891 NRIQQNv[1 FTSTSl I F41 ACQGSLLGT J F14 F27-] GSLLGTLKTj 14J F31[6FNMYA11141 371 IKSN ECLl4± F3-7-1I PLQEEIYRKECIA! F3-9-4] ETSPAJ 14] F4050PAELGVK][14 [4-01VAEGYKK 14 F4--8[KKLCDHPRLI 14 [!1-1iRLLSARACC 14E 4-1-7 LSAR ACC L-L 6 HlDVTO 14 E7 IDQVTDDTL F14 468 LLRRDG 1 496] LKNRHFKTL 14 50-31 TLRDGTVT 1114 j 47 GVGLLTM14 54-2]r GLhAA14] 573 IIIGQ-KE-NWv 114 I TabieXXljl-VI-HLA-A0201- 9mers-273P4B37 Each peptide Is a portion of SEQ ID NO: 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 11 123456789 -score 6041 [LRQTTGEK FI14] 646 1[AQ D 51114 6621 YLQSLGIAG 154 665 SLGAGI 1 68-0]CDSVEE a1 6891 OW EHY 17001 5VK~FV14 74-6] KLNKPgPQP 14] F-8-11 GEKQDLSS Eflf 184011 NEVKT 14 F8[-9 QEGPK9QEALIl- F8-60] DPLESFNYV 14 873 TKD PN 14 988 SRAGFVLHSK 14] 11049 SLFQFSV 14 Flossi SVKQF DAST 14 10-85 KSMNSRRSL 14 1091 RSLASRRSLI14 11-061 HVDE 14 1168 LAQESLGA 1 11721TSLGAPEPL 1l4] 1173 SLGAPEPLS14 12111 KECGKIQEA 14 1222]CL VLDK 14 1237 MLLLSLK 1 0019mers-273PB7 Each peptidelis 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 WGMGVKLFHGI 12 EflTPGMGLKTF I [A0201-9mers-273P4B37 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 Lposition plus eight.

Eos 123456789 MI ELSRNLI1 TableXXIV-V1-HLA- A0203-9mers- 273P4B7 Pos 12468xcr TableXXIV-VI-HLA- A0203-9mers- 273P4B7 [P0512345789 core] r~l F NoResultslound.

TableXXIV-V4-HLA- A0203-9merscN~1273P4197 FftosI 124679score] f Noesuts~und, 00 IND A0203-9mers- 273P467 I:-Po s 12468 scorel NoResultsFond TabIeXXIV-V6HA- A0203-9mers- 273P4837 PoA 124679score NogesultsFound.

TabteXXV-V1-HLA-A3- 9mers-27f3P7__ Each pepfide 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.

E~os 12345678-91 score 411 LPQHI2 519J EILFN][E F32211 PFRT 53 LID9V12 2521 RLLTP 2 F329L0] KFK 2 320 IKYFRR 2 695 YIOROK 2 1217i[QE w KI []HKKT STK 1201 41-51 RLLSARACC1120 1 1F] LVEFESQNK F2-0- 122] LVKALDIK 20 AFKLFQA 1I :190 RQRNV 19D 367 L(IWIRL VP 19l 598] QVFKDSLR 19 670] GISDHDLMY 19 717 F EQ TR 19 TabteXXV-V1-HLA-A3- 9mers-273P4B7

I

Each 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 position plus eight.

Fjo-][ F1 23456789 [srl 84-2 1AVKIE 1 119 L V 1 1 QFL SG 18u 2691 L F3G 11 48911NIEL( 856 AL1EDP lES 18 108 KISKRR 1 96106 DPPRF 1 10912SARSI 1 42O KLNL8D11 810 9 VNS2L 1 86 LLYR EH 1 1 7 l~YEHQI1 TableXXV-Vi-HLA-A3- 9mers-273P4B7.

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 1235689score 1132 EESSGEASK LF17 F1-67[ALAQELgaMLii Fj-97] TNDYETLvK IMf 1721 RVKTFHGPS -6- 186] RNLNRIQQR F16] 254 LLTGTPIQ-N ]16 29-6 RAREKD]ATP 16I 32-5]FLRRT1KEOV 16] 13281 RIEVK1161 [332 DVQKKSS 16] 3541 AICEMESLS 1116 356 CEMPSLSRK 16] 401l LELGVLKK 1 4261 NLGTFSAQD 116 1 F51[ INEQQ1N DYI E1 54-4] LMRW[ 61 ATlAQ1VO 1 5681 DRVYR IGQK 16Y 57E 1E WV 16 [57-91VV RT 1J [63891QqLSH[ i- 641 ELSL 16 7461 KLKPPQ 1 16l 1751 l ST TQI1 863 EfEFNlS 11 889 IRCPP16 913 SIEADL 1 920 DSSSL 1 969 QLHE 16 980[ SOGSArN 116 TableXXV-V1.HLA-A3- 9mers-273P4B37 Each peptide is a portion ci 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 Ithe start position plus eight.

Posf 12456789 sor 988] S G F [16 1017 AKIS[R 16 1156 KSSWLMTSK jjj6 123 EVLTS 16 36 ]DLEEAFKLF 457 AKD 1NE 54]1EKVLSQ F1 691 [LEDE] 9-1 ELNL E 1 119 1LA T GGJ[5J F1l VLLIMTNL[1 Fl-9-41 RNgVITTY iri -1- F24-4] AR AIP AS N R [3-141 EEEMIK s [3-931 LL ETP 51 (409 1 LCHPLL 14241 LL TS 1M 453 DTvEESGK 468 LLKRLREG 55RIDGTVTHL [651 It~ RE R Fi 1I [6321 RTGEK1 7SKNL 810] SIATLBEGF 8131 TLK FGS 16--1 845 k KELQPKflE 9][I NfLQSAD i-C 1025IYD ED E 's 1026_ Tabi11eXXVVNI-HLA-A3.

9mners-273P4B37 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 piseight.

FPos 123456 789 sor 826l TNSSLGMEK F147 8291 SLGMEKSFA]F7 861 PLESFNL F1-4 8-6-71 YVSRST 87 61j DIPNLDQL f 9g16fL EIDOSA fl4l 9291 QOAQAS EAK 14~ 986 PNSRAG FVHEfl1 1020 RS ARFWVS[ 1- 1030 GEDEDDSFK 14f 1089 SRRSLASRR F14 1133 ES-SGEASK [E]jf 12-19 ALNCLVKAL 14 13 LSPEQAI-Y 13 56 VLSRIQKIQ 13l F59 RIQKIQEAL 13 QLFEHQKEG 13 100 QKEGIAFLY 1 118 GILADDMGL [1-3 13 FLSGMFDAS 13 149 LLIMPTNLI 13 160 WVKEFIKWTF-1 187f NLNRIQQRN 13 E23 2J KIKSSTKS 13 2461 AIPASNRLL f13 249 ASNLLLTG 13 351 DVDAICEMP 13 383] KFVSLDHIK 13] 47 TLVFSQSRQ 13f 480 LVFSQSRQI F13] 487 QILNIIERL 13 488 ILNIIERLL 13f 502 KLRIDGTV 1 3 513 LLEREKRIN 13 524 QQNKDYSVF 13 540 GVGLTTMA 13 563, DQAV-RV 1 TableXXV-V4-HLA-A3- 9mers-27334B37 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 Po 123456789 Fscr F3MKWTPGV 23 KTJ Fi PGMG 12 Ifl G KF- PS 12 9rs273P4B7 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 123456789sor ]SLSRRRNDI 16 TableXXV-V6-HLA-A3-1 S9mers-273P4B7j 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.

I

[pos 124679sco-re] fTableXXVI-VI -HLA-A26- 9mers-273P487j 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.1 [POJ[ 124578 sco re] F8-01 DVCNSGLLL 28 102] EGIAFLYS. F2-7] 658]1 EHIAYLQSL 1(27 1 [49J DIFPNEKV 1126 1281 KTVQIIAFL J[26J 973 EHVEKEN l([ 1012 E VKAKIR F25- 3-7-9] EIF 24 689 OWESYI2-4] E~1EEAFKLFNL 2 TabteXXVI-VI -HLA-A26-1 9mers-273P4B7 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 lthe start position plus eightI 123-456789coe F5-]51 EREKRINLF J(~fJ F5-1DIPGF1(1 1133 ES EASY(2 (1233 EEVLTL(jj [16 EQAHLR(( 1 8]EQKGIFJ(-l~ 637 TQ2 LSL 2 91-3-7] EADL 2 7143 EDSEL2 12011ELKGE2 121EGQEL 1 57-9]VWRIC 2 608 TGKKF2 77 WDDPK 2 ESNYLK 1201 1195 [AN'EL 2 [93ERLLKNRHF -91 F709 EFENKEF1 7391STKKCP l 79-1]SKN L1 TableXXVi--LA-A26- 1 9mrers-273P4B37 Each peptidle 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 pepflde Is the start position plus eight.1 Po s F1234 5-6 78 9 11 061 HVOER 9- 1239 LTLSLY-KQL 19 F36 DEEFF 1 62- KQA El l1 2 72 DFCGSL1 388 DHI1KKEL LME 18 ~F 7 QtJIERL 1 F535 fl VGVL1 5-63 1AADV 18 568 D YQK1 58-9]TEKYR1 F651EKNFYF1 6281FIDQN

J

F9-2LSS-0L] I 10~[GDDS1 f2E] 117 1[TLAE F1 8- (12151 KIE lC l 1 F78 FDVNGL1 194 RGITY 1 1 97 VITTQM 17 259] PIQNNLQEL 17 E1flEDHIDQVTD-D 1.17 Ij F4-I4]DEGHQ TLV-F [17 [508 IGTVTHLLER Flff 5091 TVTHLLERE 1FI7] 1530 1 SVFLLTTQv !I F57411 GQKENVVW][ F-1 -7 F67101 GISDHDLMY~( FI7 F6-831 SVKEELDW- I-7-1 [723j RTRNEGAL 1 809] DSIArLPKG 1 7 93-01 AQSE AKL 1 7 [1009 EPEVVVKA 1 1129 EGVEESSGE F1 jjT( EKVLSR IQK r16 TableXXVI-V1-HLA-A26-1 9mers-273P4B37

I

Each peptide Is a portion of SEQ ID NO: 3; each start position is specified, the Iength'of peptIde is 9 amino acids, and the end position for each peptide is the start position lus eight.

Pos] 1-2 3456789 sor VLSRIQKI 1 ALEELAEQ I .2Th [81] V CN SGL LY[16 0fl FDA Y SLHV Ii! 6 [1 71 J- LNVEF 16- 1 1 6 WK F 16- 183] ERTRNLNRI J16 21-61RQFWY1 2201 FVWNDYVILD F16] 2751CGLGL 1 315f ELMAIIKP F16] 31-6] NLMAIIKPY 16 f F331 IEDVQKKKSS 16 36-6 DLIIWL 16 [EJI LVFSQSRQI]j16 fl RIDGTVTHL 116 517f EKRINLFQQ 16j 6261 ELFTIEDLQ 116] 733]EPVFPSK1 7 34] PVFPSSTKK 1 76"6 EDISSKMAS 16 774 SWIDDLPK 16] 816 KGFGSVEEL F16 88 DQLKDDEIL 16f 106 14 WKAKIRSK 16 39J EAFKLFNLA 15 EE RiQKIQEAL 15f 79~i TDVCNSGLL F1 127] GKTVQIIAF 15j F132] IIAFLSGMF 15 F144 LVNHVLUM 151 1-96 GVIITTYQM [15 237l STKSAICAR 1 TableXXVI-V1ILA-A26- 9mers-273P4B37

I

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.

IPos]E 123456789 [score] IFIIRTKEDVQKK is 40-5 GVLKK-LC-DH 15l 422 lCCLLNLG-TF 439MED SPD-VDHI 1 54-0GVGL-TLTAA1 550IFDPSWE F5-5][1Fsw I FiE5 F578f NVYLI 58-0] DDRIT 1 5-881 TEEKYR 5 59-8]QFDLR1 636l SVTQLQLQ-S 1 6 8-6 WES1 688f I..WEE5Y1 F69-2] EESIR F825 CTNSSLGMEIF151 841fl EAVQKETLQ I1-5 909oq ESNVSIIE I 101i 1 EEVVVKAKI 15] 1013 VVKAKIRS 15 1042 SINPFN TSL 15 1102 MVLDHVEDM 15 1218ELCVA1 12356 VLTSY1 TableXXVI-V4-HLA-A26- 9mers-273P4B37 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 pius eight Posj 123456789 scor STPGMGVKTF 14 9j GV KTPHGPS TableXXVI-V4-HLA-A26-1 9mers-273P4B7 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.

LII WTPGMGVK-T 101 FIWPGMG E Tal eXV-V-HLA-A26- 9mers-273P4B37 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.

PosT 1234567891 EM4]PSLSRRNq 12

ICEMPSLSRLI

SRRNDLIIWW

[I RND LI IW IR L I TableXXVIV6HA261 9mers27PB 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 I eight.

Pos 123456789 jFc-e 8 EVLRHCNP DQLKDDEVL 16 ELI DEVLRHCNP 12 H DDE VLRHCN W B07-mers273P4B7j 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 SP-o-s I 123456789 Iscoe 24-7] IPASNRLLL 254 L7- KMPQPQ-PSPLr 23 j399 SLEGL2 1233DPEVMLLTL 22 14 SP AHL2 111-641 KSLQT2 [41-3]I PLSR 9 [F9 8 PSR5F11 [1079ISVKS 9 [1 68 TGRK 8 [559]NADQA 8 [33-7]KSPAR 7 [3961ERPAE] 7 75-51SLSHT1 F8601DLSNV1 I 894 NWISTK1 11l45DSELS1 11-78ELSEL 7 F118 SQ5MEA 17 120 APVM 1 57]ESKIL 1 7235]RNGWL1 91 EPASPQ 1 379 EYKVL1 4021 AEGVKK 1 634 QNVTLQ 14 17 j[EPVFPSSTK 14] '7501 PPQSP 1 8141 LPK FGSV 4] 81 IK-GFG SVEE L1 14 I TableXXVIl-VI-HLA- B30702-9mers-27334B37 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. 123456789 Icoe 920]DSS SL1 F DISPPGRFF 14 10 9 6 RRLNML14- 1143 ED EL1 1212EGIEL1 17219 ANLKL1 80 DCNGLL1 5-7-8]GKERR13 F2358 13LSKN 393] LLERP 131 141-6] LLSRAC 3]4 150-61IGVIIL] F551 QUDSVL11 1F]ITQVGG 13l] F5]IP5-5 AT 13]l [F655 EHAY 1 710] EQKF I 75-1 QPQPSPLLS 13 75-3]QSLSH1 8-4-9]QGKE 1 873 KAIGNL1 F949]CFNF 13 1044 NPNTS F [j 1069 SPPGRFFSSE13 1085 KSMNSRRSL 1 101RSARRL1 [TableX)(VII-VI-HLA- B0702-9mers-273P4B7 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 Ieight.

EIZ 2456789 1161]MSPSL 13 IF-11 TSLGAPEPLI1 11776 [PPS E 3 12-31SADPEVM-LL13 291 EETNGL1 38l EEFLFL1 88-2 EYEN L 1 F61 EGAFY IE1 245 AIASRL1 F25-81 TPQNL12 272]DAQSL1 [F3 KDT0 GKA11 [322f KPYLRTK11 52-6] NDSV LL 1 576 KENVVYRL 1 63-21DQSTL1 6581AYQL1 66-81IGSHL1 739 TKCPL1 781~~F fKEQl 806] GSDIAL1121 [876] DIGPNLQ F-1 2 TableXXVII-V1-HLA- I B0702-9mers-273P4B7 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 I 123456789 scorel 930 DAQASEAKL 12 982 CGSAPNSRA 12.

1006 KDDEPEEVV 12 1042 SINPFNTSL 12 1070 PPGRFFSSQ 12 1166 SALAQETSL 12 1202 TLVKRGKEL 12 1~ 1 TableXXVII-V4-HLA- B0702-9mers-273P47 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 TPGMGVKTF 18 2 IKWTPGMGV 11 4 WTPGMGVKTI 1 B0702-9mers-273P4B7 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 scorel I IMPSLSRRND 13 PSLSRRNDL L.

LSRRNDLII 10 T SLSRRNDLI I W RRNDLIIWI 1 TableXXVII-V6-HLA- B0702-9mers-273P4B7 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 foreach peptide is the start position plus eight.

Pos 123456789 f DQLKDDEVL I 1 LDQLKDDEV 6 TableXXVIII-V1-HLA-808- 9mers-273P4B7 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 Po 123456789 lscore [739 STKKKCPKL I 32] 514] LEREKRINL 28 1791] SIKVNVTTL 28 Fo EIYRKFVSL [1018][ KIRSKARRI J(26 1202 TLVKRGKEL 26 360] SLSRKNDLI 25 S99 11HQKEGIAFL 1124 1 303 TPGEKALGF 24 318 MAIIKPYFL 24 368 IIWIRLVPL 24 416 LLSARACCL 1 495 LLKNRHFKT 24 512 HLLEREKRI 24 973 EHVEKENSL 24 1016 KAKIRSKAR J [38 EEAFKLFNL ][22 [168 TPGMRVKTF] 22 [180 SKDERTRNL 2961 RAREKDATP [457 EESGKMIFL][ 22 1619 FSKQELREL][ 22 [10141 WKAKIRSK 2] 11092 SLASRRSLI 1 2i I 1152 SSENKSSWL [2 j 29 KEATKNGDLJj 21 333 VQKKKSSNP 21 525 QNKDYSVFL 21 TableXXVIII-VI-HLA-B08- 9mers-273P4B7 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.

[PosI 123456789 score 123456789 849 QEGPKQEAL 21 1160 LMTSKPSAL 21 1209 ELKECGKIQ 21 51 FPNEKVLSR 112 RDGRKGGIL I 230 AHKKTSST 4931 ERLLKNRHF( 546 TATRWIF 699 QRVQKAQFLI 20 I 728 IGAWLREPVFII 27 EAKEATKNG 19 190 IIRIQQRNGVI 19l [344 fRLNEKNPDV 191 S396 ETRSPLAEL I19] 565 QAVDRVYRI 191 646 HAAQRKSDI 19 655 j KLDEHIAYL 1191 [769 SSKMASWI 19 780 ILPKEGEKQDI 19 [11881 SPQDKAAEA 119 1 1219 ALNCLVKAL 19 14 SPEQAAHYL 18 88 LYRELHNQL 18 170 GMRVKTFHG 18 308 ALGFKISEN 18 359 PSLSRKNDL 18 386 SLDHIKELL 18 389 HIKELLMET 18 393 LLMETRSPL 18 I 399 I SPLAELGVL 18 [464 I FLMDLLKRL 18 608 TTGEKKNPF 18 6 EKKNPFRYF 18 881 LDQLKDDEl 18 1913 SIIEIADDL 18 1020 RSKARRIVS 18 62 KIQEALEEL 17 TableXXVllI-V1-HLA-B08- 9mers-273P4B7 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 Posl 123456789 score 124 MGLGKTVQI 17 148 VLLIMPTNL 17 247 IPASNRLLL 17 282 TLKFKMEY [f] 305 GEKALGFKI 17 406 VLKKLCDHP 17 468 LLKRLRDEG 17 488 ILNIIERLL 17 551 EREKRINLF D 592 EKIYRRQVF 17 632 DLQNSVTQL 17 E8] DLSVKEELD 17] S744CPKLNKPQP 17 784 GEKQDLSSI 17 851 GPKQEALQE 17 861 PLESFNYVL 17 9 DLSASHSAL 1D 935 EAKLEEEPS 17 SINPFNTSL 17 TPKNDISPP 17 1204 VKRGKELKE 17 1221 NCLVKALDI 17 12311 SADPEVMLLI 17 1233 DPEVMLLTL 17 141 DASLVNHVL 16 204 MLINNWQQL 16 294 ITRAREKDA 16 301 DATPGEKAL 16 26 LRRTKEDVQ 16 L0]1KLCDHPRLL 16 4§8 NRHFKTLRI 16 597 RQVFKDSLI 16 1 ELRELFTIE 6 648 AQRKSDIKL 16 723 RTRNEGAWL 16 749 KPQPQPSPL 16 7721_MASWIDDL 116 TableXXVIIl-V1-HLA-B08- 9mers-273P47 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 scorel 814 LPKGFGSVE 16 841EAVQKETLQ 16 869 LSKSTKADI 16 E81 QLKDDEILR 16 1069 SPPGRFFSS 16 11 EAKGPEDYP 16 16 SALAQETSL 16 1 EATNDYETL 16 KIQEALNCL EE 6 ALDIKSADP 16 2 EASRRFP 15 36 I DLEEAFKLF 15 49 DIFPNEKVL 55 I KVLSRIQKI 15 59 RIQKIQEAL 15 86 1 LLLYRELHN 1i 118 GILADDMGL 143 SLVNHVLLI 15 ii4 FIKWTPGMR 15 182 DERTRNLNR 15 197 VIITTYQML 15 2121LSSFRGQEF 15 246 AIPASNRLL 15 259 PQNNLQEL 15 278 SLLGTLKTF 15 292 NPITRAREK 15 320 IIKPYFLRR 15 340 NPEARLNEK LKRLRDEGH 1 [8 QILNIIERL 15 505 RIDGTVTHL 15 574GQKENVVVY 15 653 DIKLDEHIA D 668 IAGISDHDL 15 721 QQRTRNEGA 15 730WLREPVFPS 15 5 YACDFNLFL 15I TableXXVll-V-HLA-BO8- 9mers-273P487 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 1002 EFSEKDDEP 112281 DIKSADPEV Tab eXXVIII-V4-HLA- B08-9mers-273P4B7 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.

Pos123456789 score SFIKWTPGMG 5 TPGMGVKTF 14 7 IGMGVKTFHGI 12 I GVKTFHGPS 1 808-9mers-273P4B7 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 Jscore [ISLSRRNDLI I 23 1 FPSLSRRNDL 181 L~1 LSRRNDLI l 2 TableXXVIII-V6-HLA- B08-9mers-273P4B7 Each peptide is a portion of SEQ 10D 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 W QLKDDEVLR 17 (II VLRHCNPWP I 14 Wl ILDQLKDEVI 12 W DQLKDDEVLI 10 DEVLRHCNPI W- ![TableXIX-VI-HLA-1 1 510-9mers-273P4B7J 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 I the start position plus eight.

Pos 123456789scr 973 EHVEKENSL 22 658 EHIAYLQSL 21 98 EHQKEGIAF 19 49 DIFPNEKVL 15 247 IPASNRLLL 15 535 UQVGGVGL 15 806 GSADSIATL 15 130 KSADPEVML 15 142 ASLVNHVLL 14 176 F PS ER14 24 RAIPASNRL 14 301 DATPGEKAL 14 337 KSSNPEARL 14 368 IIWIRLVPL 14 409 KLCDHPRLL 14 447 IDQVTDDTL 14 457 EESGKMIFL 14 488 ILNIIERLL 14 576 IKENVVVYRL 14 619 FSKQELREL 14 694 SHYIQQRVQ 14 1710 FESQNKEFL[14 i 791 SIKVNVTL 14 840 NEAVQKETL 14 873 TKDIGPNL 14 116HVEDMEERL 14 114 EEDPSGETL 14 W RRFPEAEAL 13 84 SGLLLYREL 13 99 HQKEGIAFL 13 141] DASLVNHVL 13 TableXXlX-V1-HLA- 1 6 B510-9mers-273P4B7 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.

Pol 123456789 Iscore] 180 ISKDERTRNL II i F219] EFVWDYVIL F256 TGTPIQNNL j11] 379 EIYRKFVSL II S385 )1VSLDHIKEL I1 iL 396 ETRSPLAEL 408 KKLDHPRL F 641 FLMDLLKRL 111 472 LRDEGHQTL 13 14eilv~as~ l~F13 487 QILNIIERL S499 1(RHFKTLRID 1 S525 IQNKOYSVFL fj 1 6749 ]KPQPQPSPLII 750 PQPQPSPL II S772 11MASWIDDL fj 13 S781 PKEGEKQDLI 13 S816 IKGFGSVEELI f3jJ S849 IQEGPKEAL II 861 PLESFNYLI II 1 761 DIGPNLDQL LI]3 I1108 KSMNSRRSL II i] 110915 RSLASRRSL 11096 RRSLINMVL fjI] 1105 DHVEDMEER I] I117 TSLGAPEPL I] 3 1202 TLVKRGKEL 1 212 ECGKIQEAL f3 123 1SADPEVM LLII 1233 DPEVMLLTL 14 SPE HYL 19 rsAHYLRYVKEII1 35 GDLEEAFKL E12 38 ]EEAFKLFNL l59 (RIQKIQEAL L 62 KIQEALEEL L I] [TDVCNSG LL LI TableXO(IX-V1-HLA-7 61510-9mers-273P467 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.

t Pll 11 23456789 [soe] Fl-2j KTVQIIAFL F 12 1 2Il1 AIPASNRLL lI T2 1 25[9]F PIQNNLQEL I12] 3651 NDLIIWIRL 1 393] LLMETRSPLJ( 121 39flSLA LV II1 445 DHIDQVTDD JJ 47-61GHQTLVFSQ J. I [TJ[ RIDGHL 1 2 F5-1J4[ IDGTVTHLL 2L [IT1MLLEREKRII 1 1f 12QVGGVGLTLJ 12] [596 IRRQVFKDSLJ[ 1 1 6161I FRYFSKQELII 2]1 634 IQNSVTLLJ 12]1 [I LAAQRKSDI 12 I 65[6 K12EHIAYL 7 680 CDLSVKEEi 12 739 STKKKCPKL 12 8 911 RHCNPWPII1 121 1920 )1DLSASHSALI 12 1924 ISHSALQDAQI 12 949 YACDFNLFL 12 991GFVHSKTC L 12 993 VHSKTCLSWF 12 1042 SINPFNTSL 12 1067 DISPPGRFF 12 1160 LMTSKPSAL 12 11951 EATDYETL 12 12191 ALNCLVKA 12 1229 IKSADPEVM 12 29 1KEATKNGDL 11 78 IFTDVCNSGL 11 8 E LYRELHNQL 11 TalX 1XV-HLA- BI510-Mer7P4B7 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 pl s 0eight Posl 12346789 score 920f F- AFLHN LQ 11 102 EGIAFL 11 20-41 FMLINNWQQL 11 262] NQLW SL 11-I 272f EEOGSL 1 LGF-KISENL 11' 1318 IEMIKY l lK F3 ][PSLSRKNDL]ID 1]1 S3861[ SH IK Irl 388 HIK LL1 AELGVLKKL 11 417 LAACL 1 F41 MFMDL 1 4 9 6 L K R FK T L 1 F4 NK YSVF L 1 1FITMRWI Iii 69-91 QR EEQ 1 [7QKAFL EFI4 760 HHQED 1 761

HQEDS

8 2-21 ELTL 1 F8 QALQDP I 1 Fi88-2][DLD EEi F] SIEIDO Iil 1152SEKSL1 111 KQALO 111 TableXXIX-VI-HLA- BI 510-9mers-273P4B7 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.

SP-os] F123456789 Iscorel 1235EVMLLTLSL 11 70LAUE=QGDDEF 101 [8oFD-VCNSGLLL 1101 127 DKVIIF 11 1I2711 FDA G lo-10] Fl-361 LQEEYK -010 5192 1EKIRR 101 F2I EKNPFRY 10 673 HDLMTCD 10 728 AWLEPV 10 930 AQAEAK F59471 PQYAF 959 DSDNRQN 964 QNFSQSL F72i NDISPGR 10 1166[GAl SALAETS 10 Tab4-leXFl-V4-lA 951 0-9mrs 7 EachjIRNFS petd 1s 0 oto F16 SE1DN: 0;ec start p sitin sspcfid [7239 12468 Iscr B51TPGMGVKTF 104B Tabl2XXIM-5-1-11A- B1iO -9mers-273P4B7 Each peptide is a portion of SEQ lb 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. FPos 24579 soEg TableXXIX-V6-HLA- BI 51 0-9mers-273P4B7 'Each peptide is a portion of _EQID NO 3; each start position is specified,11 the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

L259mers2734BL 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 peptlde is the start position pius eight [j5jj TPGMGVKTFI 14] FTableXXX-V5-HLA- 1 B2705-9mers-273P4B7 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.

[sI123456789sor W RRNDLIIWI 25 9 RNDLIIWIR 20 [f ICEMPSLSRRI 16 [4 PSSRRNDL 13] IL SRRNDLIIW 12 TableXXX-V6-HLA- B2705-9mers-273P4B7 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.

S123456789score] [LDQLKDDEVLI 16 II LKDDEVLRHI 13] [YIIQLKDEVLRII 11 TableXXXI-V1-HLA- 1B2709-9mers-273P413 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.

F Pos 123456789 score] W JRRFPEAEALI 28 1096 RRSLINMVL 25 1596 JRRQVFKDSLI 24 (419 ARACCLL 23 616FRYFSKQE L 22 699 QRVQKAQFL 22 183 ERTRNLNRI I 20] [472 1RDEGHQTL 20]( 14931 ERLLKNRHFI 20 498 NRHFKTLRI I 201 571 YRIGQKENV 20-1 1582] YRLITCGTV 20 582 G-7 189] NRIQQRNGVF 19fl 1019 IRSKARRIV 19 11095~ RSLM Li 89 E Q 18 F89-] E1-f F TableXXXI-Vl-HLA- I B2709-9mers-273P4B7 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.

l 123456789score 5111 YRDGRKGG I 18 515 EREKRINLF 18 S890 ILRHCNPWPII 18 245 I RAIPASNRL 11 7 [327 IRRTKEDVQK{ 17] [4o8] JKKLCDHPRL][ 17]1 [j3j ][GDLEEAFKL][ i6 [625 RELFTIEDL 1 f 5] RIQKIQEAL 15 47o9KRLRDEGHQJ[ 15] [50] RIDGTVTHL][115 571KENVWRL[ i5 1 8161KGFGSVEEL 15 110241 RRIVSDGEDI 15 110721 GRFFSSQIP 1 5 11090 RRSLASRRS jj 1 112 RDGRKGGIL 14 1 11411 GRKGGILAD I[ i. I 1142IIASLVNHVLL 11 Iis1I GVIITYQM 1L 2. j 1252 1 RLLLTGTPI II fl 1256 E1TGPQNNL 1 309 LGFKISENL ul 365 NDLIIWIR j 372 RLVPLQEEI 1fl 1382 (1RKFVSLDHI Df l4 14871 QILNIIERL [1 1] 518 KRINLFQQN F14 723 RTRN EAWL IiEI 964 R QNFSSQS LI] GFVHSKTCL9 9u 1239 LTLSLYKQL 3-9] F 42 IKLFNLAKD I 13 1 49 1[ DIFPNE F-1113-1 f TableXXXI-V1-HLA- B2709-9mers-273P4B7 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 123456789 -1ce] F1s] KVLSRIQK-4I -8i L1F81 VLLIMPTNL][1 13 1 F219]1 FVW I 11 S244ARAIPASNR] 1Y F247] I297 1FAREKDATPG][ 13 1 337 KSSNPEARL 13 343 ARLNEKNPD 13 1J 3 IRLNEKNPDVI 13 I S359 1(PSLSRKNDLI 13 13711 IRLVPLQEE 1 3 S379 )1EIYRKFVSL 1 3 S398 IRSPLAELGV I 13 S402 11AELGVLKKL 1 3 S414 11PRLLSARACI 13 S460 11GKMIFLMDLI 13 14611 KMIFLMDLL 1 3 1502 11KTLRIDGTV 1 3 S597 11RQVFKDSLI 1 3 j6801 I CDLSVKEELI 13 1 [411 KPQPQPSPLII ii i ss I GSADSIATL 111] [87l I TKADIGPNLIII3 1 88 DQLKDDoucoEILI 13 18911 RHCNPWPII j/13 191311 SIIEIADDL 11 3 j 1947 IPYADFNLI 113 1 [101811 KIRSK<ARRI 11 3 1I051FQFSSVKQFI 13 I 1089 SRRSASRR I 13 1I112 ERLDDSSEAI 13 1 230 KSADPEVMLI 13 1 i F40-21 Each peptd 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.

fp 1246 789 score [j IKTPGMGVTLI] W KiR fPGMGVKw B32709-9mers-273P4B37 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 I'o 23-456789] score] rR-N DLIIWI 22 E] PSLSRRNDL 13 SNDLIIW j TableXXXI-V6-HLA- B32709-9mers-273P4B37 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.

FPR 12468Fscorel -IIQL 7yE 13 1 1LL]DQLKDDE LII [TableXXXII-VI-HLA- B34402-9mers-273P4B37I 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.

FPosl 123456789 scr 1402 I ALGVLKL 29 I .1143 EEDPSGETL 2 TableXXXII-V1-HLA- B34402-9mers-273P4B37 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 [P os i 123456789 jscore F47 FEESGKMIFL I 25 F6[ 25FRE LFTIE DL 24 EEFKFN] 23 44F1D-EGHQTLVF 23~~ 822I EELCTNSSL 23~~ 11771 PEPLSGEQL F23 F29 FKEATKN-GDL [4 F514 LEREKRINLJ 22j 576 IKENVVVYRL L2 F610 GEKKNPFRIY j221 62211QELRELFIj 22 ESQNKEFL] 22i 84-01 NEAVQKETL 1 22 11118]SEAKGPEDYII 22 21 811 QEMDV I 3 F 11 QALEPL 21 1208 2EL2EGI]i 456 MESGMF [20 1 ElF71DI FP NE K VL Ii -3-f 1 GEKQDLS I fl NLM1IIKP 1181 F5-92]EKIYRRQVF 1W 112191ANCLVKAL 1W8 245[ RIPASNR-LL7 J 692 5f EEEIQ 1-7j 01211SAPEML 11 Tabl~eXXXIIMV-HLA- B4402-9mers-273P4B7j 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 12Efl EGIAFLYSL _6i 1,521 MPTN LI N-T-W 16 204 j(MINN WQQL E~jlfl 24611 AIPASNRLIL 1 ~~1 6581 EAY SL161 487 D16ND 16 908 IAENVII 16 9-131 SI EAD l1 1051 FQFSSVQF] Ii 11331 ESGAK 16 WKI KLFNFAKDI 1 98 [EHQKEGIAF 112811 TVIIFL ]i 1 68] T PGMRVKrTf] 1 F3 TGTPIQNN 356 CEPSSR 3:7 D EIYRKFVSL 395 METRSPLAE 115 2 41 ACCLLNL][ 4!39[ ES V 15 1 461 MFMLL 49-61jEKNRFI E TableXXXl-V1-HLA- B4402-9mers-273P4B7 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. j Pos 123456789 [521 NLFQQNKDY [15 F91 EEKIYRRQV]FJ5] 61 EKKNPFRYF 15 j 6481 AQRKSDIKL 15 654 IKLDEHIAY 15 [655 F KLDEHIAYL j15 F86 EELDVVEES 1151 71- KEFLMEQQR ii 74 KPQPQPSPL 1 76 EEDISSKMA 15 806 GSADSIATL 15 810 SIATLPKGF 15 1KGFGSVEEL[ 15] 88 ]SSLGMEKSF[ I8Z][DEILRHCNP ][Z

F]EEILRHCNPW][

PEEVWKAK 15 1 DEDDSFKDT 1 1 KDTSSINPF 15 NDISPPGRF [15j 116 DISPPGRFF 1151 1085 KSMNSRRSL 1 1 AEATNDYET 15 12111 KECGKIQEA F1?]L ECGKIQEAL 15 j3] KNGDLEEAF][1T F3DLEEAFKLF I] QEALEELAE] 14] 71 ][AEQGDDEFT 141 76IIDEFTDVCNS

LK

[i4lII SGLLLYREL ILI F4- QKEGIAFLY 1] F41 DASLVNHV 14 F LLIMPTNLI 5614 1IE][ LINTWVKEF 14 I !8Ii ERTRNLNRI 1141 I0 ~i]TYQMNNW]1W TableXXXII-V1-HLA- B4402-mers-273P4B7 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 247 IPASNRLLL 14 F288 MEYENPITR 14 3121 KISENLMAI I3T41l SENLMAIIK 1141 318 MAIIKPYFL 14 362 SRKNDLIIW 14 [36s NDLIIWRL 7ii!] [F3 SLHIKELL 14 SPLAELGVL 114] 506 IDGTVTHLL 14 F11 NKDYSVFLL[14 546 TAATRWIF 14 574 GQKENVY 14 [85 KEELOWEE 14 7078 VEFESQNKE I [7 J EFESQNKEF 14 F7i- 1I N 14 17721 MASWVIDDL 1141 791 SIKVNVTTL 14 858 ]QEDPLESFN 14 85[ EDPLESFNY 1] 8 LESFNYVLS [1W] 903NESQNAESN D [909[ ESNVSIIEI I[IW F11 IEIADDLSA 14 [941 EPSASSPQY4 [84SAPNSRAGFII 16 DEPEEVK 14 1042 SINPFNTSL 1141INPFNTSLF [1W 196RRSLINMVL 1 19 EATNYETL 14 1217 QEALNCLVK 3 VMLLTLSLY14 1LTLSLYKQL EKII 9 mers- 273 P41 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 L eight.

[Po 123456789sel ITPGMGVKTF 14 B4402-9mers-273P4B7 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.

FPos 123456789 ER iL ICEMPSLSRRI is] PSLSRRNDL 14 W SRRNDLIIW 14 5J SLSRRNDLI 13 L81 RRNDLIIWI 13 6 LSRRNDLII I111 STableXXXII-V6-HLA-1 B4402-9mers-273P47] 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.

Ios 123456789 Jlcoe ~IERHNPWI is]1 Efl ElflflI 2 l DEVLRHCNPI 12 TableXXXIIII-V1 -HLA- B5101-9mers-273P4B7 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 the start position plus eight.

Pos I123456789 TableXXXIIII-VI-HLA- B35101-9mers-273P4B7 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.

Fos]123456789 sce 5551 17NAT 1 749]KQQPP 17 816]KFSVE 17 122]DMLGT 16 309 LGFSEL 1 4 5 5 LMEG I 16 58-6 TGVE 16 61-41NPFRYFSKQ 1 6761 LYCLV 1 768 ESKAW 16 814 LKFSE 16I 1071 PRFQI16 1145 DPGETLS 16 [1168 LAQETS IE 12 18FEALNC=LVKA 16~ M1221 INCLVKALDI 6] 49fl DIFPNEKVL 15E F52] PNEKVLSRI F157 I 55 II KVSRIQKIFi75 TableXXXIIII-V1-HLA- B35101 -9mers-273P4B37 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 PosI 123456789-l 5 461 MRI F5 MRWFD fl 5-821 F6 2[ Q EL R EL FT Il F7FPSSTKKKCD151f 7 8-01 LPEEQ 18 3-6] FAKEVQ( F906]QASVI[5 120-8] EKCGI[ WF PEEASP 1 F1 0-211EGIAFLYSL 114 1 251 GLKVI~1 13 3 IFS MFD 14 1l57] DNW E1 178 PSDETR 1 P1 RQRGIL14 F1 9l ITQMIR14 1-81 QEVWY I 21 LLGP]1 256 TTIN 14 2 92f FN PTRAREK 1 340]NERNK 1 311! SKDI 14 F3-6 DLIWR611 137-41 PQER 1 382][KVLH] 14 1480 IF LVFSQSRQII[ i [F49 RFKLR-81 544] ELATW 1l 5601 ADQ 14 63-21D STL1 F683 SVELW 1 172 7 E GA WLRE P VI 141 728 AL REPF14 TableXXXtIi-VI-HLA- B51 01-9mers-273P4B7 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.

Posl 123456789 Isorel 7551 SPLLSTi-HT 14J 76-9 SMASVI 14] 11 E1VAK 14] 110441 NPNSF 4 110991 LIMLH 14]1 Tab~eXXXIIII-V4-HLA- B51 01.9mers-273P4B7 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 12468 1 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 eac peptide is the start position plus eight.

f7PosI 12568 Iso] FY31[MSLRRD.17] F]6- LSRR D EI L~ RRNDLIIWI][ 31 SLSRRNDLI 11] F]4 ES LSR R=ND L =9 Ta.eXXII--H- 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 fr each peptide is the start position plus eight.

f]DDERC F6I TableXX(XI V-V1.H LA-Al l0rners-273P4B7 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.

[Po-sI 13579 scorel [1117 SAGEY~3 16091 ITEK FR L12311, SADPEVMLLTI f 24- 11 DVCNSGLLYL IF23] DE [[~PQH 21] [1-5-1IPQAY 21] 2-81 GIKF 'MY1211 F532611 ~HKLMj 1 372][vPQEYI_0 F6-5][ ~LEAA 191 L3--3 R GH LF118] R[7-3 ADAGHQYDV F 18 11141 YIEP E F1 8 TableXXXIV-VI-HLA-AI- I0mers-273P4B37 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.

F 1234567890 s co r e E11191 DKAAN 15j CTableXXXIV-V4-HLA-AI- 10mers-273P4B7 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 peptide Is the start position pius ninej E Ta-beXXIV-V5-HLA-A1l0mers-273P4B37I 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.

I

Pos 2479 Is Lsore] [11Ro ALIR 14 Ii M PS~ SSRRNDLIIiii [~Pl SRIAL l TableXXXIV-V6-i-I1LA-A I Omers-273P4B37J 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 Ithe start position pius nine.

FPos 1234-567890scor IE NDKDEEI1 f -6EVRHCND1 [Lj DDEVJRHC El TableXXXV-V4-HLA- A0201-10mers-273P48371 Each peptide is a portion of SEQ ID NO: 3; each start position is speciffied, the length of peptide is amino acids, and the end position for each peptide is the start position pius nine.

Pos 124680 score F IK2 PMG11 W TGMVlF1 lI M GVT P 1 EliK] PG vr iii] A0201-l0mers-273P4837 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.

tPos 13579 score] F-6 LRRADII111 WMPSL EDI 11 RNLIWI L TableXXXVI-VI-HLA-A0203- 1 Omers-273P4B37 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.' ~[13579 scorel [3451 LUKN VA10]1 [-9I L-LM-ETRPLA]F10]1 I 3 II. VGGVGLILTA j[K~ 552TLQQSH 10 1 62172]~QTNG] 10 1 645 QEDISMA 1 F7 6 EGP-4A1 101 98 ADDSAHS 10j F82 I[ASSAQA I l F8 ][AFk QDAGP QAE1 F8 [Es6 sEQA17W F89211 ENFDS IW 197 1!ESS 1 0 100871 DEEVIVA[0 108-51 M~NRSL 1 F91411 EELDSSA li 119 l5F-EGESGA]1 1158 FEK-NSWLSSA 11 F)1] LQE~G rTableXXXVl-V1-HLA-A0203- 1 Omers-273PD4B37 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.

EPos 1234567890 1Iscor F11 841 QWVGSPQDKA]10_1 1210 LKECGKIQEA]1] 11217 FQEALN C-VKA][ 101 I2-0-]IHYLRYV1 EAK lIT] [[EAfKlFILK 9 I S9QIELI 122211WYI~A F2] SSK3IA 31 Ell F2][A3 AA61A ]9] F21EIW FFA][T F2-41] PIERR]9 130126A-6]A][T F2-9 93] !.SSEA

MEHSLAE]

CHPRI~SR F -9 F63 1 9ILE1A 1T 161[ALQLTGj 9 696 TableXXXVI-VI-HLA-A203-1 I Omers-273P4B37I 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.

PosI 1234567890 scr F7-6511 EEDISSKMAS ID [8-00 1QDGKGTGSADI 9T F821l SLGMEKSFAT][9] I8341[ K-sFATKN!EAv][ i [8K67][ YYSKSlAD Ii 9FI011 D~SAVSIEIAL li F9[-1 I ADDSLqD19I F 1555iwHsALQS]E F1 A.SH1LLDAQA][] I9-l PASSPQYAC[il 9821 CGSAPNSRAG jEE F1 0091 EEEV~A I 1105 3 FSVQDS[9 110-86SNR~I 1111121 LDSAif9 11130 GVES E E7 11 59 ITvTSPA 11611 ETKS EA 11168 LEESLA I 11188 S9DMAT1 11211 KCK~EL1~ STableXXXVI-V4-HLA- A0203-l0mers- 273P4B37 TabIeXXXVI-V&-HLA- A0203-1 Omers- 273P4137 Ios 1235689 score NoResutmud TableXXXVI-V6-HLA- A0203-10mers-273P4B7J IKo 234567890scr 11 NoResultsFound. ITableXXXVII-VI-HLA-A3- I Omers-273P4B7 Each pepUde 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.__ [iF 1234567890score] 119 F[LDMLK30] 172JRKTHPK 0 IFl VKAKAK I 9 Fl-2- LSEMH1[ z 60-31SIQTEKI2 48-8 INILK 2 278 SLTTK2 03 TLIGVH 2 4 00 ALVLK 2 6411QQLAR 2 [604 ILRTGEK1 [69-]SHQQQK14 [F[37 PLEEY21..

F7[PFPSTKK4 2 F-fl~[AKKST [j F3 DlIWRV 3-991SPELVK[2 471 RREHTL[~ 11011 VAISI] 2 [i10-5VKFDST5[1i F1~ ]j FL SGF1 1 F2-2 VLDAHK51L~ F3I1K9]F2RR1]~L 1 ][ACMSS Fs54-4][TTARVI 1 591 1WIGKE] 271 31 .I LTGVE]_i 676 lMCDSK[] 1683 SVKE EL DW-E 12 1! 170-0]RVA FLVE111 7061 FL EE 11 [7 32]REFSSKIL F6-][FYLKT EE Ii DIGNLDQK 11 F88][ QLKDELHIIZ [.21-ODEEW7K70DI Fi1 ATDYETL21]1 1216]1 FIQENL1 F21] 80 I DVCNSGLLLY 11 20] F871LYRLHQL20

I

325 lFLRRTKEDVQ 32-6 LRKDK2 344 RLNEKND 2 F5o 1TVT-HLLEREK 0] 81-3]i LKFSV (2 F980 FsLC GS AP NSR 12 0 2TLRGEK[Y 1235 EMLhSY1 10-6_FLYSLYRDGR1] [10-9 jSYDRKGfy [190 RQRGI 19 146111MFMDL I19 F66f E.[SLIGID fl~W 68B71~ LWEH] W I~~I1 IA D 9 9377I KLEEEPSASS 11] 10421 INFNTSLF [11 73] LAEPS [1v7j QAHLK[-181 t4-2i FKI

F[]

1155 I LNT]E I27 IRKDTPE1 1 Iz2F37EIYRKFliji P--3 U KLDHRLS 8 1 1468 LLKRLRDEGH 18- F 53-31 LLTTQVGGVG 18] F7-6-] KKNP F1YS 18 F10 82 SNSNSR1 F SGFDAL 37] F21 VIHK 17 F3-- ii KRINLNKiiiT 5-66f FA AVDR VY-R IGQ 7 580 I VVYR LITCGT [17 F6 DIl K LD EH I AY 71 11I41 wKAKIRA]1i~ 117 AAE TSLGA][M7 1203]LKGEK] T 7226ALKAPE i] F20 HYRV EAK[l 1 0-3] FGIAFLYSLYR ILE! 1491[ LLIPTl i F21511] FKTPMRK16- 32-8R T KEDVQ KKK 16] 392E LL M ETR SPL 4 MIFLMDLLKR 16] 190]1 NERLLKNR [16] I494ItRLLKNRHFKT] 2 i L49 LNRHFKTL I 502 i KTLRIDGTT 1116] 530] SVFLTTQVG 16 541VGTLTTRI 16 [5-4-1 IGQKENVVVY 16 ELFTIEDLQN ][j16jJ 62-6 ITIEDLQNSVT][)16] r629]1 I644][SLHAAQRKSD][ -6 1 669 AGISHLMY J 16 66-9 1730 IWLREPVFPSSJ 16 F7 30 16 1756 )1PLLSTHHTQE 1 6 1762 1HTQEEISSK ))16 762 1 E 773 ASWIDDLPK 16 1889 ILRHNPWPI ))1 F8 IIEIADLSA 1I]I [1076]1 SSQIPSSVNK ]Lii1i I 1O08 6NSRRSLASRR][ 1W I I11179 PLSGEQLVGS][ 1W I11206 RGKELKECGK][IW I6 112191 ALNCLVKALD][16 123VMLLTLSLYK 16] IA9i DIFPNEKVLS [Ii 531INIVSIQ 11 11 KVLSRIQKIQ 11 15]1 13 1IIAFLSGMFD ILKI5 1[31SLVNHVLLIM ][iKI 1][1 HVLLIMPTNL 1 1 1 1 3 ]PTNLINTWVK 15 1 53 1QRNGVIIT1Y 24-31R IPSR )11 HATPGEKALGF II l] 302-1 F312]1KSNM I )1 F3 551 ICEMPSLSRK ]1 1 U1LIWIRLVPL 1 F3 67 15 [i69 IWRLVPLQE I1 i. I 369- [37] LVPLQEEIYR 1 15 1 F4-2-41 PLQEEIYRKF 11151 1 T VLKKLCDHPR][ T] I 423][ CLLNLGTFSA] lW I5 (424 I[ LLNLGTFSAQ][I 1W]) I 454 II TLMEESGKMI1[ lW 1 464 I FLMDLLKRLRE 15 537 QVGGVGLTLT 15 1540 11GVGLTLTAAT ()15 1542 11GLTLT4TRV 15 F675 FDLMTCDLSV 15 697 JIQQRVQKAQF 15 F723 RTRNEGAWLR 15 177 I WIDDLPKEG 11 5 I 798 I TLQDGKGTGS 15 862 LESFNYVLSK 15 [920 I DLSASHSALQI 15 1 F968 SSQSLEHVEK 15 [992 IFVHSKTCLSW I is 997 TCLSWEFSEK is 108 KIRSKARRIV 15 1049SLFQFSSVKQ 1092 SLASRRSLIN-o 1i-2 71098 SLINMVDHV 1130 GVEESSGEAS 1WI 1131 VEESSGEASK 15 1150TLSSENKSSW 15 1159WLMTSKPSALI is] 1209 ELKECGKIQE Ills] (122j1 LVKALDIKSA liii] F11 DLEEAFKLFN 2[31 F 391EFLNA 11 1 B ALEELAEQGD lfl 102 OKIIALSYI 4 2074(LNWQ LI 4 20 IILINNWQQLS][IT] [20FWDWILDE 14I [2273DYVILDEAHK

JIZ

2][1 KIKTSSTKSA )1114 1 2673NLQELWSLFD][ 14] 275 EICQGSLLGTLK.II 11 1F2971 ENPITRAREK][14] 32701 IIKPYFLRRT 14 327 1IRRTKEDVQKK1l 14] 1382 11RKFVSLDHIK II14 1386 1[SLDHIKELM 1 4 1449 IQVTDDTMEE 14 1452 I DDTMEESGKI 141 470 I KRLRDEGHQTI 14 1 493 ERLLKNRHFK 14 505 ERIDGTVTLL 14 513] LLEREKRINL 1E] 1527 11KDYSVFLLT Dfl4 527 141 578 NW RLITC 541 r2 ELRELFTIED lIE]4 [639 EQLQLQSLH I] IEZ][LQSLHMQRK EK [ss5][WEESHYIQQ Ii i] F7401 TKKKCPKLNK IF141 74K6 FKPQPQPS I] [77Z 1I IDDLPKEGEK][114]1 [m4 GEKQIDLSSIK] IT]14 F 788 1[ DLSSIKVNVT][ 14 I 7-8[ SADSIATLPK[141 F20][SVEELCTNSS][ i4 [25][CTNSSLGMEK I[1 861 ]PLESFNYVLS 11 14 1 888 EILRHCNPWPE 1 I l95I1 NLFLEoSANI(iE1 I9I1APNSRAG FVH Ii fl F029(loSF 1 14 E097) RSLINMVLDH 1 4 102MDHVEDME 14 1 1112ERLDDSSEAI( 14 1124 EDYP EE 14 [1184 QLGSPQDKAI [14 1 [12281 DIKSADPEVM [14 I 1238 LTLSLYQL 1 1240 TLSLYKQLNN 114] TabeXXXVII-V4-HLA-A3 10mers-273P4B7 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 110] GV!TFHGPSK 26 F-3K] PGMGVK 17 [2]FIWTPGMGV 13] SIAKWIPGMGVKTI 13] I TaleXXVI HLA-A3-I 1j9 e-273P4BLJ Each peptide is a portion Sof 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.

tP-1l 1234567890 lscore] TabieXXXVII-V6-HLA-A3- I Omers-273P4B7

I

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.

po 1234567890 scorel P-QLKEVLRH 21 I7IEVLRHCNPWP II 16 [lo VLHC~WPI 1 4 [11 NLDQLDEV 10]( P3j DQ4DPVLR 10 TableXXXVIII-VI-HLA-A26-1 l0mers-273P4873 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.

1234567890 [lscore] F12351 EVMLLTLSLY I 35] 1DVCNSGLLLY I[1 33 1 112011 ETLVKRGKELI[ 27 1 1 I ETSLGAPEPL 20 1z2 7 11 -711 EIFYL 12 1 EGIAFLYSLY IL5 [636SVTQLQLQSLDI .z i F 01211 WKKR 12 [i1[EVVVKAKIRS II25] [3151 ENLMAIIKPY ][2 F6-531 DKDHA 2 I as 11 DwEEHYIQ] j] 4 196] GVIITQML j3 1 137811 EEIYRKFVSL 11 2. I P8] LVFSQSRQL 81F2 3 6313]EDLQNSVTQLI 23 j I1132 EESSGEASKYI 23 1 TableXXXVIII-VI-HLA-A26- 1 Omers-273P4B7 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.

[Posli 1234567890 Iscore] 112181 EALNCLVKAL I IM i 98 E1 HQKEGIAFL [1 22 384 FVSLDHIKEL 22 [940 2EEPSASSPQY 111051 DHVEDMEER LJ 22 77 IEFTDVCNSGL 21] [67IWTPGMRVicrFl 21 1 1657 11DEHIAYLQSL )121 [687]IELDWEESHY I 21 6- ELEQGDDEFI[

I

t36z1I LIIWIRLVPL L( 0i 54 51LTARWF 112 157811( NVVVYRLITO J1 2j] 774 1 4 IDL F1120] [80][1 DSIATLPKGF ][j20j] 1. 11 ESFYVLSKS][3] [114911 ETLSSENKSS I[ 7] I1 JEFVWV ILD I j[ j 1332DV KKKSSNPIJ!1 13791(1 EIYRKFVSLD I i9 3-7 9] ELLMETRSPL lil] F3 ETRSPLAElG 1W] 44DVDHIDQVTD][ 1W] 5981 QVFKDSLIRQ[ 1 709 1 EFESQNK-FL[ 1WQ 846 ]QEGPKQE1[ 1 [919 1DDLSASHSAL j[ ]9 VV31 VKAKIRSK It iii1 F28]DTLMEESGKM E[ 1 F8 [ILTTQVGGVGL][ 18 57911 VRLITCG 1118 1 L588 fl GTVEEKIYRR 11 18_I 1 59111( EEKIYRRQVF 1 11W1 TableXXXVIII-V1-HLA-A26- I 10mers-273P4B7 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.

[Posl 1234567890 score 1727 EGAWLREPVF 1I [7901 SSIKVNVTTL I18] 8601 DPLESFNYVL 1 0I 1872 11STKADIGPNL 1 8 958 EDSANRNFI[ *i I [119111DKAAEATNDYII18I 49 DIFPNEKVLS II iT]1 F 78 ]IFTDVCNSGLLI ri l 25181 TPIQNNLQEL[17 IATPGEKALGF][ 17]) [1I41 LRIDGTVL][) 17 1 61I((QTTGEKKNPFIVI 1 I6 11 JAGISDHDLMY II i7 l I75 1] ADIGPNLDQL IL EI L9Z3 1 EHVEKENSLCI 117 1 I100911 EPEEWVKAK]J 17]1 11039) DTSSINPFNT 1 171( 110441 NPFNTSLFQF Ii i] I1129 EGVEESSGEAII 17]1 F2mers-273P4B7 Each peptide is a portion o 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 positionplus nine.

[sI1234567890 I~e [TIIWTPGMGVKTF 21 [1J EFIKWTPGMG 16 E1GVKTFHGPSK 12 IP TableXXXVIII -HLA

I

IP A26-10mers-2731341 37 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 scre 3EMPSLSRRND 11 [iQI RNDLIIWIRL 9 W MPSLSRRNDL W [T SRRNDLIIWI 6 1 IC-EM PSLSRR Ii] LSRRNDLIIW 5j~ 9 ]JRRNDLIIWIR 1[5 TableXXXVIII-V6-HLA- A26-1Omers-273P4B7 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.

2 J1234567890Isoe [j IEVLRHCNPWP 20 [EDEVLRHCNPW I 12 TableXXXIX-VI-HLA- B0702-10mers-273P417 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.

~11234567890 (Is.o e1 749[ KPQPQPSPLL 23 [7611APEPLSGEQL IF23] [8 [MPSLSRKNDL 2 [860] DPLESFNYVL ]21j [247 IPASNRLLLT 20 1258 J[TPIQNNLQEL ]L0 17801 LPKEGEKQDL[ 2] 19411 EPSASSPQYA][ o 1946 iiSPQYACDFNL[ 20 1555 1DPSWNPATDA1[ 19 1751 QPQPSPLLST 119 J152 ]MPTNLINTWV II J F9 NPDVDAICEM 1 TableXXXIX-V1-HLA- B0702-1 Omers-273P4B7 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.

os 11234567890 scoe 441 SPDVDHIDQV1 17 107011 PPGRFFSSQI 111 -7] 1188]!SPQDKAAEAT 17 EKIPEVLR I1 1044] NPFNTSLFQF 1122 GPEDYPEEGV 16 12 IKSADPEVML 16 [135 FLSGMFDASL 115] 1178 GPSKDERTRN DE] 14181 SARAOCLLNL I15 505 RIDGTVTHLL 15 98 EHQKEGIAFL 14 F Ti DASLVNHVLL 14 27 ACQGSLLGTL i] 336KKSSNPEARL] i INEEIYRKFVSL ][I (24 [QQNKDYSVFL[ *i4 [5361TQVGGVGLT L 14 [Z3 l[ QPSPLLSTHH 11.

F5 GSDS6T 1 F14]LPKGFGSVEE 14 18751 ADIGPNLDQL ]1 19Jl QYAODFNLFL ILK I94-1 RRSLASRRSL]14 11145 DPSGETLSSE][14] 1164KPSALAQETS14 11171 ETSLGAPEPL]14 1 EEQL]41 11 KECGKIQEAL 1 121 APEVMLLTL 1 3 SRRFPEAEAL13 [28 AKEATKNGDL]1131 1111 YRDGRKGGIL Li] 1168 1TPGMRVKTFH[13 l24 ARAIPASNRL][13J 300 11KDATPGEKAL f1W] 308 ALGFKISENL 13 .TabIeXXXIX-V1 -HLA- B0702-1 mers-273P4B7 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 ine.

1234567890 Fe] 32][ KPYFLRRTKE I 1i] F67]LIIWIRLVPL Ii 139][ ELLMETRSPL[ 1] 1] SPLAELGVLK] 1W 413 HPRLLSARAC 1] 416 LLSARACCLL 13 471 RLRDEGHQTL 13 J473 IRDEGHQTLVF 113I 559 NPATDAQAVD Li 618 YFSKQELREL [3i [EDLQNSVTQL 13] E6fl 13 7 ISSKMASVVI ]13] SSIKVNVTTL 1W [B4][LQEGPKQEAL 13] [51 KQEALQEDPL 13] 1985 APNSRAGFVH 131 1990 AGFVHSKTCL 13 F1091 EPEEWKAK 13i IPSSVNKSMN 13 1 NKSMNSRRSL 13 105SRRSLINMVL 113] 114AEATNDYETL 1131 1218 EALNCLVKALI 13] 113 KSADPEVMLL 13] F98 1 PEVMLLTLSL13 111 FPEAEALSPE] 12] 148 II KDIFPNEKVL] 12] 1069 TDVCNSGLLL[12 101 KEGIAFLYSL 12 l1I GKrVQIIAFL1I12I [40 ]FDASLVNHVL 12] [2Th]QEFWDYVIL Ill 2 RAIPASNRLL 12K F194ATPGEKALGF 12 F7-TPGEKALGFK1 12 TableXXXIX-VI-HLA- B0702-1 Omers-273P4B7 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.

Pos1 1234567890 ]soe F3171[ LMAIIKPYFL [12j VDAIOEMPSL 1 364 jNLIW 121 F84]FVSLDHIKEL 1[ 12 F398 F RSPLAELGVL 1L j4J7 KNRHFKTRI I1iI 504][ LRIDGTVTHL 112 534] LTQVGGVGL[12 595YRRQVFKDS L il 615 PFRYFSKQEL 12 63-31 LQNSVTQLQL[12 [6 7 AAQRKSDIKL 12] F54IKLDEHIAYL 121 673 II1DHDLM1TCDL i F709] EFESQNKEFL IDI] 73 ]EPVFPSSTKK ][IT L3][FPSSTKKKCP[ 1 SSTKKKOPKL[ z.

736 KMASWIDDL]12 F781PKGFGVEEL]1 12 I 839 KNEAVQKETL 12 L8721 STKADIGPNL 12 9 IQDAQASEAKL i1i 1 TPKNDISPPG 12 1151LSSENKSSWL 12 115 W TSKPSAL 12] 116 PSALAQETSL 112 II ASRRFPEAEA I11 [3][NGDLEEAFKL 11 F578 SRIQKIQEAL ]F11- F--lEEFL EE [7i[ETmVCNS.GL 1W [111 FTD VCNSGLL 11 83] NSGLLLYREL 111 F1 ASLVNHVLLI 11 F471 HVLLIMPTNL 1 166 P 1179 PSKDERTRNL 11 TableXXXlX-Vi-HLA- B0702-10mers-273P4B7 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.

os1 1234567890 2551 LTGTPIQNNL 11 [701ILFDFACQGS L 11 [7I FDFACQGSLL 11 [F3 QGSLLGTLKT 11 NPIRAREKD [j1 9flITRAREKDAT j [40 ][NPEARLNEKN jif [9][METRSPLAEL [IT 397] TRSPLAELGV ]IjW 407 LKKLCDHPRLF11 F408[ KKLCDHPRLL] M45 FII RLLSARACCL][ 11 4461 QVDTL 1 45fl MEESGKMIFL [57 1EESGKMIF~M I[T 46 GKMIFLMDLL 11 463 IFLMDLLKRL I.

F480 1VSSQ L1 486 RQILNIIERL iI 14951 LLKNRHFKTL 111 51_3 LLEREKRINL 11 I 2] QNKDYSVFLL] 'ii 441 TLTMTRWI L5.I LTAATRVVIF[ 111 553 IFDPSWNPAT 11 1561 ATDAQAVDRV 11 [572 RIGQKENW j[1j [575 QKENVVYRL EI] [679 TDLSVKEEL 11 [72Q2 11] 74-CPKLNKPQPQ 11] 17481 INKPQPQPSPL EL 89 NPWPIISITN 1 89 WPIISITNES [&1 I1IIDDLSASHSAkL 11 94 IASSPQYACDF 11 958 IDSD QF11 100 KDDEPEEVW F 11 TableXXXIX-VI-HLA- B0702-10mers-273P4B7 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.

[Post 1234567890 s] 1041 SSINPFNTSL I11 1133 ESSGEASKYT ID 1167] ALAQETSLGA 11 1201 ETLVKRGKEL 11 1131DPEVMLLTLS [.i1 I TableXXXIX-V4-HLA- 1 B0702-10mers-273P4B7 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 sc TPGMGVCIFHI 13 W RFIKWTPGMGVK 7IE] B0702-1Omers-273P4B7 J 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 F [L IMPSLSRRNDL 23] [01J RNDLIIWIRL 12] TableXXXIX-V6-HLA- B0702-10mers-273P4B7] 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 13456789 TabieXL-V1-HLA--B708- 10Omers-273P4137 [PsI134679 Isc~ore] FNoResultsFound._ STableXL4V4-1-11A-B08- 1 1 Omers-273P4R7 Po s 124679 sc-o re ,FNoResuitsFound._J ITableXL-V5-HLA-B08- 1 I Omers-273P34B7 1j ,J NoResultsFound,__ STabieXL-V6-HLA-808- 1 1 Omers-273P467 j -NoResuitsFound. I TabieXLl-V1-HLA- 81 510-1 Omers- 273P4B7 [Pos 124679 score FNoResultsFoud.j TabieXLI-V4-HLA- 81510-1 Omers- [pos135780se FNoResultsFound.i 81510-1 Omers- 273P487 [Pos 12345 789 mcre] JFNoResuitsFound.j TabieX~I-V6-HLA-1 B1510-1lrners- ___273P4137 Pos 124679 icore] ,FNoResultsFound. l TabieXLII-V1 -HLA-1 82705-j1iners- 273P4137 Pos 13456890 core]~ -NoResuitsFound.j ITabieXLlI-V4-HLA- F 2705-j1iners- 273P4837 -NoResuit ond [TabieXLII-VS-HLA- B 2705-l0mers- EPs 245780score] No-ResultsFoESnd.

[TableXLII-V6-HLA- B2705-1 Omers- 273P4B37 [Pos 1245780score] -NoResultsFound.

TabieXLIII-V1-HLA-1 82709-1 Omers- 273P4B7 IHs 235790soe FNoResuitsFound.

I

TabieXLIII-V4-HLA- B2709-1 Omers- 27334B37 FPos135680 cr [NoResultsFound.j [TabieXl-V5-LHLA- B 2709-1 Omners- Ps 23479soe NjoResultsFound.j STabieXLIlI-V6-HLA- 82709-j1iners- 27P4B7 Po 135680 cr TabeXL~II-V6-HLA- B2709-j1iners- 273P4B37 FP-o s 1234567890 ,[NoResuitsFound. 1l [TableXLIV-V1-HLA-B4402.] Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, andthe end position for each peptide Is the start position plus nine.

12346789 scoe F9308][ EYKVL11f I5-4 F579-][EFSN F[ 24 (1119[4 EGK0]L] 24 [34611NKPVA [21 F1J IELLNRF 11f 6 10]]GKNFY [i 1FI-421t EDSEL1~ 145611MEGMF] 1 lF57 KNVVY6 L1 65fl] DHYQS[I F8-2-][VEC SS][21 [-9-72][LH KE L] 21 1218 EAECL L] 9 TableXLIV-V-HLA-4402- 10mers-273P4B7 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 155 jNNTWVKEF j[ 1] 245]RIANL 18 315]ELAIKY 1 402] AELGVLKKLC 18 EESGKMIFLM ][j8 [94]ASSPQYACDF 1] KDIFPNEKVL ][f 126] LGKQIIAF li F151] IMPTNLINTW 17 [246] AIPASNRLLL 1 7 302 ATPGEKALGF 17 F3-I1 SENLMAIIKP [LII [3I1 FVSHIKEL 4861 RINIR 1 [1 j[9 AGISDHDLMY j[ 17 10171 AKIRSKARRI J[ ]f 1143EE PSGES Jj fl [1176) APEPLSGEQL II ii]1 1iz3?1 ADPEVMLLTL 117 1 110 II AEALSPEQAA( 116]1 [j58j]1 SRIQKIEAL 111 [10211 EGIAFLYSLY ]j7W] 116 IWTPGMRVKT F 1LI] 274]ACQGSLLGTl F 27][ GSLLGTLKTF 16] 300 KDATPGEKAL 16 ]CEMPSLSRKN 1161 42]ACCLLNLGTF 1i j6][ AAQRKSDIKL 116] 7901 Sfl /TLII1 809[ DSIATLPKGF I 915]!IDLSS 1 1042SINPFNTSLF 16 044NPFNTSLFQF 1 12 EVMLLTLSLY 16 "34 NGDLEEAFKL 15 35] GDLEEAFKLF [15: TableXLIV-VI-HLA-B4402- 1 Omers-273P4B7 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 1 sTe] E8I EEAFKLFNLA lIZ] 54 J[ EKVLSRIQKI T [AEQGDDEFTD 15 j~j] EHQKEGIAFL lIii 142]ALNVL 15 193 RGV3P( 1 [h]j SSFRGQEFVWI ]i 224 YVILDEAHKI 15 L3ZI LIIWIRLVPL ]L15~ 91l KELLMETRSP]15 [4il LAELGVLKKL ][Y 4078 KKLCDHPRLL 49[ LLKNRHFKrL RIDGTVTHLL 1 F631 EDLQNSVTQL i 654 IKLDEHIAYL 1 F9 KPQPQPSPLL 1 76 1EEDISSKMAS 11 1 1 1851TGSADSIATL F10 11 NSSLGMEKSF] i1 I5 F1O4-11 HNWIS 11 1 939N1 EEEPSASSPQ 1[ IllW [§[AGFVHSKCL 15 110111 EEVVKAKIR j[1] 11041 SSINPFNTSL j[ 15]1 106NDISPPGRFF i5 11159 1WLMTSKPSALI is]1 111711 ETSLGAPEPL 1 F-41 112011 ETLVKRGKEL 15]1 [12141 GKIQEALNCL 11I~~ 1 SRRFPEAEAL I 26 KEAKEATKNG 14 IAII KLFNLAKDIF liii Ijj3 NEKVLSRIQK ]1]4 L1 ]I QKIQEALEEL ]14 lii] DEFTDVNSG] 14] 80 DVCNSGLLLY 14] TableXLIV-VI -HLA-B4402- 10mers-273P4B7 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.

[os l1 1234567890 1 scor [17 GGILADDMGL 14 141 DASLVNHVLL 14 Vi1I NTWVKEFIKW 141 I8 KEFIKWTPGM 14 2001 ]TQMLINNW 44I ARAIPASNRL]14 I2][ TPIQNNLQEL] 14] 290 YENPITRARE 14 I31[ FKISENLMAI lEE j30 [KEDVQKKKSS[ i1] SRKNDLIIWI I1l 1392][ ELLNITRSPL 11 14 139811I RSPLAELGVL 14 f F3 41 DEIL( L 11 463 IFLMDLLKRL ]LII I 7] RDEGHQTLVF1 .11.

487 QIIIERLL E[J LRELFTIEDL 11 630][ IEDLQNSVTQ [II14 F686] EELDWEESH j 691 VEESHYIQQR 14 71 KEF EQQRT 14 F71 KMASWIDDL 14 8221 EELCTNSSLG 14 14IIQEGPKQEALQI 14 ALQEDPLESF Ilil 912 VSIIEIADDL j [9 I EDSADNRQNF 1141 [983 IGSAPNSRAGF 1141 110!DEPEEVWKA IIii 100LFQFSSVKQF 14l F -1 ERI 1084 NKME4S][~ 1090RRSLASRRSL14 110 KELKECGKIQ IIE 12171 =QEALNCLVKA[14 1230 KSADPEVML F LLTLSLYKQL 14 TableXLIV-V1 -HLA-84402- 1 Omers-273P4B7 11 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 I 1234567890 Iscore] F313 LSPEQAAHYL 13 29 KEATKNGDLE 13~ [-68]IE-ELE-QGDDE II iii] F8-3I LYELN1] 131 19 GVITTQM 13 F1I[VIT79]IIL~ 1341-11 EA NE 13] F21 VSLHIEL F1 3 36f NEESP 13] F3]4 SQSURLNII J[131 15211QNDYVFL1 13 [F48 IQKNW3]]3 F5-I-3 VE I T][ 1-T-61YFKE EL13 TableXLIV-V1-HLA-B4402- 1 Omers-273P47 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.

Po-sl 1234567890 F 622 QLRLFIE 1 673 lDHDLMYTCDLj 13 F68-5] KEELDWVEES 1-3- 68 7]EDVEH 13 692 EESHYIQQRV 13 721] QQFN1A 1 7 27 GALRP 13 7 4 8NK QSL 1 764 QE SKA 1 8321 MESATN 13 84-51KTQGK 13 94-7]PYCFL 13 957LDANRN 1 1004SKDPEV 1 32DDSK S 1 1065 DSPGF 1 11 07VDERL 1 111 ELDSE 13 1177 P LG EQL E1 1200YETL7SKGE 13-V [TableXLIV-V4-HLA-B440 2-1 1 Omers-273P4B37 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 124680Isco~re] [Ej fPMGKT 161 XLV-V5.HLA-B402-1 10es-273P4B7 I TableXLV-VI -HIA- B35101-10mers- 273P4B37 [P051123Is56co9r e IFNoResultsFound.1 STableXLV-V4-HLA- B51 01 -1Oiners- 273P34B7 12345678 e] ,[NoResultsFound. l 85101-j1iners- 273P4B37 pos 12345 78901re ,FNoResultsFound.

TableXLV-V6-HLA- B51 01 -1 Omers- 273P4B7 Pos 2346790 re NoResultsFound.

TableXLVI-V1-HLA-DRBI-01 01- I 5mers-273P4B37 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.

7P-[ 12345 67890124 [score] [-6-7l1 DEHIAYLQSLGI-AGI ][32] _I-41 NHVLLIMPTNLNW] F2-7-7-1 GSLLGTU(TFKMEYE][ 31] F31511 ENLMAIIKPYFLRRT_11 5L I [421 JACCLLNLGTFSAQDG IL-LI 1-4511 VNHVLLMTLN 30~ 1818 IFGSVEELCTNSSLGM JU 30f 110721 GRFFS-SQIPS SVNKS [30] [8611 LLLYRELHNQLFEHQ][ 28] [616jl FIRYFKELR ELFTI 28] [F8]4 ISFNYVLSKSTAI ][8 I Ill SRRFPEAEALSPQ] 27 F77 1DDEFTDVCNSGLLLY1[TI F2-2-21 WDYVILDEAHKIKTS_ 2 F 54-0]1 GVGLTLTMATRVI 27~ Ij-7-911 VVVYRLITCGEE 1I2l 1591 I1 EKIYRRQVFKDSI ][27] [66011 IAYLQSLGIAID ][-27 [7-9-61 VTTLQDGKGTGSADS][ 7] I24-11 ASNRLLLTGTIN ][2-61 j 532][ FLLTTQVGGVGLThLT]1 Z 11681[ LAQETSLGAPEPLSG I[26I F1221][ NCLVKALDIKSADPE]jDfl F13-]1 VQIIAFLSGM F 25 li] F16-2[ KEFIKWTPGMRVKT-F 1125] [2-T8] PASNRLLLTGTPIQN J] 1452]1 DDTLMEESGKMIFLM 1[25i7 [cn7 J~ HQTLVFSQSRQILN] 5] F52-flj KDYSVFLLTTQVGGV[ JE25 ]QNSVTQLQLQLHA[ 25 F93]1 ESHYIQQRVQKAQFL 125 74411 CPKLNKPQPQPSPLL F9-7-]1 VEKENSLCGSAPS ~2 [9879I RAGFVHSKTCL EF] 10693I1 DDSFKDTSSINPN [25: li05731 FSSVKQFDASTPKND][2 [iO87]1 MNSRRSLASR-RSLIN F4-0-11 AFKLFNLAKDIFPNE 4: ,EflD NQLFEHQKEGIAFLY][T24 TableXLVl-V1..-HLA-DRBI-0101 -I I Smers-27334B37 I 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 l[ 123456789025 [core [18811j LNRIQQRNGVIT 124] [19311 QRNGVIITTYQMI E_ [20fl INN WQQLSSFRGQEF I24] I -O 9] WQQLSSFRGQE F2] 121911 EFVWDYVIL D KI2±I F 2-30][_AHKIKTSSTKSA1C-A_] 24 330][ KED VQKKKSN PER]24 F3--71 LIMWRVLEI 1 41311 HPIRLLSAROLN ]F24] F4-27] LGTFSAQDGNEGD] 24 157811_NVVVYRLITCGTE_14 1621 II QELRELFTIEDLQN jEK 163911 QLQLQSLHMRS I24] 169611 YIQQRVQKAQFLVEF124 I LVEFESQNKEFLMEQ 1124. EEDISSKMASW D E124 [82 11 VEELOTNSSGMEKS][ 24 1 [8672]I LESFNYVLSKSTKAD][ 24 [87811j GPNLDQLKDEIR ][24i [88 f[ DEILRHCNPW S 1 5]1 IEIADDLSASHSALQJ 1124 192-511 HSALQDAQASEAKLE I F9864][ RQNFSSQSLEHVEKE Ii65-01 LFQFSSVKQFDAT 1± 111281 EEGVEESSGESY 1241 1165 PSALAQETSLGAPEP]11±.

111741 LGAPEPLSG EQLVG 12321 ADPEVMVLLTLSLYKQ][ 24] [67 J LEELAEQGDD7E-TDV][ 3 [iqi]~ KEGIAFLYSLYRGR11] 112111 ADDMGLGKTQIF 123] 1l2-6][ LGKTVQIIA-FLSGMF 23] ASI.VNHVLLIMPN 23: F2-361 SSTKSAICRAPSf 23 F2-70[ _LFDFACQGSLLGTLK 123 [30611 EKALGFKISE A E(3: [30-8[ ALGFKISENL-M-AIIK ][2 F3-8-911 HIKELLMETRSPLAE ][23 I~J SRQILNIERLLKR 3 TabIeXLVI-V1-HLA-DRBI-01 01ls5mers-273P4B37 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 lieptide is the start position plus fourteen.

os([ 123456789012345 licoel 52-8]1 DYSVFLLTTQVGGVG]1231 53f11 VFLLTTQVGGVGT ]231 [s f11 TRWIFDPSW AT 11231 DRVYRIGQKE-NVV-VY 1 23] [63-7]1 VTQLQLQSLHAAQRKFI23..

1 783][ EGEKQDLSSiKVV F-23 192-3]1ASHSALQDAQSA 23] S969][ SQLEHVEKENSLCG l-0401 TSSINPFNTSLFQFS 1L-23] 1-084 NKSMNSRRSLASRRS J[23] 111541 ENKSSWLMVTSK-P-SAL][ 23 11SWLMTSKPSALAQET ]1 12181 EALNCLVKLKS 1231 122611 ALIKSADPEVMLLT]I23 f-7l1 FPEAEALSPEQAAHYII 22]1 1 111 EALSPEQMAHYLRYV II 22]1 YSLYRDGRKGIA 1122] Efi DGRKGGILADDMGLG][ F22] LE~f][ TVQIIAFLSGMFDAS 1[22i F133][ IAFLSGMFDASLVNH ]2 F221 VWDYVILDEAHKK ]221 [F26I1 QNNLQELWSLFDFAC] JF22 41 SENLMAIIKPYFLRR 1[i22 13 4-9I NPDVDAICEMPSLSR ]F 22 13651 N7DLIIWIRLVPLQEE fl i] 1384][ FVSLDHIKELM ETR 122 F3-9-]1 KELLMETRSPIAELG 22]j F4-1-0[ LCDHPRLLSACC ][2-2 VDHIDQVTDDThMEE -1 F22I [~1QTLVFSQSRQILNII ][221 50-311 TLRIDGTVTHER F5-1-9] RINLFQQNKDYSVF122] 162-511 RELFTIEDLQNST F22 F6-3-6[ SVTQLQLQSL-HM-QR F22] F6651SLGIAGISDHDMY_]22j I7676[ EDISSKMASWFD ]22 IZ-6][ SSKMASWIDLE ]12-2 F9171 I NVSIIEIADDLAH [-2 .ML931 AKLEEEPSASPY 2 TableXLVl-V1-HLA-DRBI -0101.

1 5mers-273P4B37 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.

[PosE 134578-9012345 -1eR [94201 PSASSPQYACDFNLF 22~ 1FO-811 SSVNKSMNSRRSLAS IL 22] 1109 RRSLINMVLDHVEDM [2-2j I1-11 NKSSWLMTSKPSALA] 221 l1.5711 SSWLMT SKPSA LAQE F1186]1 RNLNRIQQ-RNGVIITF1]~ 498]1 NRHFKTLRIOGTVTHI j21] 6-71][ ISDHDLM-YTCDLSVK 1121] F7T6]EFLMEQQRT RNEGAWI2 .1] F7672 HTQEEDISSKMAWI 211 [F1971 TNDYETLVKRGKELK 1[j].

F2l1 EASRRFPEAEALSPE 112T] I 411 FKLFNL.AKDIFPNEK 11 201 I 16811 TPGMRVKTFHGPSKD11 320I F23] U(TFKMEYENPIR 120] 3521VDAICEMPSLSRKND 120 [378][ EEIYRKFVSLDHIKE F21] f[3811[ YRKFVSDIEL (0 41111l CDHPRLLSAACL Li F4611KMIFLMDLLKRREI20 F4-9-01 NIIERLLKNRHFT [2 F5011FKTLRIDGTVTHLLE ]EEa I 6-01]j KDSLIRQTTGEKKNP 1[ Z9 F613]1 KNPFRYFSKQE LREL EE jji4-f[ LNKPQPQPSPLLT 120] 8-09]1 DSIATLPKGFGSVEE E[g] F8-2j91 SLGMEKSFATKNE120] F8973 CNPWPIISITNE-SQN 1L-2 19501I ACDFNLFLEDSAN ][26 10481 TSLFQFSSVKQ FDAS][ 20 [11121 ERLDDSSEAKGPEDY][ EO 112201 LNLVKALDIKSDP ]2 11223 LVKALDIKSADEV I Z F327 1 TKNGDLEEAFK-LFNL II i.

5] 1 KVLSRIQKIQE ALEE j[19 F5-8i[ SRIQKIQEALEEA 1119 I-61wvElwPMv1 AF19 TableXLVI.VI-HLA-DRB1 -0101- 1 5mers-273P4B37 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.

I~s]l 1234567890124 Fso-~ 6i][ TYQMLINNWQQLSSF ]1i9i [.212[ NUMGQFWV [2678[ WSLFDFACQGSLLGT][ 19 L3072[ ATPGEKALGFKISE] 9] [307 I TPGEKALGFK1SENL_11 ii! F3--6-41 KNDLIIWIRLVPQ 191 140411_GVKLCHRLL-S 1IF I4 6-6] MDLLKRLRDEGHT 1119 1 F4-6][ LKRLRDEGHQTLVFS 19!~ P-8[ R iLNIIRLNRH][] 1DPSWNPATDAQAD] Th] Fi602][ DSLIRQTTGEKP ]E19 F79 4I VNVTTLQDGKTA] F 19 88411 LKDDEILRHCWP 1119! T 1 SNVSIIEIADDLAS119I 1-flI VSIIEADDLSASHS l] 193211 QASEAKLEEEPSASS II ii F10-161 KAKIRSKARRIVD 1-9i 1043 INPFNTSLFQFSSVI( f19] 123 PEDYPEEGVEESSGE[ F1-9-1 1381ASKYTEEDPSGETS] 19] 11156 KSSWLMTSKPSALAQ[ EE] 11211~ KECGKIQEALN-C-LVK 1191 1122411 VKALDIKSADPEM F-9-1 [1-2361 VMLLTLSLYKQLN 1M9 EIHYLRWKEAKA11(N 1181 Ef EAFKLFNLAKDIFPN 17L8I [E 11 QEALEELAEQDDFI-8] [811 VCNSGLLLYRELHNQ ][if] [93]1[ HNQLFEHQKEGIAFL] 18 [i-0 jf 1AFLYSLYRDGRKGG 1118 EL ]io~l GGLOMLKVQ11BI 11I~ ILADDMGLGKVI 11181l I2] DMGLGKTVQIAFS181 1132] IIAFLSGMFDASLVN Jjfl~ Fl-3-7] SGFALNHLI 18 I~]1 IMPTLINTVKEF71] F16111 VKEFIKVVTPGMRVKT 1118] I12 1 EAHKIKTSSTKSAICE]l8: TableXLVI-Vl-HLA-DRBI-0101-1 15I mers-273P4B37 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 fourteen.

[Po-l 1 12345678901 2345 scoe] 12671I LWSLFDFACQGSLLG ][ia 29111 ENPITRAREKDATPG_ ][ia F3-]01 KDATPGEKAL F]18 F371I[ IKPYFLRRTKEDV7QK] 18] P32][ KPYFLRRTKEDVQKK][ 1] 363][ RKNDLIIWIRLVPLQ I 373][ LVPLQEEIYRFS 18W F3-82] RKFVSLDHIKELLME 11181 13878 DHIKELLMETRSPLA Ij 18j F39-51 METRSPLA[-IK 118 1 139811RSPLAELGVKKD ]L7W F4- IL ELGVLKKLCDHPRLL 1-18] 420IRACCLL-NLGTFSATW1i [493]I ERLLKNRHFKTLRID ][18] 5 07][ DGTVTHLLEREKRINI ][8i [5201 FINLFQQNKD)YSVLL [530] SVFLLTTQVGGVGLT]I] F5 3711 QV G GV GL T LTAATlRV a18 154-811 ATRWIFDPSWNA 18i 6i5011 -RKSDIKLDEHIAYLQ 1118 F6-5-91 HIAYLQSLGIAGISD JI-1 F67-1[ DLMYTCDLSVKEELD 1118] [i-2-][EQQRTRNEGAWLREP[ 1 [72711 EGAWLREPVF1 78] F7-28]1 GAWLREPVFPTK 1-h] MZI KKKCPKLNKQQS F 18l 17741 SWIDDLPKEEQ 118 I I7-9]1 KVNVTLQDGKGTGSII18I I !11 I IATLPKGFGSVEL FL18 1 L 11 LCTNSSLGMEKSA] 8 F84-3[ VQKETLQEGP-K-QEAL 1118] 186-611 NYVLSKSTKAIP [1 8] [9019 ESNVSIIEIAODS ]j 18 946]1 SPQYACDFNL F 1 8h F95l31 FNLFLEDSADNRQF18 IFO01211 EVVVKAKIRSKAR 1118 110711 PGRFFSSQIPSSN 1118 110931 LASRRSLINMVLDHV ]E 18 11099 LINMVLOHVED MEER F118 [I--41 RNGVIITTYQ MLINN EK9 119 TTYQMINQL 19 1- TabieXLI-VI-HL11A-DRBI1-01 01-1 I 5mers-273P4B37 J 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 fo urteen.

[pocs 12356789012345 -1scr 111-77 PEPLS G EQLVGSP Q D [-18I 11182]1 GEQLVGS KE FI-I 19 J[1 AHYLRYVKEAKEATK 1~7 1 j AKDIFPNEKVLSRIQ[i I77f] EFTDVCNSGLLLYRE117 QLFEHQKEGIAFLYS jj 1L7I FIio-][ LYRDGRKGGILAD 117] F12-711GKTVQIIAFLSGF_171 I3-4i1 AFLSGMFDASL VNHV [17] [14-111DASLVNHVLLIMPTN] 17I] PTNLINTWVKEFKW ]117] F15-4]1 TNLINTWVKEFIKWT 17 FI5I-][NTWVKEFIKWTPGMR][1 r -7 0 [G M R VK T FH G PSKD-E R][ F203 [QMLINNWQQLSSR] 171 2-42][ ICARAIPASNRLLLT 11] 25811 TPIQNNLQEL-WSL-F-D j i 2591I PIQNNLQELWSLFD-Ff111 127-311 FACQGSLLGTLKTFK 1117] F27-4]1 ACQGSLLGTLfKTFKM 171 F2-8-2] TLKTFKMEYENPT 12871!j KMEYENPITRRK ][17] F2-9-7]1AREKDATPGEKLF1 TI~ [312][ KISENLMAIIKPYFL ][17] IF][SNPEARLNEKND ][L F355]1 ICEMPS-SIRK NDUI [i I3-]81 IIWRLVPLQEEIYR lul 37-011 WiRLVPLQEEIYR77 I[T LF3-71 LDHIKELLMVETRP 1117] F-39-11 IKELLMETRSPLAEL IMAL F4-1-911 ARACCLLNLGfSAQ]117 F4-3-01 FSAQDGNEGESDI f]] 1451!TDDTLMEESGKM1FL [45-311 DTLMEESGKMIFLM-D nfl-7 [4-5-91 SGKMIFLMDLLKRLR 1 [4-6211 MIFLMDLLKRLRDEG 1 14631 IFLMDLLKRLRDG 17 75221 LFQQNKDYSV FLLTT F17 LTVGVGLTLTAA 17 TableXLVI-V1 -HLA-DRB1-0 101- 1 5mers-273P4B37 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.

fosl 123456789012345 SC-re] 542l GLTLTMATRW Fj[17 553] IFDPSWNPATDAQAV IEi.

570] VYRIGQKENVVVR-L 11171 5L881 GTVEEKIYR RQVFKD 1171 6-6111 AYLQSLGIA GISDHD I1 [6-7-]T[TDS-VKEELDWVEE 117] 17-0111 VK FLEESQNK ][W 178811 DLSSIKVNVTLD] 7 180411 GTGSADSIATPG i i 180711 SADSIATLPGFS] 17] F81-5]1 PKGFGSVEELCTN-SS 1117 1 84-811 LQEGPKQEALQEP] 17] F8701[ SKSTKADIGPNQ ][17 1 F89]1 NPWPIISITNESQNA 117] F9-3][ VH-SKTCLSWE-FSEK-D]li Flob]0 PEEVWVKAKIRSKAR 11171 111 VKAKIRSKARVS f17] 10-44 NPFNTSLFQFSK 1117 11056 VKQFDASTPKNFSP11 1063 TPKNDISPPGRFF-SS ]171 1064 PKNDISPPGRFFSSQ ][17] 11065 KNDISPPGRFFSQ1 I 1 11069 1SPPGRFFSSQIPSSV 11 107611 SSQIPSSVNSMR[ 17] 119DMEERLDDSSAKP[ 1 71 11183 EQLVGSPQDKAAAT] F17 11199 DYETLVKRGKELKEC 1117 1217QEALNCLVKLDIKS 1117! 1233 DPVLT YKQL 1I F1 235] ElVMLLTLSLYKQLNN_11 101[ AEALSPEQAAHYL-RY 116 57] [LSRIQKIQEALEELA_117] 18511 LLLYRELHNQLFEH j109][SLYRDGRKGG IT][6 11-3811 GMFDASLVNHVLLIM [16 1 Ij7ZI[ HGPSKDERTRNLNRI 1185I TRNLNRIQQRNGVI ]1] 15!NGVII1TYQMLINNW [16 TableXLVI-VI -HLA-DRB -011 I S5mers-273P4B37

J

Efach peptide is a portio'n 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 fourteen.

[Po-s 123456789012345 scorel [206211 YQMLINNWQQL-SS-FR] 1 [23311 IKTSSTKSAICARAI 1 61 12381 TKSAIOARAIPAN 16 23-911_KSAICARAIPASNRLII16-l [ljf1j AICARAIPASNRL 1161 2-44] ARAIPASNRLLLTGT [Ki~ F2 -0lI SNRLLLTGTPQN 16i F2-]l LQELWSLFDFACG 167 12-]L9 SLFDFACQGSLLGTL I][1] [3071I KALGFKISENLMAII [16] 131-0]1 GFKISENLMAIP F[1 6- [311] FKISENLMAIKP F1 6[1 RRTKEDVQKKSN 1116 I [3--581 MPSLSRKNDLIIWIR 1[ 16 13711 IRLVPLQEEIYR F 17 13791 EIYRKFVSLDIE 17] 140111 LAELGVLKKLCD-HPR] 16] 14071 LKKLCDHPRLL-SARA ]j7] 14141 PRLLSARACCLLL F][76] 424 Ij LLNLGTFSA-QDGNEG 1] F4-5-IL EESGKMIFLMO-LLKR][ 16] F4670[ GKMIFLMDLLKRR 1116] 47411 DEGHQTLVFSQSRQI ][ie] 489 LNIIERLLKNRH 6] LLKNRHFKTRDT 116] F5sii][ THLLEREKRINLFQQ ][16] TQVGGVGLTLTAAT111 F39IGGVGLTLTMATRWI_].161 1546]1 TMATRIFD N F[1~ 6 155011 RWIFDPSWNAD 116M [5761 PSWNPATDAQVDRV1 7K F56-4]1 AQA'VDRW-RI-GQKEN 1.F17 F5-6-11 VDRVYRIGQKENVW IL 67 F57-4j GQKENVVVYRLITCG 111] F5-9-3] KIYRRQVFKDSLIRQ_11716] 1~]1LFIELQNSVTQLQ F65]3 IKEH AYSLG Ilir6 F65]31 DHDLMYTCDL-SVKEE 116 F86 EELDVVEESHYIQQR]1 185.

00 TableXLVI-V1-HLA-DRB1-0101-I 1 Smers-273P4B7

I

Each peptide Is a portion of SEQ ID NO: 3; each start position i specified, the length of peptide is 15 amino acids, and the end position for each peptide Is the start position plus fourteen.

Pos~[ 12345678-90-12345 I.2L 704Q[ AQFLVEFESQN-KEFL [1-61 725 j[ RNEGAWLREPVFPSS ]i16] 727 11 NEGAWLREPVFPSST 1161 7Tz1 REPVFPSSTKKKCPK]11161 7 1 PIKEGEQDSIV Flii6] 789]I LSSIKVNVTTLQDGK 161 F799]I LQDGKGSAIT j] 800][ QDGKG TGSAD SIATL ]l16] F80-8]1 ADSIATLPKGFGSVE ]I F16] F8-]51 KETLQEPQA E] 16] GPIKQEA LQEDP LESF 1116] 1 541[ QEALQEDPLESFNYV lFIii] I85-91 EDPLESFNYVLSKST [16] I8876[ DDEILRHCNP-WPIIS 16] 92211 SASH SALQDAQASEA][T~ 1936 I AKLEEEPSASSPQY-A 11 if F94-911 YACDFNLLD D1I6I 96011 SADNSRQNFSSQS LEH 1161] F9-7-211 LENVEKENSLOSA 1I F977 I KENSLGSPRA II 1-6 9821I CGSAPNSAFVSI16] 10021 EFSEKDDEPEEVWVK 1Li-6] 110091 EPEEVVVKAKIRSKA ][16] 1109771 RSLINMVWLD-HVEDM-E]16 11104 [LDHVEDMEERLDDSS]6 11107 VEDMEERLDDSSA] 11140 KYTEEDPSGETS 6] 111-62 TSKIPSALAQESA 16 11-79 PLSGEQLVGSPQDKA 11161 11185 LVGSPQDKAE=ATND 11161 F1213] CGKIQEALNCLVKAL 11 ll IFPNEKVLSRQKQIl1 F12-]1 MGLGKTVQIIAFS ls 125][ GLGKTVQIIAFLG MFDASLVNHVLIM 17] F165][ IKWTPGMRVKTF ]15] MLINNWQQLSSRQI s [Th[ =FRGQEFWYID [I T-abeXLVI-V1-HLA-DRB1-0101l5mers-273P4B7 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of pepide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

[~[123456789012345 1Fco-e] [32-8[ RTKEDVQKKKSSNPE 15 E f[ EARLNEKNPDVDI ][jf [36-]1 SRKNDLIIWIRL-V-PLF]is] F37 4 VPLQE-EIYRKFVSLD_15 i.~ F40-11 VLKKLCDHPRLL-SAR] 1151 F5070[ HFKTLRIDGTVTL 1151 538][ VGGVGLTLTMATRVVFJ ii F541][ VGLTLTAATR IF 151 I 545][ LTAATRWIFDPSW-N F 581] jJVRILITCG3TVEEKIY I is 62-4]1 LRELIFTIEDLQN-SVT- 1[5 l[611 EDLQNSVTQLQLQ-S-L I15 633]1[ LQNsvTQLQQSH 1 is] F6-5 8[ EHIAYLQSLGIAGIS 1[5s Ef11 KEELDWVEES HYIQQ [El] 6-9911 QRVQKAQFLVEFESQ][ il 71511 KEFLMEQQRTRNE-GA][ 15 PKLNKPQPQPSPLLS 15 s F7571I QPQPSPLLSThHHTQE 15~ 75-2]1 PQPSPLLSTHQE Fl1-5I EZf][ LSTHHTQEEDISSKM '15s [761][ HHTQEEDISSMS 15W F7773][ ASWIDDLPKEGK Fll5s] F 797][ TTLQDGKGTGSADSI II is] 182611 TNSSILGMEKSFATNIls 827]I1 NSSLGMEKSFATKNE][ 15] I 8-5611 ALQEDPLESFNYV-LS 1] L86-51 FNYVLSKSTKDG 1[5~ F897]1[ PIISITNESQNAESN ][5s F90-21 TNESQNAESNSE ][is F91]6 EIADDLSASHSALQD ][5l F93-41 SEAKLEEEPSAP ]1]1 1 952]I DFNLFLEDSADNQ [E] LF99 KTLSEFE 15I 10081 DEPEEVWKAKIRSK 15 i 110131 VKKIRSKA-RRI-V 11j51 iF10 2[DE-DDSFKDTSSNPF] 15~ 11047 TSLFQFSSVKQFDA T5 ]I TableXLVI-1-1HLA-DRB31-01101- 1 Smers-273P4B37 Each peptidle 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 plu s fourteen.

Ejos 12345678901 234-5 rIFO6211 STPKNDISPPGRF-F-S lF-l 111011 NMVILDHVEDMEERLD fl 11127 PEEGVEESSGEASKY Flis] 111421TEEDIPSGETILSSE1151 11169 AQETSLGAPEPLSGE Di] 11171i ETSLGAPEPLSGEQLaIl] 112311 SADPEVMLTSY 1234 PEVMLLTILSLYKQLN fTableXLVI-V44ILA-DRB1-O1 01- 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. I Igo 123456789012345- score FI5-I KEFIKWTPGMGVKF 7T [I7] [TJ ]WVKEFIKWTPMGK19 []VKEFiKwrPGMGK 8 Ti [P GM G VK TF HGPS KD -181 NTWVKEFIKWTPGMGl17] [13 GMGVKTFHGPSKER17] 1 l IKWTPGMGVKTFHGP .16]j ITableXLVI-VS-HLA-DRB1-0 101- 1 Smers-273P34B7 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 1 cr [T3] VDAICEMPSLSRRND I El] RNDLIIWIRLVPLQEE][fi RRNDLIIWIR PQ ]l 1 ICIEMPSILSRRNDI [W l[ MPSLSRRNDLI-IWIR[ 16] INi I SRRNDLIIWIRLP js ITableXLVI-5-H1-ADRB31-0101-1 I Smers-273P4B73 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 fourteen.

[Posl 123456789012345 ]score [-2-1DVDAICEMPSLRN 1 4 ILPDVDAICEMPSLR 12 [11fl SLSRRNDLIIWIRLV F10] [7l CEMPSILSRRNDLI F 9] 112 LSRRNDLIIWIRLV= I [TableXLVI-6-HL11A-DRBI-01011 I 5mers-273P4B7 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 fourteen.

q oI1234678901235~~e R4I GPNLDQLKDDEVR j~ f-1311 DEVLRHCNPWPIISIl 24 I -O]ILKDDEVLRHCNPP 7& FlflDDEVLRHCNPWPiS16] g lVLRHCNPWPII=SITN F 14 ableXLVll-V1-HLA-DRB1-0301- 115mers-273P4B37 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 1234567890124 scoe _j Dfl~ 461[ KMIFLMDLLKRLRDE 30~W 854 ]FQEALQDPESNV ][30 382][ RKFVSLDHIKELLME_] W 1023 ]1 ARRIVSDGEDEDDF] 9 L 1 JFLYSLYRDGRKGGILA[ Ef] F-30I1 IKELLMETRSPLAEL 7 F478]EQTLVFS QSRIL NII F[2-7- F-485i]E SRQILN IIERLKNR iLZLI I EATKN GDLEEAFKLF rf 2- 24411 ARAIP ASNRLLLTT 1261 F-370 WIRLVPLQEEIYRKF 1j 26] 1 5 DlMEESGKMIFLMD I 6 TableXLVI-V6-HLA-DRBI -0101- 15mers 273P4B7 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.

Po 1 3468012345 F511] THLLEREKRINFIQQ]1 26 11103 VLDHVEDMEERLDDS][ -2I b~ I LIDIKSADPEVMLT 26~ [67131] KNPFRYFSKERL12 [I68i][ KEELDWEESHYIQQ F25 946][ SPQYACDFNLLD 25 F40][1 AFKLFNLADFPNE41I] F222][j WDYVILDEKIT F-2-4] F31-4 I1_SENLMAIIKYR_] 24] F-7-5[ DDEFTDVCNSLL] 3 [T-1-61 KGGILADDMGG Ff23- L7501I1 FKTLRIDGTHLLE ][231 F549-11 TRVVIFDPSWNPATD 23~W [707 ]j LVEFESQNKEFL E I 23 F881]1 LDQLKDDEILR HCNP j[2] F-531FNLFLEDSADNQFJ 23] 145][ VNHVLLIMPTNLN ]j22- [-2W68][ WSLFDFACQGLT r-2-2] F 772 MASWIDDKEE ]22~ [773 If ASWIDDLP KEGEKQ ]22] F-9V4-[ IIEIADDLSASHSAL 22] F-10-9-9] LINMVLDHVEDMEER 1[7272] 1-43i1 LFNLAKDIFPEV ][21 78]IFTDVCNSGLLLYE f~ 111711 GGILADDMVGILGKV 211 F20-17] 1YQMLINNWQQLSSF i L F-406][ VLKKLCDHPRLLSAR i] [43]lHPRLLSARACCLLNL 1I 21] IDQVTDDTLMEG IT [469 ]LRRDEGHQTLVFSJFl 21] [596 ][RRQVFKDSLIRQTTG 21V [-667[GAGISDHDLMYTCD EE 2] [Th23y6 VMLLT LSLYKQL NNN E][1 1o I0 1AEALSP EAHYR 20 19-]I AHYLRYVKEAKEATK ]fl 22 J[ LRYVKEAKEATKNGD][ 201 I14 AKDIFPNEKVLSRIQ 20 TableXLVI-V6-HLA-DRBI .0101- 1 Smers-273P4B7 I 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.

So 123456789012345]Hsor Tj[ LEELAEQGDEFD120 831 -85 11 GLLLYRELHNQ LFEH 1IifIi 129I[1TVQIIAFLSGMFDAS_]1 194 j1 RNGVIiTTYQMLN j -26- 209 IIWQQLSSFRGQE F 20][7.

217 ]1 GQEFVWDYV7L DEAH] 36 ]1 NDLIIWIRLVPLE ii 7T73]1.LVPLQEEIYRKFVSL ][207 50][ VTHLLEREKRIN FY ELFTIEDLQNSVTQL. ]f [634 QNSVTQLQLQS-LH-AA [803 KGTGSADSIATL-PKG F870 SKSTKADIGPNL-D-QL F898 j[ IISITNESQNAESNV 1026 I IVSDGEDEDDS F1040][ 1l157IISSWLMTSKPSALAQE 1 5I20-]1 ETLVKRGK ELKECGK [1 233f ]F DPEVMLLTLSLYKQL] NEKVLSRIQKIQEAL ig] [i6-0i][ IQKIQEALEELAQ ]19 F-133If IAFLSGMFDASLVH_119 [-147j][ HVLLIMPTNLI-N-TWV [.19] 15-3 ]I PTNLINTWKFK 1191 YQMLINNWQQLSSFR] 119] F2-76 QGSLLGTLK-TFKMEY119 F306]1[ EKALGFKISENLMAI ][iE f315 ENLMIVIKPYFLRRT ]f F326_]1 LRRTKEDVQKKKSSNj 19 ii I 3-55 ICEMPSLSRKDII1 If 19] F3584j11FVSLDHIKELMT ]f 19] F47]lLKKLCDI-PRLSR 11.!.

Li4i14-71 PRLLISARACCLLN LG_1 IFLMDLLKRLRDEFH1191 F-45] LMDLLKRLRD-EGHQT [19] [478]F RQILNIIERLLKNRH 1191 [91 ERLLKNRHFKTLD ]19]_ LTabieXLVI-V6-HLA-DRBI-0101l5mers-273P4B37 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 fourteen.

Po 12345678901234 soe _0T TLRIDGTVTHLLERE 19] [5i1 l EKRINLFQQNKDS FIi-] F522]1 LFQQNKDYSVFLLT-TIJ19W] [-64 I I MAQRKSDIKLDEHIA 19 I_66ill SLGIAGISDHDLMYT ]L19I I70751 QFLVEFESQNKE Ftv I19] 176911 SSKMASVVIDDK ][19] I il5I 1[NVTTLQDGKGTGSAD III19] F--4I1 KETLQEGPKQEALQE I[1~ II886011 NLDQLKDDEILRH-CN]F 19] SHSALQDAQASAL] i [TilT EERLDDSSEAKGPED 1119_I [T-152h1 ]GEQLVGSPQDKME flI YETLVKRGKEL KECG 119I fl-207][ GKELKEOGKQE ALN F[ 1 [TI21-][ CGKIQEALNCLVAjliW F34 NGDLEEAFKLFNLAK 1118 I 57 LSRIQKIQEALEELA II ij.

F641[ QEALEELAEQGDE 118] F_86-1 LLLYRELHNQLFEHQ UJA I -158 II NTWNKEFIKTPM 1118 11-9511 NGVIITTYQMLIN L-2--4-1 YVILDEAHKIKTS 1 F243 J[ CARAIPASNRLLLTG jF][ I2-L711 GTPIQNNLQELWSLF J[18 I -3311 PYFLRRTKEDVQK 1-8 364 ]1 KNOLl! WIRLVPLQE][ 18] 137111j IRLVPLQEEIYRKF-V 18 L40_1 LAELGVLKKLCDHPR 18 I VDHIDQVTDDTLMEE F-4672 MIFLMDLLKRLRD][18 [489][ LNIIERLLKNRHFKT ][18 [_507It DGTVTHLLEREKRIN 18 [_52t] YSVFLLTTQVGGVGL]=8i [i I GLTLTMATRWWFD 118 i-570-i It RIGQKENVVVRL 1118 67 TCDLSVKEELDVVfE 118: 778 jDDLPKEGEKQDLSSI E 18 TabeXLVI-V6-HLA-DRB1 -0101- I Smers-273P4B37 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.

188611 DDEILRHCNPWPIIS 181 I 969 11 SQSLEHVEKENLG] 18] 1012 1l EVVVKAKIRSKARRI 18]~i L1i0ii][ KAKIRSKARRSD 5FIi~] F-106I5 KNDISPPGRFFSQ [jfj F1100 I[ INMVLDHVEDMEERLjIt I [Fi_10 It LDH VEDMVEERDS It 8II t-1 9-3]1I AAEATN DYETVR I ti12i77t QEALNCLVKALDI1KS liii 1222][ CLVKALDIKSADE [8 HNQLFEHQKEGAL1Y LFI-0-1 IAFLYSLYRDGRKGG F117] [176 IFHGPSKDERTRNLNR 1117 [1-85-1 TRNLNRIQQRNGVII ~[ID] [5 20[] QMLINNWQQLSSFRG 117_ [283j[l LKTFKMEYENTR 129T11 ENPITRAREKDATPGll] Fj308jj ALGFKISENLMAIIK 1]117] F334-11 QKKKSSNPEARLN-EK 17] I I8-l i KFVSLDHIKELLMET IL 1] I 428 ]1 GTFSAQDGNEEP] ff] L--iiI SPDVDHIDQTDh E][L F49j]] NIIERLLKNRHFKL[I-7] F--5-1 [SWNPATDAQVR] F17 [_567[ VDRWYRIGQKENVW [605][ IRQTTGEKKNPFYF ][I7 S6-16 FIRYFSKQELRELTI ][17 S617_11 RYFSKQELREL FIE]17 694 ]l SHYIQQRVQKA FLV[17 [_744 ]NKEFLMEQQRTRNEG[ i F715 ]I[KEFLMEQQTRNEGA][IT 1 774]~ SWIDDLPKEGEQ 1117 [11111] IDDLPKEGEKDS [i1i7 F-808 ADSIATLPKGFGSVE_1117 815I t PKGFGSVEELCTNSS Ir117 j j[QEOPLESFNYVLSKS 1117

SNYVLSKSTKIPNV~

[Tab~eXLVI-V6-HLA.DRB1-0101- I 5mers-273P4B7 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 Iq the start position plus fourteen.

1o 13457901 2345]H [927 1 ALQDAQASEAKLE 171 F981 IILCGSAPNsRAGF][71 [:i0b_5]1 PEEWvvKAKIRKR[7 [i_04]8 TSLFQFSSVKQF-DASl 17 SSQIPSSVNK MNSR 7] [Ti1-81GELSSENKSSWLMT F171 [-5471 EKVLSRIQKIQEAl1 VLSRIQKIQEAEL 6 [-697[ ELAEQGDDEFTDVCN 116 1 [lil DMGLGKTVQI FLS _16] 1-i11 TNLINTWVKE FIKT116] F-5--1 LLLTGTPIQNNLQEL 1161 II j1 LLGTLKFFKMEYENP 1]_I [291[REKDATPGEKAL F J6i 132211j KPYFLRRTKEDVQKK 1161 F349 II NPDVDAICEMSS a]I F35-6- OEMPSLSRKNLI 6W L-51--1 KRINLFQQNKDYSVF 111j 1519 1 RINLFQQNKDYSVFL [16] [ssi_1]l WIFOPSWNPAT-AQ ][16] E K]j AQAVDRVYRIGQKEN J[16] F_589§ ]TVEEKIYRRQVF161 F-627]I LFTIEDLQNsvTQLQ I[ 1-6I 31IfEDLQINSVTM LS I1 67~.

F-62][ LQSLHAAQRKDK 11111 F-703T][ KAQFLVEFESQNKEF 1116 F73-J[ EPVFPSSTKKKOPKL II ~fl F It_] ETLQEGPKQEALQED 1-16] F-896-[ WPIISITNESQNE 167 F-9507[ ACDFNLFLEDSAN 116] F-970-It QSLEHVEKENSLG I i6] 11-0011] WEFSEKDDEPEEVWV IJ-1 [ioso_]j LFQFSSVKQFDASTP 1516] 1080 I PSSVNKSMNSRRSLA IDE6 1I-0Ifl[SVNKSMNSRRSLASR]F16] l1_08I[ NSRRSLASRRSLINM I16 7 liFI[] LASRRSLINMLH F16] i:i J KEAKEATKNGDLEEA ITableXLVI-V6-HLA-DRB1-01 01- I 5mers-273P34B7 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 fourteen._ I o] 1 123456789012345 soe F A6 II LAKDIFPNEKVLSRI J[ 151 r-94 1j NQLFEHQKEGAY 1151 I 18-j GMFDASLVNHV)7LIM 15i F-4-jNHVLLIMPTNLIN T-W 15i I_173_][ VKTFHGPSKDE-RT-RN 15~ IA479][ TLVFSQSRQILNIIE ]151 t 4.1I[LITCGTVEEKIYRRQ 15 i~ [i593 II KIYRRQVFKDSLIRQ F-677-] MYTCDLSVKEELV ]l1 5- [-695]Ij HYIQQRVQKAQFLVElls] F7041] AQFLVEFESQNKEFL 1 15 I i70671i FLVEFESQNKEFLME I.Jkl1 F819 ]1 GSVEELOTNSSLGME Ill1- Ii F-825]1 CTNSSLGMEKFT 15ii I 3511 EKSFATKNEA-VQKETl 15s F865-11 FNYVLSKSTKADIG-P 1i r 97881] SRAGFVHSKTCLW 15 I 1039]i DTSSINPFNTSLQ ii F1158 ]1 SWLMTSKPSAAQET iI I163][ SKPSALAQETS-LGAP ]111 1120-91 ELKECGKIQEALNCL]115 jPEVMLLTLSLY-KQLN ]F-isj TableXLVII-V4-HLA-DRBI-0301- 1 Smers-273P4B7 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 fourteen.

Ilo-s1 123456789012345- score l]iiiNTWVKEFIKTMGI11 KEFiKwTPGmG~vKrfl 12] [1-flGMGVKTFHGPSKE M1 I1-fI TPGMGVKIFHGPSKD 10j] F-4]1 VKEFIKWTPGMGVKT]Oq] ,[II IKWTPGMGVKTFHG j iTableXLV114V5--11ADRBI-00-l I Smers-273P4B37 I Each peptide is a portion of SEQ ID NO: 3; each start 7position is specified, the length of peptide is 15 amino acid s, and the end position for each peptide is the start position plus fourteen.

P051~ 123456789012345_Fore 671 ICEMPSLSRRNDl19 15][ RNDLIIWIRL-VPLQE 1111] I7E1CEMPSLSRRNDLIIW W[ [EVDAICEMPSLsRRNDFi [_Tl SLSRRNDLIIWIRLV M12 [9Q] MPSLSRRNDLIIWIR II ii [-8-11EMPSLSRRNDLIIWI 110 I J]JIPDVDAI CEMPSLSRR]1~ E13 IRRNDlWRLVPLWF--7 Tab~eXLVII-V6-HLA-DRBI-0301-1 I 1 mers-273P4B7 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.

[13031 123456789012345 1 r I 7] LDQLKDDEVLHN 23 -II NLDQLKDDEVLRHCNI z201 I IIDDEVLRHCNPWPI 1181 121DIGPNLDQLKDVI 11 ±G P NLD QLK D DE-VL RHI 11 01 DEVLRHONPW PIISI 1 TableXLVIII-VI-HLA-DR-0401- I Smers-273P4B7 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.

1 ll123456789012345 [-1 1 75 DDEFTDVCNSLY t [T9-91 II.I1YQMLINNWQQLS j[28 8]j WSLFDFACQGSLG j[ 28 F8-6-]1 LESFNYVLSKSTKAD jE 28 946][ SPQYACDFNLFLES 28~ F001LFQFSSVKQFAT ]j28] I811GLLLYRELHNQLFEH 6] El[ DYVILDEAHKIKTSS I-] TableXLVIII-V1-HLA-DR-0401- I Smers-273P4B7 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 fourteen.

Ps 12345678024 scor [27f7LL7GTLKTFKMEYE [261 IKELLMETRSPLAE-L1126] [421][ ACCLLNLGTFSAQDG jf 26] [46-91 LKRLRDEGHQTLVF-S l26i [48611 RQILNIIERLLNR ]L26iI liii THLLEREKRINLF ]2-6] F54-2I1 GLThTMATRWI-FDP [26] 56411[AQAVDRVYR1G-QKEN [2-6] Ej] VDRVYRIGQKEV 26 L5-7-]1 NWVYRLITCG TVEE[ 26 F6-2]1 LFTIEDLQNSVTL ][2i6 DEHIAYLQSLAI F2-6- [68-71[ ELDVVEESHYIQR 6] [70411 AQFLVEFESQNKF ]l6z 181811 FGSVEELCTNSG F-2i-6 1 182711 NSSLGMEKSFATK-NE III iii F89-]1 PWPIISITNESQA F]2-6I 195ItEKLEEEPSASSPQY][2 F5flj DFNLFLEDSNRN[ 26] [1047][ NTSLFQFSSVKQFDA 26 F11-6-]1 PSALAQETSLGE F-2-6-1 12-1-31 CGKIQEALNCLVKALJ 2 [l~I YLRYVKEAKEATKNG 1I 22] [ElI LLLYRELHNQLFEHQ F-22-] 115711 INTWVKEFIWTG F22] 116411j F-KWTPGMRVKTFH-G III 22i] I2076 IINNWQQLSSFRGQEF] 22] F21--]1 EFVWDYVILDEAHKI ][22] VWDYVILDEAHKIKT 1122 F2--]1QELWSLFDACQ21 [283 LKTFKMEYENTA II 22]1 [30811 ALGFKISENLMAI [2 13-7811 EEIYRKFVSLDH IKE 2 381 11 YRKFVSLDHIKELLM IL 22 461j~ KMIFLMDLLKRLRDE] IF22 F4791 TLVFSQSRQILNIIE ][F22 F49]1NRHFKTLRIDGTVTH_12 52611j NKDYSVFLLTTQVGG If 2-2 [l~i]l WIFDPSWNPATDAQ [L2: FTableXLVIII-Vl-HLA-DR-0401- [1 mers-273P4B37 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.

[P-os]l 1234567890124 score] [5-5]5 DPSWNPATDAQAD I.22.1 5911] WVYRLITCGTVE [22 [173~] KNPFRYFSKQELREL 1 22] F62]5 RELFTIEDLQNSVTQ ][i7i 1675] DLMYTCDLSVKEELD 22 F7073 KAQFLVEF ESQNKEF 22~i 174iI NKEFLMEQQRTRNEG I1221 F7-2-7][ EGAWLRPFST II 2l 81-511 PKGFGSVEELCTNSS]1Ii2i F8--41 SFNYLS KSTKA DIG 122] 95011 ACDFNLEDAN ]22i F987 I RAGFVHSTLWF] 2 11 231 PEDYPEEG EESG LI] [1197]l TNDYETLVKRGKELK] 22] [2-2]1 LRYVKEEANG ]20Y L34]1 NGDLEEAKLFLAK ]0 F6[1 1~ IQK IQEALEE LA-E QG- I20 [10111j KEGIAFYYRG]20 [104]I IAFLYSRDKG ][20 107]I LYSLYRRKGA ][E0 F 177 GGILADDMGLGKTVQ[ I 121][ ADDMG-GKTVQIAF110 F1231 DMGLGKTVQI-IAF-L-S []W [i 279 TVQIIAFLSGMFDAS j 2 [1475 _VNH-VLLIMPTNN 11201 F46NHVLLIMPTNLI1NTW 11201 147][ HVLLIMPTNLIN~V I 2] L17I[GMRVKTFHGPKEI21 F185][ TRNLNRIQQNGI II 20 [i1 9-5 NGV I I TryQ mLNN-w-I 120211 YQMLINNWQQSR ]17K [2073[ QMLINNWQQUSFRG][ Y] 2 22]I DVLEHIT ]20 224]j 2=0EHKKST 230] I AHKIKTSSTKSAICA ]20] 244]j ARAIPASNRLLLTGT ]20 S N R LLLTGTP IQNN C F261]I QNNLQELWSLFDFAc[ 25 0 2657 ]JLWSLFDAQSL] 0 TabieXLVI Il-V1 -HLA-DR-0401 I Smers-27334B37 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 scr [2 91 ENPITPAREKDATPG I[-2o ENLMAIIKPYFL-RRT []F0 34911 NPDVDAICEMPSS 20w 3-55]1 ICEMPSLSRKNDLI1Ff] F3[51 NDLIIWIRLVPLE ]LKi RKFVSLDHIKELE] 20 13841 FVSLDHIKELET 1I20i F39-]1 KELLMETRSPEL ]l270 [39811 RSPLAELGVLKKLD f]20 [4-07] LKKLCDHPRL-LSARA][ 20 F41T3 I HPRLLSARAC CLLNL] 20l 14-4111 SPDVDHIDQVDT 144411 VDHIDQVTDDTLMEE[ 45211 DDTLMEESGK MIFLM 2[0~ F45--91 SGKMIFLML L] F20] F4-6-]1 IFLMDLLKRLRDEH ]20] [477] HQTLVFSQSR-QILNIl ]20] QTLVFSQSRQLI l~i SRQILNIIERLLN F207 4_92-]_IERLLKNRHKTR [50-11 FKTLRIDGVHE j[20] F50] TLRIDGTVTHEEI 201I [50-71 DGTVTHLLEREKI F1201] [Tfl[ EKRINLFQQNKDS 11207 [527 I DYSVFLLTTQ VGGVG F20- F53-5II TTQVGG3VGLTTA ]F20 F5-38[ VGGVGILTLTAAR 1201 15481! ATRWIFDPS WNPAT ]L207 E141 TRIFDPSWNPT] 20f 159611 ]RKSLRTG 1 9 160 11]KSIQTGKN 6-21][ KQELRELFTIEOLQN][ 20] 624]1 LRELFTIEDLQNSVT 1630 IEDLQNSVTQLQLQS M[i F6341 QNsvTQLQLQSLHM][ I20 [642][ LLQSLHMAQRKSDIKL I[20] F651][ KSDIKLODEHIAYQS 660] IAYLQSLGIAGISDH ][20] EEJ __o TableXLVIII-V1-HLA-DR-040 1 Smers-273P34B37 0 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 eahpeptide Is the start position pius fourteen.

FP__ 123456789012345 jrsTe) 7051 QFLVEFESQNKEFLM 1 F717 IKEFLMEQQRTRNG] [73211 REPVFPSSTKKKP ff SSKMASWID-DLPKE [78611 KQDLSSIIKVNVTL 7879I LSSIKVNVTLDK]ii F8-2-11 f-8-91 EDPLESFNYVLSKST] F87781 GPNLDQLKDD EILH[ 0 F8-9-8]1 IISITNESQNAESNV ]1 F9--11 NVSIIEIADDLSH 1[20] I 1L VSIIEIADDLSASHS] I 918][ADDLSASHS ALQDAQ II I HSALQDAQAEAE 1 201 I9679[ SQSLEHVEKENSLCG II 2P.

[972][1 LEHVEKENSLCGSAP ][20-1 9-96][ KTOLSWEFSEKDDEP] 1FO-1-1 EEWVVKAKIRSKARR I[ 10401 TSSINPFNTSLFQFS 1 F165 11076 SSQIPSSVNKSMS F2[01 11099 LINMVLDHVEME 11177 PEPLSGEQLVGSP-Q-D] I11i81 GEQLVGSPQDKAAEA] 1220 LNCLVKALDIKSADP 112211 NCLVKALDISAE 112-261 ALDIKSADPEVMLLT 1234 PEVMLLTLSLYKQLN 11-2-0- 12351 EVMLLTLSLYQN FI207 L-67][ RFPEAEALSPEQAAH 11_ [E]j LEEAFKLFNLAKDIF 15181 E][l NLAKDIFPNEKVLSR 1181 [5-0]l IFPNE-KVLS-RIQKI7Q 1-iI 5-il1 FPNEKVLSRIQKIQE lEE]l 11351FLSGMFDASLVNHVL ][18] [13811 GMFDASLVNHVLLIM ]I1 [13911 MFD-ASLVNHVLLIMP I[E8I F1h j[LiPT0iTvKF 8 rabeXLVIlI-VI-HLA-DR-0401- I Smers-273P4B37I 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 pius fourteen.

7osL 1234 545 sI-ore F1fl IMPTNLINTWVK F1181 1761I FHGPSKDERTNN] 18] F1mfll HGPSKDERTRNLRR 1 FI-8-1 F187 I DERTRNLNRIQQRNG][1 I 19211 QQRNGVIITTY QMLl 18iW F1973 I QRNGVIITrYQMLIN_]F-18]1 20011 TTYQMLINNWQQLSS] I1 1227]1 LDEAHKIKTSSTKSA_11-8] 243](CARAPASNIRILLG 1 18] 125411 LLTGTPIQNNLQELW [274]1 ACQGSLLGTLKTFKM]fj I9] AIIKPYFLRRTKEDV 18i 3261 LRRTKEDVQKKKS 134fIl PEARLNEKNP DVDAI [18] 37411 VPLQEEIYRKFSD] 18] 1410]1 LCDHPRLLSARACCL11i 4_8QSARACCLLNLGTS [8 W311 QDGNEGEDSPDH] F43]8[ GEDSPDVDHIDQT 18 rZ1I[ TDD)TLMEESGKMFL 118]I 4761I GHQTLVFSQSRQILN F4__51 LLKNRH-FKTLR DGT Kj8 [-611fl REKRINLFQQN-K-DYS 118 F572I LFQQNKDYSVFLT ]1181 F5-27] KDYSVFLLTTQVGGV 11-81 F511 VGLTLTMATRWF1181 F5--75] QKENVWVYRLI1TCGT ]i [587 Ij GTVEEKIYRRQVFKD]18 F5_95_8[ QVFKDSLIRQTGK] W 16-2611 ELFTIEDLQNSVQ ][18] F11[EDLQNSVTQLQLQSL][178 F6]fl SVTQLQLQSLHMQR][ iR] F641 QLQSLHMAQRKSDI 18] F6_6][ EELDWVEESHYIQQR~l l1-8 F6_911 'EEHiQQRVQKAQ 1118 F73l01 WLREPVFPSSTkKKC 1118 F75l1 PQPSPLLST-HTQEE ]1 F7-6-I] HHTQEEDISSKMASV 1118 ,[L6J[HTQEEDISSKMASV~v E18 TableXLVIII-VI-HLA-DR-0401- 1 Smers-27334B37 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 pius fourteen.

[Po-1 123456789012345 [1soe] [7-7-91 DLPKEGEKQDLSSIK I ~1 F7 82] K E GE KQ D LS SIKV NV 1181 F78I31 EGEKQDLSS1KVNVT 11181] I8-021 GKGTGSADSI1ATLPK ]181 l0f][ TGSADSIATLPK F ]ia 83]f FATKNEAVQKETLQE ]18 8873][ QLKDDEILRH CNPWP][-18] 789-2][ HONPWPIISITNESQ [18] F9--21 TNESQNAESNVSIIE I][8 F9--11 NESQNAESNVSIIEI 1F_8] F9-1l] IADDLSASHSALQDA ]18] FIl LSASHSALQDAQaSE][18] F95-][ LFLEDSADNRNFSS118] F96-1 [ADNRQNFSSQEV 181 1966 11 NFSSQSLEHVEKES118 r979][ NSLOGSAPNSRAF18 il 9-861[ PNSRAGFVHSKTCLS 1I8 1 0171 AKIRSKARRIVSDGE 1]18] 11033 EDDSFKDTSSINPFN ]1181] F10_4 NPFNTSLFQFS-SVKQ I18 110541 SSVKQFDASTPKNI][18] 1105 5 SVKQFDASTPKNDIS ]Ie]l 110681 ISPPGRFFSSQlPSS 18 1 110731 RFFSSQIPSSVN-KSM1 1[8W 1I087 MNSRRSLASRRSLIN 18] 110881 NSRRSLASRRSLINM]118] 110_93 ILASRRSLINMLDHV1118] 11109I DMEERLDDSSEAKGP 1118 1111 MEERLDDSSEKE 1181 1F127 PEEGVEESSGEASKY I1181 11131]I VEESSGEASKEE 18 11139 SKYTEEDPSGETLSSj_ 1118 111-45 DPSGETLSSENKSSW ][187 1_621 TSKPSALAQETSLGA][ 18 L131ADPEVMLhL 18] [ti4l SRFEEAPEQAQ][ W] 11AAHYLRYVKEAKEATi][] 1 38F EEFLNADF 16] 1 4 IIFKLFNL.AKIFP E 116] TableXLVIIl-V I-HLA-DR-0401- I Smers-273P4B37 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 fourteen.

Pos] 123456789012345 [coe] 8]l KDIFPNEKVLSRIQK I116 1 9]1 NQLFEHQKEGIAFLY II NW AFLYSLYRDGRKGGI 1116] [-0-811 YSLYRDGRKGIA 5j16W] 13-2]1 iIAFLSGMFDASV li16] [IJEJ SGMFDASLVNHVLI 167W [Ell1 GQEFVWDYLEA ][166] 127011 LFDFACQGSL K] F1 6] [28711 KMEYENPITRRK [16] F3-6-71 LIIWIRLVPLQEEIY ][16] 152-011 INLFQQNKDYVL 1! F529][ YSVFLLTTQVGGG 16 1 568]1 DRWYRIGQKENWWV-Y 1116 1 EKIYRRQVFKDI 1116] [5977[ RQVFKDSLIRQTG 1161 [616][ FRYFSKQELRLT 116 [693 ESHYIQQRVQKAQFL7 ]16 [fILVEFESQKMEQ 116] F8-931 CNPWPIISITNEQ [9_5l1 FNLFLEDSADNRQNF 1[16] 19_6411 RQNFSSQSLEHVEKE] 16] [{I1 DDSFKDTSSINPFT ][16] 11043 INPFNTSLFQ FSS 1 1071] PGRFFSSQIPSSVNK [16] 110721 GRFFSSQIPSNS F[16]1 11138 ASKYTEEDPSGETLS ][161 [117561 KSSWLMTSKPSALQ][ 16 1 F53]1 NEKVLSRIQKIQEAL IllS 306][ EKALGFKISENLA 155] F557-01 WYRIGQKENVVVYRL 1 169411SHYIQQRVQKAQFL Fioi-2] EVWKAKIRSKARRI 5-1 11016 KAKIRSKARRVD 1084 NKSMNSRRSASRRS[ 100RRSLASRRSLINMVL] .1 is 111581 SWLMTSKPSAAE] F-1 I 110 FIA-EALSPEQMAHYLRY][ 14 lI AHYLRYVKEAKEATK 1(14] F4_0]AFKLFNLAKDIfPNE II TableXLVIII-Vi-HLA-DR-0401 1 5mers-273P4B37 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptide i's 15 amino acids, and the end position for each peptide Is the start position p us fourteen.I [P osll1 123456-789012345] scr L7!II LFNLAKDIFPNEV ]F14 (F477 1 AKDIFPNEKVLSRIQ Ilil] 14 1 EKVLSRQIEL 14] LSRIQKIQEALEELA114 QEALEEEQ DE]14 NSGLLLYRELHNQLF[ 14] F891 YRELHNQLFEHQKEG][ j14j f[i3II1 HNQLFEHQKEGIAFL I1141 1!!6I1 KGGILADDMGLGKTV 1 14 12711j GKTVQIIAFLSG-MFD 11141] 13301 VQIIAFLSGMFDASL ]714] F13731 IAFLSGMFD-ASLVNH 1114 1 I 136]I LSGMF DASLV NHVLL ]1141 I1-4h]1 DASLVNHVLLIMPTN 1[14] Il-411 ASLVNHVLLIMPTNL L[1[ VLLIMPTNLINTWVK ][14 TNLINTWV KEFIKWT ]14] I 5-][NTWVKEFIKWTPGMR[ IEC] Fl621KEFIKWTPGMRVKF1 34 RNGVITTYQMLIN ][14 [19611 GVIITTYQMLINNW-Q 141 F2-011 TYQMLINNWQQLSSF] 14~ F207 IIWQQLSSFRGEFW] i 125111 NRLLLTGTPIQNNLQ1i] 2521[ RLLLTGTPIQNNL-Q-E 1[I] F2-571[ GTPIQNNLQELWSLF 14i F26]4[ LELWSLFDFACG ]4 F27]6[ QGSLLGTLKTFKM-EyIl] F280][ L-GTL-KTFKMEYNPI -[14 285]1 TFKMEYENPITRR ][14 3101I GFKISENLMAIP ][i14 [F31I SENLMAIIKPYFLRR ]F1141 F3178 J MAIIKPYFLRREDI i 13231 PYFLRRTKEDVQKKK II T 135211 VDAIOEMPSLSRKND ]LF± I F36-]1 KNDLIIWIRLVPL-QE lIE] 368]1 IIWIRLVPLQEEIYR IiI 137011 WIRLVPLQEEIYK ]flf I3f31 LVPLQEEIYRKFSL ]14 TableXLVIII-VI-HLA-DR-0401 I 5mers-273P4B37 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.

P osjl 1234567890124 [core [387][ LDHIKELLMETS_[H4] 40111l LAELGVLKKLODHPR E11] LGVLKKLCHRL 11141 CCLLNLGTFSAQD-GN] 1141 [42411 LLNLGTFsAQDGE F -u [44fl[ IDQvTDDTLMEESGK 14 1462]1 MIFLMDLLKRLRE 141 14661I MDLLKRLRDE=GHQTL EE]± F48] LNIIERLLKNRHFKT 41 151911 RINLFQQNKDYS F ]14 F53011 SVFLLTTQVGG L 1114] [5470 GVGLTLTAARW F]4 [55011 RWIFDPSWNAD 14 581][ VYRLITCGVEI j[I4T] 582][ YRLITCGTEIY 114 [602][ DSLIRQTTGEKKNPF1 I14~ vTQLQLQSLHAAQ-RK 14 k [63j9[ QLQLQSLHAAQRKSD 1114 [65-]1 DIKLDEHIAYLQ=SLG 114] I66731 LQSLGIAGISD=HDLM 1114] 66811 IAGISDHDLMYTCDL 14I 673]1 DHDLMYTCDLSVKEE 1C4± F6-7411 HDLMYTCDLSVK=EEL 1141 F6-i 11 DLSVKEELDWES 1114] 685][ KEELOWVEESHYQ ]4 F6-8-1[ LDWVEESHYIQQRVQ] l141 F698][ QQRVQKAQFLVEFES II ii F1i]1EFLMEQQRTRNEGAWII 14 F7278[ GAWLREPVFP=SSTKK 1141 1 744] IOPKLNKPQPQPSPLL]EI 1 [754]1 PSPLLSTHHTQEEDI 11 14] 1NflI SPLLSTHHTQ-EE-DIS ]jj 1721 MSWDDPKGE ]14 I ZZl1 ASWIDDLPKEGEKQ Il] SIDD LPKEGE KQD 114] IDDLPKEGEKQDLSS J1 F F79-3 KVNV1TTLQDG GTGS][ 1W [~IVTTLQDGKGT SADS IEi4 5a ADSIATLPKGFGSVE [114 TabieXLVIII-VI -HLA-DR-0401 1 Smers-273P4B37 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 fourteen.

Tjj11 123456789012345- scoe 782]fl SLGMEKSFATKNEAv] 1±1 F8--01 NEAVQKETLEGK 114 1 F8--51 KETLQEGPKQEAL-QE 141 1 854][ QEALQEDPL1ESFNYV] 14] 866][ NYVLSKSTKDGN1 I87Z4l1 KADIGPNLDQL-KDDE IFI4 8-81 I~ LDQLKDDEILRHN 114] F88l61 DDEILRHCNPWPIIS 11141 I88-11 DEILRHCNPWPISI 11141 F8 9]I WPIISITNESQNAES_]ILL- F9-09] ESNVSIIEIADLSA II141 [95-411 NLFLEDSADNRQNFS II ]l I [10231 ARRIVSDGEEDS114 110531 FSSVKQFDAS-TPKND ][14] 10801 PSSVNKSMNSR-RSLA11141 10961 RRSLINMVLDHVEDM ][14] 11097 RSLINMVLDHEM [1 4] 101101 NMVLDHVEDEEL[ F-14 111281 EEGVEESSGEASKYTf 114 1148 GETLSSENKSSWLMT[ 1TC 1115771 SSWLMTSKPSLQ 114 11-8311 EQLVGSPQDKAEA I1-4 1711 QEALNCLVKADK F14 12331 DPEV/MLLTLSL Q 1114 1236 VMLLTLSLYK QLNNN EC TabieXLVIl-V4-HLA-DRBI-0401- 1 5mers-273P4B37 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.

[P-os 13456789012345 [score] 1 1IGMGVKTFHGPSKDERI1 j- FIKWVTPGMGVKTFHG I116~ IL ]NTWVKEFlKNPG j 14: [fl EFIKWTPGMGVKTF I114 11 WPMVKFGSI1 TableXLVII-V4-HLA-DRBI-0401-1 1 5mers-273P34B7 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 fourteen, I Pos 1246789012345 Jscorel 12 IPGMGVKTFHGPSKDE jj1-2j ~jj VKEFIKWTPGMGVKT I0- STableXLVII-V5-HLA-DRBI-0401- I 5mers-27334B37 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 fourteen.

Posi 123456789012345 score] I ICEMPSLSRRNDLI L7I I 3] VDAICEMPSLSRRND 14i F11 RNDLIIWIRLVPLQE I]14] [11 jDVDAICEMPSLSR I AICEMPSLSRRNDLI D] I T]ICEMPSLSRRNDLII 12[I [lIi]l SLSRRNDLIIWIRLV IT] SRRNOLIIWIRL=VPL ]Efl 14][ RRNDLIIWIRLVL] 12 Ej ~(MPSLSRRNDLIWR[EW ITableXLVII-V6-HLA-DRB1-0401- I Smers-273P4B37 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 fourteen.

[I 123456789012345 Iscorel F4]GPNLDQLKDDEVLRH 20] W -]QLKODEVLRHCNW E7j] 71I LDQLKDDEVLRHCNP]I41i IDDEVLRHCNPW PIIS 1114] fTableXLVII-V6-HLA-DRBI-0401] I Smers-273P4B37 J 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 Istart position plus fourteen.

M oO11234567890124 ~e [1-3]1 DEVLRHCNPWPIS ]14] [1]i ADIGPNLDQLKE F] 2-] I lol LKDDEVLRHCNPPI]I12 TableXLIX-V1-HLA-DRBI-1 101- 1 Smers-273P34B7 f I Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 each peptide is the start position plus fourteen.

[s-o-s 12345678024 [core TNDYETLVKRGEKj 7 [80]fl ADSIATLPKG F 26EIi [§96 ][SQSLEHVEKENSLCGj 26 [381]I YRKFVSLDHIKLMj~ [207 ]I NN WQQLSSFREF[ (i11561I KSSWLMTSKPS-ALA QIj.i [10101 PEEVVVAKIRSKA 1231 [-1217fl QEALNCLVKADK 1123 1 [4-0]l AFKLFNLAK-DIF-PNE 2 I A7-l AKDIFPNEKVSI l22I [219j EFVWDYVILDEAHKI 22 F46211 MIFLMvDLL-KRLEG 122 4-63]1 IFLMDLLKRLRDEGH][ 2 48-91 LNIIERLLKNRHFKT J 22 11501LFQFSSVKQFDSP]IZ I [1764 ]j FIKWTPGMRVKTH] [327 I RRTKEDVQKKKSN] Y [367 I. KNDLIIWIRLVPLQE 1[ 21] I][LAELG VLKKLODP]CY [40-7[ LKKLCDHPRLLSRA I 21] 16] RQILNIIERLLKR [5179[ RINLFQQNKDYSVFL II21] [79-31 KVNVTTLQDGKGG] i [10121 EVWKAKIRSKARRI 1211 [18 ]AAHYLRYVKAET10 EKVLSRIQKIQEALE ]20j 1 0-4 ItIAFLY SLY RD GR KGG 107QI LYSLYRDGRKGIA[2 [31-4 IISENLMAIIKPYFLRR]_ 20 I355 J IOEMPSL SRKN DL II [20 46-5]f L-MDLLKMRLRDEGHQT[~ F528 ]DYSVFLLTTQVGGVGk 20 [I QAVDRWYRIGQKEN1L20 [74111 KKKCPKLNPQP [77-4j SVVIDDLPKEGEKQ] [826 I TNSSLGMEKSFAK] F845]1 KETLQEGPKQELQE] F884] LKDDEILRHONPWPI I1065]1 118511 TRNLNRIQQRNGVII 19i~ 733][ EPVFPSSTKCK II 9l NVSIIEIADDLAH 19I 2111 [YLRYVKEA TN F, 18 DDEFTDVCNSGLLY11 [1081 YSLYRDGRKGGILAD 1118I -1271 GKTVQIIAFLSGMFD 1118 F14It ASLVNHVLLIMTNL 1 I8 FIr. j(l VKVFHGPSKDERTRN[ 18 F327 1I KPYFLRRTKEDVFK1][8iW F3-4 [1 NPDVDAICEMPSLSR][ 18- 42-111 AOCLLNLGTFSAQDG[ 1 18 F56-]1 DRVYRIGQKENWW][V-F18] 159211 EKIYRRQVFKDSLI ][Z [6131 [KNPFRYFSKQELRE1 i8i F625 RELFTIEDLQNSVTQ 1(18] F7077][LVEFESQNKEFLM1118 F8-1811 FGSVEELCTNSSG 1181 F-98] ][LSWEFSEKDDEPEE 1118 I [i-008[ DEPEEVVVAIS 118 I 1221][ NCLVKALDIKSADPE .58-ll LLLYRELHNQLFEHQ 11 [94] NQLFEHQKEGIAF-LYI1117 I 1073I GIAFLYSLYRDGRKG 117- F-27-0-1[ LFDFACQGSLLGLK17 I 2979I EKDATPGEKALGFKI ][177J F53111 VFLLTTQVGGGT] F1 7] F54211~ GLTLTAATRWIFDP17] [69 3 ESHYiQQRVQKAQFL][F17 18-]4] SFNYVLSKSTKADIG ][17] I9879]IRAGFVHSKTCL WE [17 FI0-7-1][ PGRFFSSQIPSSN 117 [11-98][ NDYETLVKRGKEK 1117 T4 1[SRRFPEAEALSPQ I 1.26 F4-1]1 FKLFNLAKDIFPNEK I 116 Fl321IIAFLSGMFDASLVN 1116 lIT 1[ TNLINTWVKEF1KV -16 ,EIi~ ITTYQMLINNWQQLS EiC6 22111 VWDYVILDEAHKIKT 1 6 288 I[ MEYENPIT RREKDA j*16 ZLILGTFSADNGSI 16] 498 ]NRHFKTLRIDGTVTH I 16 1 710VTHLLERERNF 1161 [52-9[ YSVFLLTTQVGGVGL U E LTMATRWFPN F1i -6- ][DPSWNPATDAQAVDR][ Ii [5775 I QKENVVYRLITCGT] 16 I 57911 VVVYRLTGEE ][16] [604 I LIRQ1TGE-KKNPFRY I 16 659][l HIAYLQSIAISD 1 715 ][KEFLMEQQRTRN Fl 1 f 727][EGAWLRPPST 161 F8175]IPKGFGSELTS F16 862ILESFNY VLSKS TKAD I £6I [893]I CNPWP IISITNE SQN ]16] 1 950 ACDFNLFLEDSADNR] 1I6] 17i1 KAKIRSARVD l -6] 10341 DDSFKDSNPT ]16 1 1104311 INPFNTSLFQFSSVK 1116] Il 2i3 1 PEDYPEGESE 1116] F- jFPNEKVSIKQ 15 F821 CNSGLLRLHQ 115 [-93 ff HNQLFE HQKEG IAFL] 115] LADOMGLGK1QI ][is5] DMGLGKVIAL Di15 [T13]9 MFDASLVNHVLLM] 15 [2231jDYVILDE AHKIKT SS 1[5~ 1 2-24]1 YVILDEAH-KIKTSST_-l15 l [2371 STKSAICARAIPAN 15 F24411 ARAIPASNRLLLTGT II15 F371IIKPYF LRRTK EDVQ I l [3-8 ]RTKED VKKSP] [375]1 PLQEEIYRKFVSLDH ][15 [405f][ PLAELGVLKKLCDHP] I15 DTLMEESGK IF] 15i F49-1[]IERLLKNRHFKTR ][15 1 IKNRHFKTLRIDGT 115] F504 -I RIOGTvTHLLER E I1] F507~ DGTVTHLLEREKRIN j] 1i50I8 [GTVTHLLEREKRIN_ J15-], 156881[qGTVEEKIYRRQVFKD 1[I15 687 LDWESHYIQRV][IF75 863 ESFNYVLSKSTKADI 15 10 AIRSKARRIVSDEE1 10811 [SSVNKSMNSRRSIAS.1I 1083][VN-KSMNSRRSLASRR]L15I 1087][ MNSRRSLASRRSLIN 15 [1098[ SLINMVLDHVEDMEE j Fli158 1I SWLMTSKPSAAE] F-1-5 [i0O[[ YETLVKRGKELKECG 1 15] F-3411 NGDLEEAFKL FKI 4i] 1TEj ]GLLLYRELHNQLF114] F16-f]jPGMRVKTFHGPSKDE] 14 1 FI 1 [DERTRNLNRIQQN ff14 1 ~226][ ILDEAHKIKTSSTKS ][1 127611[ QGSLLGTLKTFKMEY i II fl1 ENPITRAREKDATPG]4-1 [-37-41 VPLQEEIYRK F 14D i7 388]1 DHIKELLMETR-SPLA] 14] [3-90J[ IKELLMETRSPL E 11I 410 ]I LCDHPRLLSARAC F[ 1-4 1 4381 GEDSPDVDHIDQVTD F460 11GKMIFLMDLLKRL-RD 114I I4679]ILKRLRDEGHQDlV 14 [4-7-4]1 DEGHQTLVFSQSRQI][ 14 SRQILNIIERLLKNR][4 THLLEREKRINLFQQ][ W F5-6- ATDAQAVDRWYRIGQ[ E1 [5973] KIYRRQVFKDSLR F-14] 597 I[ QVFKDSLIRQTTGEK 1 F609 l[TGEKKNPFRYFSQ 14ii 6341] Q(NSVQQLSH 1114 1 642 JD QU IMQRKKL][ 14 I] KsDIKLDEHIAYLQS] 14] [69§f][H-YIQQRVQKAQF=LVE]

DW]

I 70 1 VQKAQFLVEFESQNKl 141 1717 IFLMEQQRTRNE-GA-WL 114] F77 r.[GAWLREPVFPSSTKK I W S734 IIPVFPSSTKKKCPKLN )[14] 17 74 1 SLLSTHHTQEEDI F-141 [763 TQEEDISSKMASWI F1-4- 176911 SSKMASVVIDDLPKE i [770] SKMASWVIDDLPKEG

W

F79-5] NVTTLQDGKGTGSAD][14] F.831] GMEKSFATKNEAVQKE][ [8371 ATKNEAVQKETLQEG [14 866[ NYL TAIGPN IT 88 QLKIDEIRCNW 114] 966 11NFSSQSE VE E qq31VHS-ROCLSWEFSEKD] 14i 10370] GEDEDDSFKDTSSIN]jF4 1044 1NPFNTSLFQFSSVKQ[ 14] 10771 SQIPSSVNKSMNSRR] 1T 10961 RRSLINMVLDHVEDM 1 ii [1104 LDHVEDMEERLDS II ill 148 1GETLSENSWM 114 [11681 LAQETSLGAPEPS 114 1 [12-03] LVKRGKELKECGKIQ 1 14 1 11207 GKELKECGKIQEAL-N 14ll [i-23]1 SADPEVMLLTLSLYK ]F[14]1 Iifi AHYLRYVKEAKEATK][ 1 I Ifi[ LSRIQKIQEAEL 3T] Ii~i][ IQKIQEALEELAEQG 13II 1161 rKGGILADDMGLGKTV][ 13] [126][ LGKTVQIIAFLSGMF liii] [l-11l NHVLLIMPTNLIT 13I [l16-8 I1PMVTHP SKD] 13 flI1YQMLINNWQQLSF1 1 I -6 7I LWSLFDFACQGSLLGFf13]1 F-27-7Jj GSLLGTLKTFKMEYE][ 13 I Lj TLKTFKMEYENPITR][1 1 [TPGEKALGFKISENL IT]- I32[KISENLMAIIKPYFL 13IT L37zO]l WIRLVPLQEEIRFl3T] F38FVSLDHIKELMT 1113]1 I38-91 HIKELLMETRS-PLAE 113 I [Th KELLMETRSPLAELGI f13] RSPLAELGVLKKLCD] 1 I4 5-9IL SGKMIFLMDLLKRLR] I13 F 53511 TTQVGGVGLTL-TAAT][ 13] F55][TAATRWIFDPSWNP IT]13 F550 ]1 RWIFDPSWNPATDA] I-]3 Ili PFRYFSKQELRELFT 11.Ii F6-2-11 LRELFTIEDLQNSVT 11_ F627 I LFTIEDLQNSVTQLQJ113 I F65jf[l RKSDIKLDEHIAYLQ J1711 [1 flj ]DLMYTCDLSVKEELD]IF 1-3] 68-5Q KEELDWEESH YIQQ[ IT]7 F7-8][ KQDLSSIKNTQ ]1 3] 791]1 SIKVNVTT-LQDGKGT][1] 82][ LCTNSSLGMEKSA] I F85-911 EOPLESFNYVLSKST 113] I 949 I YACDFNLFLEDSD 1113] Ii_71SINMDHVEDME [E's 1111 EERLDDSSEAKGPED 1[ 13 114I ENKSSWLMTSKPSAL F[ 13] 1182 GEQLV SP F13[ 12 201 -L.CI KLDIKADP 1233 FVLLLLYQ j3- 1 11235 EVMLLTLS=LYKQLNN 5731 i TalXLIX-V4-HLA-DRBl-1 101-1 Tbe1 Smers-273P4B7 I 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 fourteen.

[Pos 123456789012345 score] [F-1-1NTWVKEFIKWTPGMGI2 VKEFIKWTPGMGVK1 1 [I1 PGMGVKTHPKE 1 [TJ IKWTGMGVTFHG 1311 TableXLIX-V4-HLA-DRBI-1 101- I I 5mers-273P4B37 Each peptide is a portion of SEQ 10) 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 fouteen F~sj123456789012345][ IIiITPGMGVKTFHGPSK-D 13 III[TWVKEFKWTPGMGv 12] H WT~fPGMGVKTFHGS 12] j KVTPGMGVKTFHGPS 10K TableXLIX-V5-HLA-DRB1 -1101-1 I Smers-273P4B37j 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 I start position plus fourteen.

Is 123456789012345:] E] ICEMPSLSRRNDI I[21-1 [isfl RNDLIIWIRLVPLQE EK] I 3-]VDAICEMPSLSRN FT1 2I ITableXUIX-V6-HLA-DRBl -1 101- 1 Smers-27P8 Each peptide is a portion of SEQ1 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 I123456789012345 [Ec-oe [10J LKDDEVLRHCNPPI21 III jQLKDDEVLRHCNPWP j 14 [I4] GPNLDQLKDDEVLR F[12 F11 DDEVLRHCNPWPIIS [12 Table L: Protein Characteristics of 273124137 Bioinformatic URL Outcome Program ORF ORF finder 95-3847 Protein length 1250 aa Transmembrane region TM Pred http:/Iwww.ch.embnetorg/ 2TM, aa132 -157, aa532-554 HMMTop http://www.enzim.hu/hmmtop/ 2TM, aal 32-1 51, aa 53B-558 Sosui http:llwww.genome.ad.jp/SOSui/ soluble protein TMHMM http:Ilwww~cbs.dtu.dklservicesTMHMM no TM Signal Peptide Signal P hftp://www.cbs.dtu.dkIservices/SignalP1 no pI p1/MW tool http://www.expasy~ch/tools/ 5.1 pi Molecular weight p 11MW tool http://www.expasy.chltoolsl 141.1 kDa Localization PSORT http://psort.nibb.ac.jp/ 50% cytoplasmic, mitoctiondriall PSORT 11 http://psortnibb.ac.jp/ 65% nuclear, 21 %cytoplasmic Motifs Pfam http:/Iwww.sanger.ac.uk/Pfam/ SNF2-N-terminal domain, Helicase-C-terminal Prints http://www~biochem.ucl.ac.uk/ none found Blocks htp://www.blocks~fhcrc.org/ SNF2 related domain Table LI: Exon boundaries of transcript 273124137 v.1 Table 1-lI(a). Nucleotide sequence of transcript variant 273134137 v.2 (SEQ ID NO: 1 atgcgcgggg cgggagtgag cgaaattcaa gctccaaact ctaagctcca tc caagctcc aagctccaaa ctcccgccgg ggtaactgga acccaatccg aggcatcccg aaggtttccg gaagccgagg ccttgagccc agagcaggct taagggtctt gctgtgtcgc ccagactgga attcagtggc ctgatcatag cctcgaactc ctgggctcaa gcagtcctcc tgccccagcc tccctagtag agatatgtga aagaggccaa agaagcaact aagaatggag acctggaaga cttttcaatt tggcaaagga catttttccc aatgaaaaag tgctgagcag atacaggaag ccttggagga gttggcagaa cagggagatg atgaatttac aactctggct tgctacttta tcgagaactg cacaaccaac tctttgagca ggcatagctt tcctctatag cctgtatagg gatggaagaa aaggtggtat gatatgggat tagggaagac tgttcaaatc attgctttcc tttccggtat tcacttgtga atcatgtgct gctgatcatg ccaaccaatc ttattaacac gaattcatca agtggactcc aggaatgaga gtcaaaacct ttcatggtcc gaacggacca gaaacctcaa tcggattcag caaaggaatg gtgttattat caaatgttaa tcaataactg gcagcaactt tcaagcttta ggggccaaga gactatgtca tcctcgatga agcacataaa ataaaaacct catctactaa tgtgctcgtg ctattcctgc aagtaatcgc ctcctcctca caggaacccc aatttacaag aactatggtc cctatttgat tttgcttgtc aagggtccct ttaaaaactt ttaagatgga gtatgaaaat cctattacta gagcaagaga accccaggag aaaaagcctt gggatttaaa atatctgaaa acttaatggc ccctattttc tcaggaggac taaagaagac gtacagaaga aaaagtcaag gccagactta atgaaaagaa tccagatgtt gatgccattt gtgaaatgcc aggaaaaatg atttaattat ttggatacga cttgtgcctt tacaagaaga aaatttgtgt ctttagatca tatcaaggag ttgctaatgg agacgcgctc gagctaggtg tcttaaagaa gctgtgtgat catcctaggc tgctgtctgc tgtttgctaa gatgtggacc atattcctaa tctcaatcga acattgcgaa ttccagcaaa ttaacattaa gatgctcaag aggctaatca tcattaataa gaattaagag cagtctttgc ctgcagtctt tctgttaaag aaagctcaat agaactagaa aaatgcccta actcaggaag gagggtgaga aaaggtacag gaactttgta gtacaaaaag gaaagtttta caactaaagg aatgaaagtc gcatcccata tc ag at ct t aac agacaaa ggctctgcac atcttgggac atattgatca tggacctact ggcaaattct tcgatgggac ataaagatta ctgcagcaac ctgtggatag cttgtgggaC gacaaactac agctctttac atgctgctca tggggatagc aagagcttga tcctcgttgz atgagggggt aattgaataE aagatatcac aacaagatct gtagtgctgz ctaactctt( agacattaci attatgtact atgatgagal aaaatgcagi gtgcactgc caccacagti atttttcca(.

ctaattcca(.

attctctgct agtaactgat taagaggctg aaacatcatt agttactcat ctctgttttt tagagtggtc agtttaccga tgtagaggaa tggtgaaaaa aatcgaggat Lgaggaaatct :tggaatctce Ltgtggtagaz tattcgagtct ctggctaagz iaccacagcct ttccaaaatc ctccagtat2 i ctctatagct attgggaatc i agaggggccl :tagcaaatci :tttacgtcal i atcaaatgti 3, ggatgctca; a tgcatgtgal a agcagggttcaagatggaa gacacattga cgagatgagg gaacgcctct cttttggaac ctgcttacca atttttgacc Lattggacaaa Laaaatataca Laagaaccctt cttcagaact gatataaaac Lgaccatgatt Lgaatctcact :caaaataaag x gaacctgtat :cagccttcac Igcaagtgtag i aaggtgaatg actttaccaa Sgaaaaaagct aagcaagagg i. accaaagctg :tgcaatcctt a gcaagtgagg a gagcatgttg t gtgcatagca atgaggggga tggaagaatc gacatcaaac taaagaatag gagaaaaaag ctcaagtagg ctagctggaa aagagaatgt gaagacaggt tccgatattt ctgtaacca tagatgaaca tgatgtacac atattcaaca agttcctgat ttccttcttC ctcttctaag tcattgatga ttaccacctt aggggtt tgg ttgcaactaa cactgc aaga atattgggCC ggcccattat aaatagctga ccaagttgga tcttggaaga agaaagaaaa aaacatgtct Lgctccaagc tgggtcatgg 120 ~ctcattacc 180 :tcactgcag 240 ~tgggactta 300 ~gcatttaaa 360 xatccaaaaa 420 xgatgtgtgc 480 -cagaaggaa 540 3ttggCtgat 600 3tttgatgca 660 3tgggtaaaa 720 :agcaaggat 780 cactacatac 840 gtttgtgtgg 900 gtcagcaata 960 aatccagaLat 1020 gctg~jgaaca 10B0 gaaggatgct 1140 aatcataaaa 1200 caacccagag 1260 ttccctttcc 1320 aatatacagg 1380 acctttggct 1440 acgggcttgt 1500 agattcccca 1560 tggaaaaatg 1620.

tctggtgttt 1680 gcactttaag 1740 aattaactta 1800 tggtgtcggt 1860 tcctgcaact 1920 tgtggtttat 1980 tttcaaggac 2040 tagtaaacaa 2100 gctgcagctt 2160 tattgcctac 2220 atgtgatctg 2280 aagggttcag 2340 ggaacaacaa 2400 aacaaagaag 2460 tactcatcat 2520 tctgcccaaa 2580 gcaagatggt 2640 aagtgtagaa 2700 aaatgaaLgct 2760 ggatcctctg 2820 aaatttagat 2880 ttccataaca 2940 tgacctttca 3000 agaggaacct 3060 ctcagcagac 3120 tagcttgtgt 3180 cagttgggag 3240 ttttctgaga gctagaagga ataaatccat actcccaaaa gtaaataagt gttttagacc cctgaagatt gaagaggatc tctaagccta gaacagttgg cttgtaaagc ttagttaaag ttgtataagc actgaatatg ggcttaaggc agtc tggtat catattctat ttcttaattt aagacgatga, ttgtttcaga tcaacacatc atgacatcag ctatgaactc acgtggagga atccagaaga cttccggaga gtgctctagc ttggttctcc gtggaaaaga cgcttgacat aacttaataa agggaat ttt aagaaagatc tctgagcact aaatttaact tactctgaaa accagaagaa tggcgaagat tctctttcaa tccaccagga tagaagatct catggaggaa aggggtggag aacactgtct tcaagagacc ccaggataag actaaaagag aaaaagtgca caattgagaa tgttcccata tcaaaaagca agcttaatat gtgttgtttc gtgatcatct gtag tagtta gaagatgat t ttctcatctg aggttctttt ctggcttcta agacttgacg gaaagcagtg t cagaaaac a tctcttggtg gcggcagagg tgtggaaaaa gatcctgaag tgtaacctgt attggattct acttctgccc ttcttcactt ttggaaagtt ttgtatataa aagcaaaaat cttttaaaga tgaaacaatt catctcaaat ggaggtctct acagcagtga gcgaagcctc agtccagctg cccctgagcc ctacaaatga tccaggaggc ttatgctctt ttattgtatt ttgggaacat tgcaacgccc gaatattctt ttgtaaaatt cagttcagat .cagaagtaaa tacctcaagc tgatgcttca acc cagtagt tattaatatg agcaaagggt caagtataca gttaatgacg tttgtctggt ctatgagact cctaaactgc gactttaagt ttaaagtgaa gaagcattca cccactccat atattttagg attctggtca aagaaaatta 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4334 aagttacttt tctc Table 1-III(a). Nucleotide sequence alignment of 273P4137 v.2 (SEQ ID NO: 111) and 273124137 vM (SEQ ID NO: 112) v.2 23 aaattcaagctccaaactctaagctccaagctccaagctccaagctccaa 72 v.1 1 aaattcaagctccaaactctaagctccaagctccaagctccaagctccaa v.2 73 gctccaaactcccgccggggtaactggaacccaatccgagggtcatggag 122 v.1 51 gctccaaactcccgccggggtaactggaacccaatccgagggtcatggag 100 v.2 123 gcatcccgaaggtttccggaagccgaggccttgagcccagagcaggctgc 172 v.1 101 9catcccgaaggtttccggaagccgaggccttgagcccagagcaggctgc 150 V.2 173 tcattacctaagggtcttgctgtgtcgcccagactggaattcagtggcct 222 v.1 151 tcattacc 158 v.2 223 gatcatagttcactgcagcctcgaactcctgggctcaagcagtcctcctg 272 V.1 158 v.2 273 ccccagcctccctagtagctgggacttaagatatgtgaagaggccaaag 322 v.1 taagatagtgaaagaggccaaag 182 v.2 .323 aagcaactaagaatggagacctggaagaagcatttaaacttttcaatttg 372 v.1 183 aagcaactaagaatggagacctggaagaagcatttaaacttttcaatttg 232 v.2 373 gcaaaggacatttttcccaatgaaaaagtgctgagcagaatccaaaaaat 422 v.1 233 gcaaaggacatttttcccaatgaaaaagtgctgagcagaaccaaaaaat 282 v.2 423 acaggaagccttggaggagttggcagaacagggagatgatgaatttacag 472 V.1 283 acaggaagccttggaggagttggcagaacagggagatgatgaatttacag 332 v.2 473 atttcattgtgtctttggatccacat 522 v.1 333 atttcattgtgtctttggatccacat 382 v.2 523 tttgagcaccagaaggaaggcatagctttcctctatagccgtataggga 572 v.1 383 tttgagcaccagaaggaaggcatagctttcctctatagcctgtataggga 432 v.2 573 tggaagaaaaggtggtatattggctgatgatatgggattagggaagactg 622 v.1 433 tggaagaaaaggtggtatattggctgatgatatgggattagggaagactg 482 V.2 623 ttaactgttctcgtagtgtctatgga 672 v.1 483 ttaactgttctcgtagtgtctatgga 532 v.2 673 caggtcgtagcacattataaaggaag 722 v.1 533 caggtcgtagcacattataaaggaag .582 v.2 723 atctaggatcgataggcaactctgct 772 0 v.1 583 atctaggatcgataggcaactctgct 632 C1 v.2 773 gcaaggatgaacggaccagaaacctcaatcggattcagcaaaggaatggt 822 v.1 633 gcaaggatgaacggaccagaaacctcaatcggattcagcaaaggaatggt 682 v.2 823 gtattatctcaagtatatatgacatt 872 v.1 683 gttattatcactacataccaaatgttaatcaataactggcagcaactttc 732 v.2 873 aagctttaggggccaagagtttgtgtgggactatgtcatcctcgatgaag 922 v.1 733 aagctttaggggccaagagtttgtgtgggactatgtcatcctcgatgaag 782 v.2 923 caaaataactacatagcgatttcctc 972 v.1 783 caaaataactacatagcgatttcctc 832 v.2 973 atctc~gatgcctcccgaccatcgaa 1022 v.1 833 attcctgcaagtaatcgcctcctcctcacaggaaccccaatccagaataa 882 v.2 1023 ttaagattgccatgattcttaggccg 1072 v.1 883 ttaagattgccatgattcttaggccg 932 v.2 1073 tgggaacattaaaaacttttaagatggagtatgaaaatcctattactaga 1122 v.1 933 tggaataacttaagggagaacttatg 982 v.2 1123 gcaagagagaaggatgctaccccaggagaaaaagccttgggatttaaaat 1172 v.1 983 gcaagagagaaggatgctaccccaggagaaaaagccttgggatttaaaat 1032 V.2 1173 attaactagcactaaccatttaggat 1222 V.1 1033 attaactagcactaaccatttaggat 1082 V.2 1223 aaagctcgaaaataacacaagcgcta 1272 v.1 1083 aaagctcgaaaataacacaagcgcta 1132 V.2 1273 gaaaagaatccagatgttgatgccatttgtgaaatgccttccctttccag 1322 V.1 1133 gaagacaagtagcttggatctcctca 1182 v.2 1323 gaaagttatttgtcgcttcttcaaga 1372 v.1 1183 gaaaaatgatttaattatttggatacgacttgtgcctttacaagaagaaa 1232 v.2 1373 tatacaggaaatttgtgtctttagatcatatcaaggagttgctaatggag 1422 v.1 1233 tatacaggaaatttgtgtctttagatcatatcaaggagttgctaatggag 1282 v.2 1423 acgcgctcacctttggctgagctaggtgtcttaaagaagctgtgtgatca 1472 v.1 1283 acgcgctcacctttggctgagctaggtgtcttaaagaagctgtgtgatca 1332 v. 43tcagcgtttccgctgtttcaattggct 12 v.2 1473 tcctaggctgctgtctgcacgggcttgttgtttgctaaatcttgggacat 1582 00 v.2 1523 tctctgctcaagatggaaatgagggggaagattccccagatgtggaccat 1572 1111111111111111 t 111111 v.1 1383 tctctgctcaagatggaaatgagggggaagattccccagatgtggaccat 1432 v. 53atacagacgtaaatgtgaattgaaagt 12 v.2 1573 attgatcaagtaactgatgacacattgatggaagaatctggaaaaatgat 1482 v.2 1623 attcctaatggacctacttaagaggctgcgagatgagggacatcaaactc 1672 v. 1 1483 attcctaatggacctacttaagaggctgcgagatgagggacatcaaactc 1532 v.2 1673 tggtgttttctcaatcgaggcaaattctaaacatcattgaacgcctctta 1722 v.1 1533 tggtgttttctcaatcgaggcaaattctaaacatcattgaacgcctctta 1582 v.2 1723 aagaataggcactttaagacattgcgaatcgatgggacagttactcatct 1772 v.1 1583 aagaataggcactttaagacattgcgaatcgatgggacagttactcatct 1632 v.2 1773 tttggaacgagaaaaaagaattaacttattccagcaaaataaagattact 1822 v.1 1633 tttggaacgagaaaaaagaattaacttattccagcaaaataaagattact 1682 v.2 1823 ctgtttttctgcttaccactcaagtaggtggtgtcggtttaacattaact 1872 v.1 1683 ctgtttttctgcttaccactcaagtaggtggtgtcggtttaacattaact 1732 v. 2 1873 gcagcaactagagtggtcatttttgaccctagctggaatcctgcaactga 1922 v.1 1733 gcagcaactagagtggtcatttttgaccctagctggaatcctgcaactga 1782 v.2 1923 tgctcaagctgtggatagagtttaccgaattggacaaaaagagaatgttg 1972 v.1 1783 tgctcaagctgtggatagagtttaccgaattggacaaaaagagaatgttg 1832 v.2 1973 tggtttataggctaatcacttgtgggactgtagaggaaaaaatatacaga 2022 v.1 1833 tggtttataggctaatcacttgtgggactgtagaggaaaaaatatacaga 1882 v.2 2023 agacaggptttcaaggactcattaataagacaaactactggtgaaaaaaa 2072 v.1 1883 agacaggttttcaaggactcattaataagacaaactactggtgaaaaaaa 1932 v.2 2073 gaaccctttccgatattttagtaaacaagaattaagagagctctttacaa 2122 v.1 1933 gaaccctttccgatattttagtaaacaagaattaagagagctctttacaa 1982 v.2 2123 tcgaggatcttcagaactctgtaacccagctgcagcttcagtctttgcat 21~72 v.1 1983 tcgaggatcttcagaactctgtaacccagctgcagcttcagtctttgcat '2032 v.2 2173 gctgctcagaggaaatctgatataaaactagatgaacatattgcctacct 2222 v.1 203gctgctcagaggaaatctgatataaaactagatgaacatattgcctacct 2082 v.2 2023gatttggtgtgaccgcagttagaaa 2272 v.1 083gcagtctttggggatagctggaatctcagaccatgatttgatgtacacat .2132 v. 23ggttgcgtagaactgttgaagattatt 22 v.1 2273 gtgatctgtctgttaaagaagagcttgatgtggtagaagaatctcactat 232 00 v.2 2323 attcaacaaagggttcagaaagctcaattcctcgttgaattcgagtctca 2372 v.1 2183 attcaacaaagggttcagaaagctcaattcctcgttgaattcgagtctca 2232 v.2 2373 aaataaagagttcctgatggaacaacaaagaactagaaatgagggggcct 2422 v.1. 2233 aaataaagagttcctgatggaacaacaaagaactagaaatgagggggcct 2282' v.2 2423 ggctaagagaacctgtatttccttcttcaacaaagaagaaatgccctaaa 2472 v.1 2283 ggctaagagaacctgtatttccttcttcaacaaagaagaaatgccctaaa 2332 v.2 2473 ttgaataaaccacagcctcagccttcacctcttctaagtactcatcatac 2522 v.1 2333 ttatacaactactcaccttatcctaa 2382 v.2 2523 tcaggaagaagatatcagttccaaaatggcaagtgtagtcattgatgatc 2572 v.1 2383 tcaggaagaagatatcagttccaaaatggcaagtgtagtcattgatgatc 2432 v.2 2573 tgcccaaagagggtgagaaacaagatctctccagtataaaggtgaatgtt 2622 v.1 2433 tgcccaaagagggtgagaaacaagatctctccagtataaaggtgaatgtt 2482 v.2 2623 accaccttgcaagatggtaaaggtacaggtagtgctgactctatagctac 2672 v.1 2483 accaccttgcaagatggtaaaggtacaggtagtgctgactctatagctac 2532 v.2 2673 tttaccaaaggggtttggaagtgtagaagaactttgtactaactcttcat 2722 v.1 2533 tttaccaaaggggtttggaagtgtagaagaactttgtactaactcttcat 2582 v.2 2723 tgggaatggaaaaaagctttgcaactaaaaatgaagctgtacaaaaagag 2772 v.1 2583 tgggaatggaaaaaagctttgcaactaaaaatgaagctgtacaaaaagag 2632 v.2 2773 acattacaagaggggcctaagcaagaggcactgcaagaggatcctctgga 2822 v.1 2633 acattacaagaggggcctaagcaagaggcactgcaagaggatcctctgga 2682 v.2 *2823 aagttttaattatgtacttagcaaatcaaccaaagctgatattgggccaa 2872 v.1 2683 aagttttaattatgtacttagcaaatcaaccaaagctgatattgggccaa 2732 v.2 2873 atttagatcaactaaaggatgatgagattttacgtcattgcaatccttgg 2922 v.1 2733 atttagatcaactaaaggatgatgagattttacgtcattgcaatccttgg 2782 v.2 2923 cccattatttccataacaaatgaaagtcaaaatgcagaatcaaatgtatc 2972 v.1 2783 cccattatttccataacaaatgaaagtcaaaatgcagaatcaaatgtatc 2832 v.2 2973 cattattgaaatagctgatgacctttcagcat~ccatagtgcactgcagg 3022 v.1 2833 cattattgaaatagctgatgacctttcagcatcccatagtgcactgcagg 2882 v.2 3023 atgctcaagcaagtgaggccaagttggaagaggaaccttcagcatcttca 3072 V.1 2883 atgctcaagcaagtgaggccaagttggaagaggaaccttcagcatcttca 2932 v.2 3073 ccacagtatgcatgtgatttcaatcttttcttggaagactcagcgacaa 3122 00 v.1 2933 ccacagtatgcatgtgatttcaatcttttcttggaagactcagcagacaa 2982 IND13cgcaatttcg~g~ttggagtagagaaa 37 v.1 3283 cagacaaaatttttccagtcagtctttagagcatgttgagaaagaaaata 3032 v.2 3173 gcttgtgtggctctgcacctaattccagagcagggtttgtgcatagcaaa 3222 v.1 3033 gcttgtgtggctctgcacctaattccagagcagggtttgtgcatagcaaa 3082 v.2 3223 acatgtctcagttgggagttttctgagaaagacgatgaaccagaagaagt 3272 v.1 3083 acatgtctcagttgggagttttctgagaaagacgatgaaccagaagaagt 3132 v.2 3273 agtagttaaagcaaaaatcagaagtaaagctagaaggattgtttcagatg 3322 v.1 3133 agtagttaaagcaaaaatcagaagtaaagctagaaggattgtttcagatg 3182 v.2 3323 gcgaagatgaagatgattcttttaaagatacctcaagcataaatccattc 3372 v.1 3183 gcgaagatgaagatgattcttttaaagatacctcaagcataaatccattc 3232 v.2 3373 aacacatctctctttcaattctcatctgtgaaacaatttgatgcttcaac 3422 v.1. 3233 aacacatctctctttcaattctcatctgtgaaacaatttgatgcttcaac 3282 v.2 3423 tcccaaaaatgacatcagtccaccaggaaggttcttttcatctcaaatac 3472 v.1 3283 tcccaaaaatgacatcagtccaccaggaaggttcttttcatctcaaatac 3332 v.2 3473 ccagtagtgtaaataagtctatgaactctagaagatctctggcttctagg 3522 v.1 3333 ccagtagtgtaaataagtctatgaactctagaagatctctggcttctagg 3382 v.2 3523 aggtctcttattaatatggttttagaccacgtggaggacatggaggaaag 3572 v.1 3383 aggtctcttattaatatggttttagaccacgtggaggacatggaggaaag 3432 v. 2 3573 acttgacgacagcagtgaagcaaagggtcctgaagattatccagaagaag 3622 v.1 3433 acttgacgacagcagtgaagcaaagggtcctgaagattatccagaagaag 3482 v.2 3623 gggtggaggaaagcagtggcgaagcctccaagtatacagaagaggatcct 3672 v.1 3483 gggtggaggaaagcagtggcgaagcctccaagtatacagaagaggatcct 3532 v.2 3673 tccggagaaacactgtcttcagaaaacaagtccagctggttaatgacgtc 3722 v.1 3533 tccggagaaacactgtcttcagaaaacaagtccagctggttaatgacgtc 3582 v.2 3723 taagcctagtgctctagctcaagagacctCtcttggtgcccctgagcctt 3772 v.1 3583 taagcctagtgctctagctcaagagacctctcttggtgcccctgagcctt 3632 V.2 3773 tgtctggtgaacagttggttggttctccccaggataaggcggCagaggct 382 2 v.1 3633 tgtctggtgaacagttggttggttctccccaggataaggcggcagaggct 3682 v.2 3823 acaaatgactatgagactcttgtaaagcgtggaaaagaactaaaagagtg 3872 v.1 3683 acaaatgactatgagactcttgtaaagcgtggaaaagaactaaaagagtg 3732 v.2 3873 tggaaaaatccaggaggccctaaactgcttagttaaagcgcttgacataa 3922 00 11111 t11111111111111111 l ID v.1 3733 tggaaaaatccaggaggccctaaactgcttagttaaagcgcttgacataa 3782 v.2 3923 aaagtgcagatcctgaagttatgctcttgactttaagtttgtataagcaa 3972 v.1 3783 aaagtgcagatcctgaagttatgctcttgactttaagtttgtataagcaa 3832 v.2 3973 cttaataacaattgagaatgtaacctgtttattgtattttaaagtgaaac 4022 v.1 3833 ctataatggagactgtattttaataa 3882 v.2 4023 tgaatatgagggaatttttgttcccataattggattctttgggaacatga 4072 v.1 3883 tgaatatgagggaatttttgttcccataattggattctttgggaacatga 3932 v.2 4073 agcattcaggcttaaggcaagaaagatctcaaaaagcaacttctgccctg 4122 v.1 3933 agcattcaggcttaaggcaagaaagatctcaaaaagcaacttctgccctg 3982 v-.2 4123 caacgccccccactccatagtctggtattctgagcactagcttaatattt 4172 v.1 3983 caacgccccccactccatagtctggtattctgagcactagcttaatattt 4032 v.2 4173 cttcacttgaatattcttatattttaggcatattctataaatttaactgt 4222 v.1 4033 cttcacttgaatattcttatattttaggcatattctataaatttaactgt 4082 v.2 4223 gttgtttcttggaaagttttgtaaaattattctggtcattcttaatttta 4272 v.1 4083 gttgtttcttggaaagttttgtaaaattattctggtcattcttaatttta 4132 v.2 4273 ctctgaaagtgatcatctttgtatataacagttcagataagaaaattaaa 4322 v.1 4133 ctctgaaagtgatcatctttgtatataacagttcagataagaaaattaaa 4182 v.2 4323 gttacttttctc 4334 v.1 4183 gttacttttctc 4194 Table LIV(a). Peptide sequences of protein coded by 273P4137 v.2 (SEQ ID NO: 113) MGLGKTVQII AFLSGMFDAS LVNHVLLIMP TNLINTWVKE FIKWTPGMRV KTFHGPSKDE RTRNLNRIQQ RNGVIITTYQ MILINNWQQLS SFRGQEFVWD YVILDEAHKI KTSSTKSAIC 120 ARAIPASNRIJ LLTGTPIQNN LQELWSLFDF ACQGSLLGTL KTIFKMEYENP ITRAREKDAT 180 PGEKALGFKI SEN1LMAIIKP YFLRRTKEDV QKKKSSN~PEA RLjNEKlNPDVD AICEMPSLSR 240 KUqDLIIWIRL VPLQEEIYRK FVSIJDHIKEL LMETRSPLAE LGVLKKLCDH PRIJLSAPACC 300 LLNLGTFSAQ DGbIEGEDSPD VDHIDQVTDD TLMEESGKMI FLMDLLKRLR DEGHQTLVFS 360 QSRQILN~IIE RLLKNRHF1CT LRIDGTVTHL LEREKRINLF QQNKDYSVFL IiTTQVGGVGL 420 TLTAATRVVI FDPSWNPATD AQAVDRVYRI GQKENVVVY. LITCGTVEEK IYRRQVFKDS 480 LIRQTTGEKK NPFRYFSKQE LRELFTIEDL QNSVTQLQLQ SLHAAQRKSD IKLDEHIAYL 540 QSIJGIAGISD HDLMYTCDLS. VKEELDVVEE SHYIQQRVQK AQFLVEFESQ NKcEFLMEQQR 600 TRNEGAWIJRE PVFPSSTKKK CPKLNKPQPQ PSPLaLSTHHT QEEDISSKMA SVVIDDLjPKE 660 GEKQDLSSIK VNVTTLQDGK GTGSADSIAT LPKGFGSVEE LCTNSSLGME KSFATKN'EAV 720 QKETLQEGPK QEALQEDPLE SFNYVLSKST KAJJIGPNIJDQ LKDDEILRHC NPWPIISITW 780 ESQNAESNVS IIEIADDLSA SHSALiQDAQA SEAKIJEEEPS ASSPQYACDF NLFLEDSADN 840 RQNFSSQSLE RVEKENSLCG SAPNSRAGFV HSXTCLSWEF SEKDDEPEEV VVKAKIRSKA 900 RRIVSDGEDE DDSFKDTSSI NPFNTSLFQF SSVKQFDAST PKNDISPPGR FFSSQIPSSV 960 NKSMNSRRSL ASRRSLINMV LDHVEDMEER LDDSSEAKGP EDYPEEGVEE SSGEASKYTE 1020 EDPSGETLSS ENKSSWLMTS KPSALAQETS LGAPEPLSGE QLVGSPQDKA AEATNDYETL 1080 cI VKRGKELKEC GKIQEALNCL VKALDIKSAD PEVMLiLTLSL YKQLNNN 1127 Table LV(a). Amino acid sequence alignment of 273P4137 v.2 (SEQ ID NO: 114) and 273P4137 v.1 (SEQ ID NO: 115) CI v.2 1 MGLGKTVQIIAFLSGMFDASLVNHVLLIMPTNLINTWVKEFIKWTPGMRV 00 v.1 124 MGLGKTVQIIAFLSGMFDASLVNH1VLLIMPTNLINTWVKEFIKWTPGMRV 173 v.2 51 KTFHGPSKDERTRNLNRIQQRNGVIITTYQMLjINNWQQLSSFRGQEFVD 1.00 v.1 174 KTFHGPSKDERTRNI 1 NR~IQQRNGVIITTYQMLINNWQQLSSFRGQEFVWD 223 v.2 101 YVILDEAHKIKTSSTKSAICARA-IPASNRLLLTGTPIQNNLQELWSLFDF 150 v.1 224 YVILDEAHKIKTSSTKSAICARAIPASNRLLLTGTPIQNNLQELWSIJFDF 273 v.2 151 ACQGSLLGTLKTFKMEYENPITRAP.BKDATPGEKAIJGFKISENLaMAI IKP 200 v.1 274 ACQGSLLGTLKTFKMEYEN~PITRAREKDATPGEKA3JGFKISENLMAI IKP 323 v.2 201 YFLRRTKEDVQKKKSSNPEARLNEKNPDVDAICEMPSLSRK=LIIWIRL 250 v.1 324 YFLRRTKEDVQKKKSSNPEARINENPDVDAICEMPSLSRKNDLIIWIRL 373 v.2 251. VPLQEEIYRKFVSLDHIKELLMETRSPLELGVLKKLCDHPRLLSARACC 300 v.1 374 VPLQEEIYRKFVSLDHIKELLMETRSPLAELGVLKKLiCDHPRLLSA.ACC 423 v.2 301 LLNLGTFSAQDGNBGEDSPDVDHIDQVTDDTLMEESGKMIFLMDLLKRLR 350 v.1 424 LLNLGTFSAQDGNEGEDSPDVDHIDQVTDDTLMEESGKMIFIJMDLLKRLR 473 v.2 351 DEdHQTILVFSQSRQILNiIERLLKNRFKTLRIDGTVTHLLEREKRINLF 400 v.1 474 DEGHQTLVFSSRQILNIIERLLIGIRHFKTLRIDGTVTHLjLEREKRINLF 523 v.2 401. QQNKDYSVFLLTTQVGGVGLTLTAATRVVIFDPSWNPATDAQAVDRVYRI 450 v.1 524 QQNKDYSVFLLTTQVGGVGLTLTATRVVIFDPSWN'PATDAQAVDRVYRI 573 v.2 451 GQKENVVVYRLITCGTVEEKIYRRQVFKDSLIRQTTGEKKUPFRYFSKQE 500 v.1 574 GQKENVVVYRIITCGTVEEKIYRRQVFKDSLIRQTTGEKKUPFRYFSKQE 623 v.2 501 LRELFTIEDLQNSVTQLQLQSLHAAQRKSDIKL 1 DEHIAYLQSLGIAGISD 550 v.1 624 LRELFTIEDLQNSVTQLQLQSLHAAQRKSDIKLDEHIAYLQSLGIAGISD 673 v.2 551 IiDLMYTCDI 4 SVKEELDVVEESHYIQQRVQKAQFLVEFESQNKEFLMEQQR 600 v.1 674 HDLMYTCDLSVKEELDVVEESHYIQQRVQKAQFLVEFESQNKEFLMBQQR 723 v.2 601 TRNEGAWLREPVFPSSTKKKCPKLNKPQPQPSPLLSTHflTQEEDISSKMA 650 V.1 724 TRNEGAWLREPVFPSSTKKKCPKLICPQPQPSPLLSTHHTQEEDISSKMA v. 2 V.1I v.2 V. 1 v. 2 V.1I v. 2 V.1I v.2 V. 1 v.2 V.1I v. 2 V.1I V. 2 V. 1 v. 2 V. 1 v. 2 V.1I 651 SVVIDDLPKEGEKQDILSS IKVNVTTLQDGKGTGSADS IATLPKGFGSVEE 774 SVVIDDLPKEGEKQDLSSIKVNVTTLQDGKGTGSADSIATLPKGFGSVEE 701 LCTNSSLGMEKSFATKNEAVQKETLQEGPKQEALQEDPLESFNYVTJSKST 824 LCTNSSLGMEKSFATKNEAVQKETLQEGPKQEALQEDPLESFNYVLSKST 751 KADIGPNLDQLKDDEILRHCNPWPI IS ITNESQNAESNVS IIEIADDLSA 874 KDIGPNLDQLKDEILRCNPWPIISITNESQNAESNVSIIEIADDLSA 801 SHSALQDAQASEAKLjEEEPSASSPQYACDFNLFLEDSAlI'RQNFSSQSLE 924 SHSALQDAQASEAKLiEEEPSASSPQYACDFNLFLEDSAlNRQN~FSSQSLE 851 HVEKENSIJCGSAPISRAGFVHSKTCI.SWEFSEKDDEPEEVVVKAIRSKA 974 HVEKENSTLCGSAPNSRAGFVHSKTCLSWEFSEKDDEPEEVVVKAKIRSKA 901 RRIVSDGEDEDDSFKDTSSINPFNTSLFQFSSVKQFDASTPK=DISPPGR 1024 RRIVSDGEDEDDSFKDTSSINPFNTSLFQFSSVKQFDASTPKNDISPPGR 951 FFSSQIPSSVNKSMNSRRSLASRRSLINVLDHVEDMEER.LDDSSEAXGP 1074 FFSSQIPSSVNKSMNSRRSASRSLINMVLDHVEDMEERJDDSSEAKGP 1001 EDYPEEGVBESSGEASKYTEEDPSGETLSSENKSSWLMTSKPSALAQETS 1124 EDYPEEGVEESSGEASKYTEEDPSGETLSSENKSSWLiMTSKPSALAQETS 1051 LGAPEPLSGEQLVGSPQDKAAEATNDYETJVKRGKELKECGKIQEAIJNCL 1174 LGAPEPIJSGEQLVGSPQDKAAEATNDYETLVKRGKELKECGKIQEALNCL 1101 VKALDIKSADPEVMLLTLSLYKQLNNN 1127 1224 VKALDIKSAflPEVMLLTLSJYKQLNNN 1250 700 823 750 873 800 923 850 973 900 1023 950 1073 1000 1123 1050 1173 1100 1223 Table 1.1I(b). Nucleotide sequence of transcript variant 273154137 v.9 (SEQ ID NO: 116 aaaatgaatc atgtgctgct gatcatgcca accaatctta ttaacacttg ttcatcaagt ggactccagg cggaccagaa acctcaatcg atgttaatca ataactggca tatgtcatcc tcgatgaagc gctcgtgcta ttcctgcaag ttacaagaac tatggtccct aaaactttta agatggagta ccaggagaaa aagccttggg tattttctca ggaggactaa agacttaatg aaaagaatcc.

aaaaatgatt taattatttg tttgtgtctt tagatcatat ctaggtgtct taaagaagct ttgctaaatc ttgggacatt gtggaccata ttgatcaagt ttcctaatgg acctacttaa caatcgaggc aaattctaaa ttgcgaatcg atgggacagt aatgggagtc gattcagcaa gcaactttca acataaaata taatcgcctc atttgatttt tgaaaatcct atttaaaata agaagacgta agatgttgat gatacgactt caaggagttg gtgtgatcat ctctgctcaa aactgatgac gaggctgcga catcattgaa tactcatctt aaaacctttc atggtcctag aggaatggtg agctttaggg aaaacctcat c tc ctcacag gcttgtcaag attactagag tctgaaaact cagaagaaaa gccatttgtg gtgcctttac ctaatggaga cctaggctgc gatggaaatg acattgatgg gatgagggac cgcctcttaa ttggaacgag ttattatcac gccaagagtt ctactaagtc gaaccccaat ggtccctgct caagagagaa taatggcaat agtcaagcaa aaatgccttc aagaagaaat cgcgctcacc tgtctgcacg agggggaaga aagaatctgg atcaaactct agaataggca aaaaaagaat ggtaaaagaa caaggatgaa tacataccaa tgtgtgggac agcaata tgt ccagaataat gggaacatta ggatgctacc cataaaaCc cccagaggcc cctttccagg atacaggaaa tttggctgag ggcttgttgt ttccccagat aaaaatgata ggtgttttct ctttaagaca taacttattc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 cagcaaaata acattaactg gctcaagctg ctaatcactt ttaataagac ttaagagagc tctttgcatg Cagtctttgg gttaaagaag gctcaattcc actagaaatg tgccctaaat caggaagaag ggtgagaaac ggtacaggta ctttgtacta Caaaaagaga agttttaatt ctaaaggatg gaaagtcaaa tcccatagtg gcatcttcac agacaaaaft tctgcaccta tctgagaaag agaaggattg aatc Catt Ca cccaaaaatg aataagtcta ttagaccacg gaagattatc gaggatcctt aagcctagtg cagttggttg gtaaagcgtg gttaaagcgc tataagcaac gaatatgagg ttaaggcaag ctggtattct attctataaa ttaattttac ttacttttct aagattactc cagcaactag tggatagagt gtgggactgt aaactactgg tctttacaat ctgctcagag ggatagctgg agcttgatgt tcgttgaatt agggggcc tg tgaataaacc atatcagttc aagatctctc gtgctgactc actcttcatt cattacaaga atgtacttag atgaggtttt atgcagaatc cactgcagga cacagtatgc tttccagtca attccagagc acgatgaacc tttcagatgg acacatctct acatcagtcc tgaactctag tggaggacat cagaagaagg ccggagaaac ctctagctca gttcccccca gaaaagaact ttgacataaa ttaataacaa gaatttttgt aaagatctca gagcactagc tttaactgtg tctgaaagtg

C

tgtttttctg agtggtcatt ttaccgaatt agaggaaaaa tgaaaaaaag cgaggatctt gaaatctgat aatctcagac ggtagaagaa cgagtctcaa gctaagagaa acagcctcag caaaatggca cagtataaag tatagctact gggaatggaa ggggcctaag caaatcaacc acgtcattgc aaatgtatcc tgctcaagca atgtgatttc gtctttagag agggtttgtg agaagaagta cgaagatgaa ctttcaattc accaggaagg aagatctctg ggaggaaaga ggtggaggaa actgtcttca agagacctct ggataaggcg aaaagagtgt aagtgcagat ttgagaatgt tcccataatt aaaagcaact ttaatatttc ttgtttcttg atcatctttg cttaccactc tttgacccta ggacaaaaag atatacagaa aaccctttcc cagaactctg ataaaactag catgatttga tctcactata aataaagagt cctgtatttc ccttcacctc agtgtagtca gtgaatgtta ttaccaaagg aaaagctttg caagaggcac aaagctgata .aatccttggc attattgaaa agtgaggcca aatcttttct catgttgaga catagcaaaa gtagttaaag gatgattctt tcatctgtga ttcttttcat gcttctagga cttgacgaca agcagtggcg gaaaacaagt CttggtgCCC gcagaggcta ggaaaaatcc cctgaagtta aacctgttta ggattctttg tctgccctgc ttcacttgaa gaaagttttg tatataacag aagtaggtgg gctggaatcc agaatgt tgt gacaggtttt gatattttag taacccagct atgaacatat tgtacacatg ttcaacaaag tcctgatgga.

cttcttcaac ttctaagtac ttgatgatct ccaccttgca ggtttggaag caactaaaaa tgcaagagga ttgggccaaa ccattatttc tagctgatga agttggaaga.

tggaagactc aagaaaatag ca tg t Ct cag Caaaaatcag ttaaagatac aacaatttga ctcaaatacc ggtctcttat gcagtgaagc aagcctccaa ccagctggtt ctgagccttt caaatgacta aggaggccct tgctcttgac ttgtatttta ggaacatgaa aacgCccc tattcttata taaaattatt ttcagataag tgtcggttta tgcaactgat ggtttatagg caaggactca taaacaagaa.

gcagcttcag tgcctacctg tgatctgtct ggttcagaaa, acaacaaaga aaagaagaaa tcatcatact gcccaaagag agatggtaaa tgtagaagaa.

tgaagctgta tcctctggaa tttagatcaa.

cataacaaat C Ctt t Cagca, ggaacct tCa agCagacaac Cttgtgtggc ttgggagttt aagtaaagct ctcaagcata tgcttcaact cagtagtgta taatatggtt aaagggtcct gtatacagaa aatgacgtct gtctggtgaa tgagactctt aaactgctta tttaagtttg aagtgaaact gcattcaggc actccatagt ttttaggcat ctggtcattc aaaattaaag 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3671 Table 1.111(b). Nucleotide sequence alignment of 273P4137v.9 (SEQ ID NO: 117) and 273P4137v.1 (SEQ ID NO: 118) v.1 501 tttCCggtatgtttgatgcatCacttgtgaatcatgtgCtgCtgatcatg 550 V.9 1 aaaa tgaatcatgtgctgctgatcatg 27 v.1 551 CCaaCCaatCttattaaCaCatgggtaaaagaattCatCaagtggactcc 600 v.9 28 CCaaCCaatcttattaaCaCttgggtaaaagaattcatcaagtggactcc 77 v.1 601 aggaatgagagtcaaaacctttCatggtCctagCaaggatgaacgga~cc 650 v.9 78 aggaatgggagtcaaaaCCtttcatggtcctagCaaggatgaacggacca 127 v.1 651 gaaccacgtcgaagatggttaccaaa 700 V.9 128 gaaccacgtcgaagatggttaccaaa 177 v.1 701 caaatgttaatcaataactggcagcaactttcaagctttaggggccaaga 750 III I 11111 Htiiiitllllililit illll tIillIII V.9 178 caaatgttaatcaataactggcagcaactttcaagctttaggggccaaga 227 v.1 751 gtttgtgtgggactatgtcatcctcgatgaagcacataaaataaaaacct 800 V.9 228 gtttgtgtgggactatgtcatcctcgatgaagcacataaaataaaaacct 277 v.1 801 catctactaagtcagcaatatgtgctcgtgctattcctgcaagtaatcgc 850 v.9 278 catctactaagtcagcaatatgtgctcgtgctattcctgcaagtaatcgc 327 v.1 851 ctcctcctcacaggaa~cccaatccagaataatttacaagaactatggtc 900 V.9 328 ctcctcctcacaggaaccccaatccagaataatttacaagaactatggtc 377 v.1 901 cctatttgattttgcttgtcaagggtccctgctgggaacattaaaaactt 950 00 V.9 378 cctatttgattttgcttgtcaagggtccctgctgggaacattaaaaactt 427 v.1 951 ttaagatggagtatgaaaatcctattactagagcaagagagaaggatgct 1000 V.9 428 ttaagatggagtatgaaaatcctattactagagcaagagagaaggatgct 477 V. 01accgggaagctgattaaacgaactagc V.1 1001 accccaggagaaaaagccttgggatttaaaatatctgaaaacttaatggc 1507 v.1 1051 aatcataaaaccctattttctcaggaggactaaagaagacgtacagaaga 1100 V.9 528 aatcataaaaccctattttctcaggaggactaaagaagacgtacagaaga 577 v.1 1101.aaaagtcaagcaa~ccagaggccagacttaatgaaaagaatccagatgtt 1150 V.9 578 aaaagtcaagcaacccagaggccagacttaatgaaaagaatccagatgtt 627 v.1 1151 gatgccatttgtgaaatgccttccctttccaggaaaaatgatttaattat 1200 V.9 628 gatgccatttgtgaaatgccttccctttccaggaaaaatgatttaattat 677 v.1 1201 ttggatacgacttgtgcctttacaagaagaaatatacaggaaatttgtgt 1250 V.9, 678 ttggatacgacttgtgcctttacaagaagaaatatacaggaaatttgtgt 727 v.1 1251 ctttagatcatatcaaggagttgctaatggagacgcgctcacctttggct, 1300 V. 9 728 ctttagatcatatcaaggagttgctaatggagacgcgctcacctttggct 777 v.1 1301 gagctaggtgtcttaaagaagctgtgtgatcatcctaggctgctgtctgc 1350 v.9 778 gagctaggtgtcttaaagaagctgtgtgatcatcctaggctgctgtctgc 827 v.1 1351 acgggcttgttgtttgctaaatcttgggacattctctgctcaagatggaa 1400 V. 9 828 acgggcttgttgtttgctaaatcttgggacattctctgctcaagatggaa, 877 v.1 1401 atgagggggaagattccccagatgtggaccatattgatcaagtaactgat 1450 V.9 878 atgagggggaagattccccagatgtggaccatattgatcaagtaactgat 927 v.1 1451 gacacattgatggaaLgaatctggaaaaatgatattcctaatggacctact 1500 V.9 928 gacacattgatggaagaatctggaaaaatgatattcctaatggacctact 977 V.1 1501 taagaggctgcgagatgagggacatcaaactctggtgttttctcaatcga 1550 V.9 978 taagaggctgcgagatgagggacatcaaactctggtgttttctcaatcga 1027 v.1 1551 ggcaaattctaaacatcattgaacgcctcttaaagaataggcactttaag 1600 v.9 1028 ggcaaattctaaacatcattgaacgccecttaaagaataggcactttaagj 1077 V.1 1601 actggacaggcgtctacttgaggaaa 1650 v.9 1078 acattgcgaatcgatgggacagttactcatcttttggaacgagaaaaaag 12 v.1 1651 aatatatcgaatagatccgttcgtac 1700 V.9 1128 aatatatcgaatagatccgttcgtac 1177 00 v.1 1701 ctcaagtaggtggtgtcggtttaacattaactgcagcaactagagtggtc 1750 1111 Il1111 ii111111 11111111 V.9 1178 ctcaagtaggtggtgtcggtttaacattaactgcagcaactagagtggtc 1227 v.1 1751 atttttgaccctagctggaatcctgcaactgatgctcaagctgtggatag 1800 V.9 1228 atttttgaccctagctggaatcctgcaactgatgctcaagctgtggatag 1277 v.1 1801 agtacatgaaaaaaagttgtaagtac 1850 V.9 1278 agtacatgaaaaaaagttgtaagtac 1327 v.1 1851 cttgtgggactgtagaggaaaaaatatacagaagacaggttttcaaggac 1900 V.9 1328 ctgggcgaagaaaatcgaaagttaga 1377 v.1 1901 tctataaaatcgtaaaagacttcaat 1950 V.9 1378 tctataaaatcgtaaaagacttcaat 1427 v.1 1951 tatacaatagggtttaatggactaac 2000 V.9 1428 tatacaatagggtttaatggactaac 1477 v.1 2001 cttacacgactatttgagtccggaac 2050 V.9 1478 cttacacgactatttgagtccggaac 1527 v.1 2051 gaaaacaagaaatccacgatttggtg 2100 V.9 1528 gaaaacaagaaatccacgatttggtg 1577 v.1 2101 tgattaactatgttaaaggtttttaa 2150 V.9 1578 tgattaactatgttaaaggtttttaa 1627 v.1 2151 aaactaggtgaattccaatacaggta 2200 V.9 1628 aaactaggtgaattccaatacaggta 1677 v.1 2201 aagtatctgtatcagccaaaaatcta 2250 V.9 1678 aagtatctgtatcagccaaaaatcta 1727 v.1 2251 ggaacagatgatagggcgcaggacga 2300 v.9 1728 ggaacagatgatagggcgcaggacga 1777 v.1 2301 ttcttcaaaagatcctatgaaaccgc 2350 v.9 1778 ttccttcttcaacaaagaagaaatgccctaaattgaataaaccacagcct 1827 v.1 .2351 cagccttcacctcttctaagtactcatcatactcaggaagaagatatcag 2400 V.9 1828 cagccttcacctcttctaagtactcatcatactcaggaagaagatatcag 1877 v.1 2401 ttccaaaatggcaagtgtagtcattgatgatctgcccaaagagggtgaga 2450 V.9 1878 ttccaaaatggcaagtgtagtcattgatgatctgcccaaagagggtgaga 1927 v.1 2451 aacaagatctctccagtataaaggtgaatgttaccaccttgcaagatggt 2500 00 v.9 1928 aaagtttcgaaaggattacctgagtg 1977 v.1 2501 aagtcgtggtatttacattcaaggtg 2550 v.9 1978 aagtcgtggtatttacattcaaggtg 2027 v.1 2551 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 2600 v.9 2028 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 2077 v.1 2601 ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 2650 v.9 2078 ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 2127 v.1 2651 aagcaagaggcactgcaagaggatcctctggaaagttttaattatgtact 2700 V.9 2128 aagcaagaggcactgcaagaggatcctctggaaagttttaattatgtact 2177 v.1 2701 tagcaaatcaaccaaagctgatattgg9ccaaatttagatcaactaaagg 2750 V.9 2178 tagcaaatcaaccaaagctgatattgggccaaatttagatcaactaaagg 2227 v.1 2751 atgatgagattttacgtcattgcaatccttggcccattatttccataaca 2800 V.9 2228 atgatgaggttttacgtcattgcaatccttggcccattatttccataaca 2277 v.1 2801 aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 2850 V.9 2278 aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 2327 v.1 2851 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 2900 V.9 2328 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 2377 v.1 2901 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 2950 v.9 2378 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 2427 *v.1 2951 ttcaatcttttcttggaagactcagcagacaacagacaaaattttt0cag 3000 v.9 2428 ttcaatcttttcttggaagactcagcagacaacagacaaaatttttccag 2477 v.1 3001 tcagtctttagagcatgttgagaaagaaaatagcttgtgtggctctgcac 3050 V.9 2478 tcagtctttagagcatgttgagaaagaaaatagcttgtgtggctctgcac 2527 v.1 3051 ctaattccagagcagggtttgtgcatagcaaaacatgtctcagttgggag 3100 111111111 l11111111111111111.1111111 ill I I1 III v.9 2528 ctaattccagagcagggtttgtgcatagcaaaacatgtctcagttgggag 27 2577 v.1. 3101 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat 3150 V.9 2578 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat 2627 v.1 3151 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt 3200 V.9 2628 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt 2677 v.1 3201 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa 3250 V.9 2678 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa 2727 v.1 3251 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag 3300 00lilll 111ll11111ii iili iiIIli ID V.9 2728 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag 2777 C1 v.1 3301 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt 3350 V.9 2778 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt 2827 v.1 3351 ctatgaactctagaagatctctggcttctaggaggtctcttattaatatg 3400 V. 9 2828 ctatgaactctagaagatctctggcttctaggaggtctcttattaatatg 2877 v.1 3401 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 3450 V.9 2878 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 2927 v. 1 3451 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg 3500 V. 9 2928 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg 2977 v.1 3501 gcgaagcctccaagtatacagaagaggatccttccggagaaacactgtct 3550 V.9 2978 gcgaagcctccaagtatacagaagaggatccttccggagaaacactgtct 3027 v. 1 3551 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 3600 V.9 3028 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 3077 v.1 3601 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 3650 v.9 3078 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 3127 v.1 3651 ttggttctccccaggataaggcggcagaggctacaaatgactatgagact 3700 V.9 3128 ttggttccccccaggataaggcggcagaggctacaaatgactatgagact 3177 v.1 3701 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 3750 V.9 3178 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 3227 v.1 3751 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag 3800 V.9 3228 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag 3277 v.1 3801 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 3850 V.9 3278 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 3327 v.1 3851 tgtaacctgtttattgtattttaaagtgaaactgaatatgagggaatttt 3900 v.9 3328 tgtaacctgtttattgtattttaaagtgaaactgaatatgagggaatttt v.1 3901 tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc v.9 3378 tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc V.1 3951 aagaaagatctcaaaaagcaacttctg~cctgcaacgccccccactccat v.9 3428 aagaaagatctcaaaaagcaacttctgccctgcaacgccccactccat V.1 4001 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt v.9 3478 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt V.1 4051 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt V.9 3528 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt 3377 3950 3427 4000 3477 4050 3527 4100 3577 v.1 4101 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 4150 V.9 3578 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 3627 v.1 4151 ttgtatataacagttcagataagaaaattaaagttacttttctc V.9 3628 ttgtatataacagttcagataagaaaattaaagttacttttctc 4194 3671 Table LIV(b). Peptide sequences of protein coded by 273P4137 v.9 (SEQ ID NO: 119) MNHVLLIMPT NTJINTWVKEF IKWTPGMGVK TFHGPSKDER TRNLNRIQQR LINNWQQLSS FRGQEFVWDY VILDEAHKIK TSSTKSAICA RAIPASNRLL QELWSLFDFA CQGSLLGTLK TFKMEYENPI TRAREKDATP GEKAILGFKIS FLRRTKEDVO KKKSSNPEAR LNEKNPDVDA ICEMPSLSRK NDLIIWIRLV VSILDHIKELL METRSPLAEL GVLKKLCDHP RLLSARACCL LNLGTFSAOD DHIDQVTDDT LMEESGKMIF LMDLLKRLRD EGHQTLVFSQ SRQILNI IER RIDGTVTHLL EREKRINLFQ QNKDYSVFLL TTQVGGVGLT LTAATRVVIF QAVDRVYRIG QKENVVV'IRL ITCGTVEEKI YRRQVFKDSL IRQTTGEKKU~ RELFTIEDLQ 1NSVTQLQLQS LHAAQRKSDI KLDEHIAYLQ SLGIAGISDH KEELDVVEES HYIQQRVQKA QFLVEFESQN KEFLMEQQRT RNEGAWLREP PKLNKPQPQP SPLLjSTHHTQ EEDISSKMAS VVIDDLPKEG EKQDLSSIKV TGSADSIATL PKGFGSVEEL CTNSSLGMEK SFATKNEAVQ KETLQEGPKQ FNYVLSKSTK ADIGPNLDQL KDDEVLRHCN PWPIIS ITNE SQNAESNVSI HSALQDAQAS EAKLEEEPSA SSPQYACDFN LFLEDSAflNR QNFSSQSLEH APNSRAGFVH SKTCLSWEFS EKDDEPEEVV VKAKIRSKAR RIVSDGEDED PFNTSLFQFS SVKQFDASTP KNDISPPGRF FSSQIPSSVN KSMNSRRSLA DHVEDMEERL DDSSEAKGPE DYPEEGVEES SGEASKYTEE DPSGETLSSE PSALiAQETSL GAPEPLSGEQ LVGSPQDKAA EATNDYETLV KRGKELKECG KALDIKSADP EVMLLTLjSLY KQIfLN NGVI ITTYQM LTGTPIQNNLi ENLMAI IKPY

PLQEEIYRKF

GNEGEDS PD V LLKN1RHFKTL

DPSWNPATDA

PFRYFSKQEL

DLMYTCDLSV

VFPSSTKKKC

NVTTLQDGKG

EALQEDPLES

IEIADDLSAS

VEKENSLCGS

DSFKDTSSIN

SRRSLINMVL

NKSSWLMTSI(

KIQEALNCLV

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1106 Table LV(b). Amino acid sequence alignment of 273P4137 v.9 (SEQ ID NO: 120) and 273P4137 v.1 .(SEQ ID NO: 121) v.1 101 KEGIAFLYSLYRDGRKGGILAflDMGIGKTVQIIALSGMFDASLKRLL 150 V.9 1 MNHVLL 6 v.1 151 IMPTNLINTWVKEFIKWTPGMRVKTFHGPSKDERTRNLNRIQQNGVIIT 200 V.9 7 IMPTNLINTWVKEFIKWTPGMGVKTFGPSKDERTNLNRIQQNGVIIT 56 v.1 201 TYQMLINQQLSSFRGQEFVWDYVILDEAHKIKTSSTKSAICRAIPAS 250 V.9 57 TYQMLINNWQQLSSFRGQEFVWDYVILDEAHKIKTSSTKSAIRAPA 106 v.1 251 NLLLTGTPQN~NLQELWSLFDFACQGSLLGTLKFKMEYENPITRREK 300- V.9 107 NRLLLTGTPIQNNLQELWSLFDFACQGSLLGTLKTFMEYE1NPITRAREK 156 v.1l 301 DATPGEKALGFKISENAIIKPYFLRTKDVQKKKSSPEARLNEKNP 350 V.9 157 DATPGEKALGFKISENLMAIIKPYFLRRTKEDVQKKKSSNPEARLNTEKNP 206 v.1 351 DVDAICEMPSLSRKNDLIIWIRLVPLQEEIYRKFVSLDHIKELLMETRSP 400 C1 v.9 207 DVDAICEMPSLSRKMDLIIWIRLVPLQEEIYRKFVSLDHIKELLMETRSP 256 v.1 401 LAELGVLKKLCDHPRLLSARACCLLNLGTFSAQDGNEGEDSPDVDHIDQV 450 0 V.9 257 LAELGVLKKLCDHPRLLSAPACCLLNLGTFSAQDGNEGEDSPDVDHIDQV 306 00 0 v.1 451 TDDTLMEESGKMIFLMDLLKRLRDEGHQTLVFSQSRQILNIIERLLNR 500 V.9 307 TDDTLMEESGKMIFLMDLLKRLRDEGHQTLVFSQSRQILNIIERLLKNRH 356 v.1 501 FKTLRIDGTVTHLLEREKRINLFQQ.KYSVFLLTTQVGGVGLTLTAATR 550 V.9 357 FKTLRIDGTVTHLLEREKRINFQQNKDYSVFLLTTQVGGVGLTLTAATR 406 v.1 551 VVIFDPSNNPATDAQAVDRVYRIGQKENVVVYRLITCGTVEEKIYRRQVF 600 V.9 407 VVIFDPSWNPATDAQAVDRVYRIGQKENVVVYRLITCGTVEEKIYRRQVF 456 v.1 601 KDSLIRQTTGEKKNPFRYFSKQELR ELFTIEDLQNSVTQLQLQSLHAAQR 650 V.9 457 KDSLIRQTTGEKKNPFRYFSKQELRELFTIEDLQNSVTQLQLQSLHAAQR 506 v.1 651 KSDIKLDEHIAYLQSLGIAGISDDLMYTCDLSVKELDVVEESHYIQQR 700 V. 9 507 KSDIKLDEHIAYLQSLGIAGISDHDLMYTCDLSVKEELDVVEESHYIQQR 556 v.1 701 VQKAQFLVEFESQNKBFLMEQQRTRNEGAWLREPVFPSSTKKKCPKJNKP 750 V.9 557 VQKAQFLVEFESQNKEFLMEQQRTRNEGAWLREPVFPSSTKKCCPKLNKP 606 v.1 751 QPQPSPLLSTHITQEEDISSKv1ASVVIDDLPKEGEKQDLSSIKV1NTTLQ 800 V.9 607 QPQPSPLLSTHHTQEEDISSKMASVVIDDLPKEGEKQDLSSIKVNVTTLQ 656 v.1 801 DGKGTGSADSIATLPKGFGSVEELCTNSSLGMEKSFATKNEAVQKETLQE 850 .9 657 DGKGTGSADSIATLPKGFGSVEELCTNSSLGMEKSFATKNEAVQKETLQE 706 v.1 851 GPKQEALQEDPLESFNYVLSKSTKAflIGPNLDQLKDDEILRHCNPWPIIS 900 V.9 707 GPKQEALQEDPLESFNYVLSKSTKAflIGPNLDQLjKDDEVTLRHCPWPIIS 756 v.1 901 ITNESQNAESNVSIIEIADDLSASHSALQDAQASEAKLEEEPSASSPQYA 950 V.9 757 ITNESQN~AESNVSIIEIAfDLSASHSALQDAQASEAKLEEEPSASSPQYA 806 v.1 951 CDFNLFLEDSADNRQNFSSQSLEVEKEN~SLCGSAPNSRAGFVSKTCLS 1000 V.9 807 CDFNLFLEDSADNRQNFSSQSLEHVEKENSLCGSAPNSRAGFVHSKTCLS 856 v.1 1001 WEFSEKDDEPEEVVVKAKIRSKARRIVSDGEDEDDSFKDTSSflNPFNTSL 1050 V.9 857 WEFSEKDDEPEEVVVKAKIRSKARRIVSDGEDEDDSFKDTSSINPFNTSL 906 V. 1 1051 FQFSSVKQFDASTPKN~DISPPGRFFSSQIPSSVNKSMNSRRSLASRRSLI V.9 907 FQFSSVKQFDASTPKNDISPPGRFFSSQIPSSVNKSM1NSRRSLASRRSLI v.1 1101 NMVLDHVEDMEERLDDSSEAKGPEDYPEEGVEESSGEASKYTEEDPSGET V.9 957 NMVLDHVEDMEERLDDSSEAKGPEDYPEEGVEESSGEASKYTEEDPSGET v.1 1151 LSSENKSSWLI4TSKPSALAQETSLGAPEPIJSGEQLVGSPQDK-AAEATNDY V.9 1007 LSSENKSSWLMTSKPSALAQETSLGAPEPLSGEQLVGSPQDKAAEATNDY v.1 1201 ETLVKRGKELKECGKIQEALN~CLVKA~LDIKSADPEVMLLTLSLYKQLNNN v.9 1057 ETLjVKRGKELKECGKIQEALNCLVKAXDIKSAfPEVMLLTLSLYKQLNN Table 1-I1(c). Nucleotide sequence of transcript variant 273P4137 v.1 0 (SEQ ID NO: 122) 1100 956 1150 1006 1200 1056 1250 1106 tcattaataa gacaaactac tggtgaaaaa gaattaagag agctctttac aatcgaggat cagtctttgc atgctgctca gaggaaatct ctgcagtctt tggggatagc tggaatctca tctgttaaag aagagcttga tgtggtagaa aaagctcaat tcctcgttga attcgagtct agaactagaa atgagggggc ctggctaaga aaatgcccta aattgaataa accacagcct actcaggaag aagatatcag ttccaaaatg gagggtgaga aacaagatct ctccagtata taaggtacag gtagtgctga ctctataact gaactttgta ctaactcttc attgggaatg gtacaaaaag agacattaca agaggggcct gaaagtttta attatgtact tagcaaatca caactaaagg atgatgagat tttacgtcat aatgaaagtc aaaatgcaga atcaaatgta gcatcccata gtgcactgca ggatgctcaa tcagcatctt caccacagta tgcatgtgat aacagacaaa atttttccag tcagtcttta ggctctgcac ctaattccaa agcagggttt ttttctgaga aagacgatga accagaagaa gctagaagga ttgtttcaga tggcgaagat ataaatccat tcaacacatc tctctttcaa actcccaaaa atgacatcag tccaccagga gtaaataagt ctatgaactc tagaagatct gttttagacc acgtggagga catggaggaa cctgaagatt atccagaaga aggggtggag gaagaggatc cttccggaga aacactgtct tctaagccta gtgctctagc tcaagagacc gaacagttgg ttggttctcc ccaggataag cttgtaaagc. gtggaaaaga actaaaagag ttagttaaag cgcttgacat aaaaagtgca ttgtataagc aacttaataa caattgagaa actgaatatg agggaatttt tgttcccata ggcttaaggc aagaaagatc tcaaaaagca agtctggtat tctgagcact agcttaatat catattCtat aaatttaact gtgttgtttc ttcttaattt tactctgaaa gtgatcatct aagttacttt tctc aagaaccctt cttcagaact gatataaaac gaccatgatt gaatctcact caaaataaag gaacctgtat cagccttcac gcaagtgtag aaggtgaatg actttaccaa gaaaaaagct aagcaggagg accaaagctg tgcaatcctt tccattattg gcaagtgagg ttcaatcttt gagcatgttg gtgcatagca gtagtagtta gaagatgatt ttctcatctg aggttctttt ctggcttcta agacttgacg gaaagcagtg tcagaaaaca tctcttggtg gcggcagagg tgtggaaaaa gatcctgaag tgtaacctgt attggattct acttctgcc ttcttcactt ttggaaagtt ttgtatataa tccgatattt ctgtaaccca tagatgaaca tgatgtacac atattcaaca agttcctgat ttccttcttc ctcttctaag tcattgatga ttaccaczctt aggggtttgg ttgcaactaa cactgcaaga atattgggcc ggcccattat aaatagctga ccaagttgga tcttggaaga agaaagaaaa aaacatgtct aagcaaaaat cttttaaaga tgaaacaatt catctcaaat ggaggtctct acagcagtga gcgaagcctc agtccagctg cccctgagcc ctacaaatga tccaggaggc ttatgctctt ttattgtatt ttgggaacat tgcaaccc gaatattct ttgtaaaatt cagttcagat tagtaaacaa gctgcagctt tattgcctac atgtgatctg aagggttcag ggaacaacaa aacaaagaag tactcatcat tctgcccaaa gcaagatggg aagtgtagaa aaatgaagct ggatcctctg aaatttagat ttccataaca tgacctttca agaggaacct ctcagcagac tagcttgtgt cagttgggag cagaagtaaa tacctcaagc tgatgcttca acccagtagt tattaatatg agcaaagggt caagtataca gttaatgacg tttgtctggt ctatgagact cctaaactgc gactttaagt ttaaagtgaa gaagcattca cccactccat atattttagg attctggtca aagaaaatta 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2294 Table LIII(c). Nucleotide sequence alignment of 273P4137 v.10 (SEQ ID NO: 123) and 273P4137 v.1 (SEQ ID NO: 124) v.1 1901 tcattaataagacaaactactggtgaaaaaaagaaccctttccgatattt 1950 1 tcattaataagacaaactactggtgaadaaaagaaccctttccgatattt v.1 1951 tagtaaacaagaattaagagagctctttacaatcgaggatcttcagaact 2000 51tagtaaacaagaattaagagagctctttacaatcgaggatcttcagaact v.1 2001 ctgtaacccagctgcagcttcagtctttgcatgctgctcag&99aaatct 2050 101 ctgtaacccagctgcagcttcagtctttgcatgctgctcagaggaaatct 150 C1 v.1 2051 gatataaaactagatgaacatattgcctacctgcagtctttggggatagc 2100 151 gatataaaactagatgaacatattgcctacctgcagtctttggggatagc 200 v.1 2101 tggaatctcagaccatgatttgatgtacacatgtgatctgtctgttaaag 2150 00 I1111111111lillilllllliltl ID v.10 201 tggaatctcagaccatgatttgatgtacacatgtgatctgtctgttaaag 250 v.1 2151 aagagcttgatgtggtagaagaatctcactatattcaacaaagggttcag 2200 251 aagagcttgatgtggtagaagaatctcactatattcaacaaagggttcag 300 agtatccctgatggccaataggtcgt 2201 aaagctcaattcctcgttgaattcgagtctcaaaataaagagttcctgat 2250 3051 gaaccaacgctgtaatgagtggctcataaagagaacctgtat 2300 2251 ggaacaacaaagaactagaaatgagggggcctggctaagagaacctgtat 2300 v.1 2302. ttccttcttcaacaaagaagaaatgccctaaattgaataaaccacagcct 2350 401 ttccttcttcaacaaagaagaaatgccctaaattgaataaaccacagcct 450 v.1 2351 cagccttcacctcttctaagtactcatcatactcaggaagaagatatcag 2400 4521 cagccttcacctcttctaagtactcatcatactcaggaagaagatatcag 500 v.1 2401 ttccaaaatggcaagtgtagtcattgatgatctgcccaaagagggtgaga 2450 501 ttccaaaatggcaagtgtagtcattgatgatctgcccaaagagggtgaga 550 v.1 2451 aacaagatctctccagtataaaggtgaatgttaccaccttgcaagatggt 2500 551 aacaagatctctccagtataaaggtgaatg ttaccacctt-gcaagatggg 600 v.1 2501 aaaggtacaggtagtgctgactctatagctactttaccaaaggggtttgg 2550 601 taaggtacaggtagtgctgactctataactactttaccaaaggggtttgg 650 v.1 2551 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 2600 651 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 700 v.1 2602. ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 2650 701 ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 750 v.1 2651 aagcaagaggcactgcaagaggatcctctggaaagttttaattatgtact 2700 751 aagcaggaggcactgcaagaggatcctctggaaagttttaattatgtact 800 v.1 2701 tagcaaatcaaccaaagctgatattgggccaaatttagatcaactaaagg 2750 I HIM11111 III1111 111 HIM i tII Ill I III I111 I I I I 801 tagcaaatcaaccaaagctgatattgggccaaatttagatcaactaaagg 850 v.1 2751 atgatgagattttacgtcattgcaatccttggcccattatttccataaca 2800 851atgatgagattttacgtcattgcaatccttggcccattatttccataaca v.1 2801 aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 2850 901 aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 950 v.1 2851 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 2900 951 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 1000 v.1 2901 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 2950 00 tl! 111111111111 111li! 111 NO v.10 1001 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 1050 v.1 2951 ttcaatcttttcttggaagactcagcagacaacagacaaaatttttccag 3000 1051 ttcaatcttttcttggaagactcagcagacaacagacaaaatttttccag 1100 V. 01tatttaacttggaaaatgtggtgttcc 3001 tcagtctttagagcatgttgagaaagaaaatagcttgtgtggctctgcac 3050 v.1 3051 ctaattccagagcagggtttgtgcatagcaaaacatgtctcagttgggag 3100 1151 ctaattccaaagcagggtttgtgcatagcaaaacatgtctcagttgggag 1200 v.1 3101 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat 3150 1201 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat 1250 v.1 3151 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt 3200 1251 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt. 1300 v.1 3201 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa 3250 1301 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa. 1350 v.1 3251 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag 3300 1351 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag, 1400 v.1 3301 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt 3350 1401 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt ,1450 v.1 3351 ctatgaactctagaagatctctggcttctaggaggtctcttattaatatg ,3400 1451 ctatgaactctagaaga~tctctggcttctaggaggtctcttattaatatg I1500 v. 1 3401 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 3450 1501 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 1550 v.1 3451 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg 3500 1551 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg .1600 v.1 3501 gcgaagcctccaagtatacagaagaggatccttccggagaaacactgtct 3550 1601 gcgaagcctccaagtatacagaagaggatccttccggagaaacactgtct 16510 v.1 3551 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 3600 1651 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 1700 C v.1 3601 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 3650 1701 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 1750 v.1 3651 ttggttctccccaggataaggcggcagaggctacaaatgactatgagact 3700 1751 ttggttctccccaggataaggcggcagaggctacaaatgactatgagact 1800 00 ID v.1 3701 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 3750 1801 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 1850 v.1 3751 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag 3800 1851 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag 1900 v.1 3801 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 3850 1901 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 1950 v.1 3851 tgtaacctgtttattgtattttaaagtgaaactgaatatgagggaatttt 3900 1951 tgtaacctgtttattgtattttaaagtgaaactgaatatgagggaatttt 2000 v.1 3901 tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc 3950 2001 tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc 2050 v.1 3951 aagaaagatctcaaaaagcaacttctgccctgcaacgccccccactccat 4000 2051 aagaaagatctcaaaaagcaacttctgccctgcaacgccccccactccat 2100 v.1 4001 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt 4050 2101 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt 2150 v.1 4051 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt 4100 2151 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt 2200 v.1 4101 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 4150 2201 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 2250 v.1 4151 ttgtatataacagttcagataagaaaattaaagttacttttctc 4194 2251 ttgtatataacagttcagataagaaaattaaagttacttttctc 2294 Table LIV(c). Peptide sequences of protein coded by 273134137 v.10 (SEQ ID NO: 125) MEKSFATKINE AVQKETLQEG PKQEALQEDP LESFNYVLSK STKAflIGPNLj DQLKDDEILR HCNPWPIISI TkNESQNAESN VSIIEIADDL SASHSALQDA QASEAKLEEE PSASSPQYAC 120 DFNLFLEDSA DNRQNFSSQS LEHVEKENSL CGSAPN\SKAG FVFISKTCLSW EFSEKDDEPE 180 EVVVKAKIRS KARRIVSDGE DEDDSFKDTS SINPFNTSLF QFSSVKQFDA STPKNDISPP 240 GRFFSSQIPS SVNKSMNSRR SLASRRSLIN MVLDHVEDME ERLDDSSEAK GPEDYPEEGV 300 EESSGEASKY TEEDPSGETL SSENKSSWLM JTSKPSALAQE TSLGAPEPLS GEQLVGSPQD 360 KAABATNDYE TIJVKRGKELK ECGKIQEALN CLVKALDIKS ADPEVMIJLTL SLYKQLNNN 419 Table LV(c). Amino acid sequence alignment of 273P4137 v.1 0 (SEQ ID NO: 126) and 273P4137 v.1 (SEQ ID NO: 127) v.1 801 DGKGTGSADSIATLPKGFGSVEELCTNSSLGMEKSFATKN'EAVQKETLQE 850 1 MEKSFATKNEAVQKETLQE 19 v.1 851 GPKQEALQEDPLESFNYVLSKSTKADIGPNLDQLKDDEILRHCNPWPIIS 20 GPKQEALQEDPLESFNYVLSKSTKADIGPNLDQLKDDEILRHCNPWPI

IS

v.1 901 ITNESQNIAESNVSIIEIADDLSASHSALQDAQASEAKIJEEEPSASSPQYA 70 ITNESQlNAESNVSIIEIADDLSASHSALQDAQASEAKIJEEEPSASSPQYA v.1 951 CDFNLFLEDSADNRQNFSSQSLEHVEKENSLCGSAPNSRAGFVHSKTCLS 120 CDFNTLFLEDSADNRQNFSSQSLEHVEKENSLCGSAPNSKAGFVHSKTCLS v.1 1001 WEFSEKDDEPEEVVVKAKIRSK-ARRIVSDGEDEDDSFKDTSSINPFNTSL.

170 WEFSEKDDEPEEVVVKAKIRSKARRIVSDGEDEDDSFKDTSSINPFNTSL v.1 1051 FQFSSVKQFDASTPKNDISPPGRFFSSQIPSSVNKSMNSRRSLASRRSI 220 FQFSSVKQFDASTPKNDISPPGRFFSSQIPSSVNKSM&SRRSLASRRSLI v.1 1101 NMVLDHVEDMEERLDDSSEAKGPEDYPEEGVEESSGEASKYTEEDPSGET 270 NMVLDHVEDMEERLIDSSEaKGPeDPEixEGVEESSGEASKYTEEDPSGET v.1 1151 LSSENKSSWLMTSKPSALAQETSLGAPEPLSGEQLVGSPQDKAAEATNDY 320 LSSENKSSWLMTSKPSALAQETSLGAPEPLSGEQLVGSPQDKAAEATNDY v.1 1201 ETLVKRGKELKECGKIQEALNCLVKALDIKSADPEVMLLTLSLYKQLNNN 370 ETLVKRGKELKECGKIQEALNCLVKALDIKSADPEVMLLTLSLYKQLNNN 900 69 950 119 1000 169 1050 219 1100 269 1150 319 1200 369 1250 419 Table LII(d). Nucleotide sequence of transcript variant 273P4B37 v.11 (SEQ ID NO: 121 ggcacgaggc taccaaaggg aaagctttgc aagaggcact aagctgatat atccttggcc ttattgaaat gtgaggccaa atcttttctt atgttgagaa atagcaaaac tagttaaagc atgattcttt catctgtgaa tcttttcatc cttctaggag ttgacgacag gcagtggcga aaaacaagtc ttggtgCCCC caccttgcaa gtttggaagt aactaaaaat gcaagaggat tgggccaaat cattatttcc agctgatgac gttggaagag ggaagactca agaaaatagc atgtctcagt aaaaatcaga taaagatacc acaatttgat tcaaataccc gtctcttatt cagtgaagca agcctccaag cagctggtta tgagcctttg gatggtaaag gtagaagaac gaagctgtac cctctggaaa ttagatcaac ataacaaatg ctttcagcat gaacc ttc ag gcagacaaca ttgtgtggct tgggagtttt agtaaagcta tcaagcataa gCttcaactc agtagtgtaa aatatggttt aagggtcctg tatacagaag atgacgtcta tctggtgaac gtacaggtag tttgtactaa aaaaagagac gttttaatta taaaggatga aaagtcaaaa cccatagtgc catcttcacc gacaaaattt ctgcacctaa ctgagaaaga gaaggattgt atccattcaa ccaaaaatga ataagtctat tagaccacgt aagattatcc aggatccttc agcctagtgc agttggttgg tgctgactct ctcttcattg attacaagag tgtacttagc tgagatttta tgcagaatca actgcaggat acagtatgc~a ttccagtcag ttccagagca cgatgaacca ttcagatggc cacatctctc catcagtcc a gaac tctaga ggaggacatg agaagaaggg cggagaaaca t c tagctcaa ttctccccag atagctactt ggaatggaaa gggcctaagc aaatcaacca cgtcattgca aatgtatcca gctcaagCaa tgtgatttca tctttagagc gggtttgtgc gaagaagtag gaagatgaag tttcaattct ccaggaaggt agatctctgg gaggaaagac gtggaggaaa ctgtcttcag gagacctctc gataaggcgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 cagaggctac aaatgactat gagactcttg taaagcgtgg aaaagaacta aaagagtgtg 1260 gaaaaatcca ggaggcccta aactgcttag ttaaagcgct tgacataaaa agtgcagatc 1320 ctgaagttat gctcttgact ttaagtttgt ataagcaact taataacaat tgagaatgta 1380 acctgtttat tgtattttaa agtgaaactg aatatgaggg aatttttgtt cccataattg 1440 gattctttgg gaacatgaag cattcaggct taaggcaaga aagatctcaa aaagcaactt 1500 ctgccctgca acgcccccca ctccatagtc tggtattctg agcactagct taatatttct 1560 tcacttgaat attcttatat tttaggcata ttctataaat ttaactgtgt tgtttcttgg 1620 aaagttttgt aaaattattc tggtcattct taattttact ctgaaagtga tcatctttgt 1680 CA atataacagt tcagataaga aaattaaagt tacttttctc 1720 Table 1.I11(d). Nucleotide sequence alignment of 273P4137 v.11 (SEQ ID NO: 129) and 273P4137 v.1 (SEQ ID NO: 130) v. 1 2451 aacaagatctctccagtataaaggtgaatgttaccaccttgcaagatggt 2500 v.11 10 ccaccttgcaagatggt 26 00 v.1 2501 aaaggtacaggtagtgctgactctatagctactttaccaaaggggtttgg 2550 IND itIIIi 11111111111111111 v.11 27 aaaggtacaggtagtgctgactctatagctactttaccaaaggggtttgg 76 v.1 2551 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 2600 v.11 77 aagtgtagaagaactttgtactaactcttcattgggaatggaaaaaagct 126 v.1 2601 ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 2650 v.11 127 ttgcaactaaaaatgaagctgtacaaaaagagacattacaagaggggcct 176 v.1 2651 aagcaagaggcactgcaagaggatcctctggaaagttttaattatgtact 2700 v.11 177 aagcaagaggcactgcaagaggatcctctggaaagttttaattatgtact 226 v.1 2701. tagcaaatcaaccaaagctgatattgggccaaatttagatcaactaaagg 2750 v.11 227 tagcaaatcaaccaaagctgatattgggccaaatttagatcaactaaagg 276 v.1 2751 atgatgagattttacgtcattgcaatccttggcccattatttccataaca 2800 v.11 277 atgatgagattttacgtcattgcaatccttggcccattatttccataaca 326 v.1 2801. aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 2850 v.11 327 aatgaaagtcaaaatgcagaatcaaatgtatccattattgaaatagctga 376 v.1 2851 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 2900 v.11 377 tgacctttcagcatcccatagtgcactgcaggatgctcaagcaagtgagg 426 v.1 2901 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 2950 v.11 427 ccaagttggaagaggaaccttcagcatcttcaccacagtatgcatgtgat 476 v.1 2951 ttcaatcttttcttggaagactcagcagacaacagacaaaatttttccag 3000 v.11 477 ttcaatcttttcttggaagactcagcagacaacagacaaaatttttccag 526 v.1 3001 tcagtctttagagcatgttgagaaagaaaatagcttgtgtggctctgcac 3050 v.11 527 tcagtctttagagcatgttgagaaagaaaatagcttgtgtggctctgcac 576 v.1 3051 ctaattccagagcagggtttgtgcatagcaaaacatgtctcagttgggag 3100 v.11 577 ctaattccagagcagggtttgtgcatagcaaaacatgtctcagttgggag 626 V. 1 3101 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat .3150 v.11 627 ttttctgagaaagacgatgaaccagaagaagtagtagttaaagcaaaaat 676 v.1 3151 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt 3200 v.11 677 cagaagtaaagctagaaggattgtttcagatggcgaagatgaagatgatt 726 V.1 3201 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa 3250 v.11 727 cttttaaagatacctcaagcataaatccattcaacacatctctctttcaa 776 v.1 3251 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag 3300 v.11 777 ttctcatctgtgaaacaatttgatgcttcaactcccaaaaatgacatcag 826 00 ID v.1 3301 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt 3350 v.11 827 tccaccaggaaggttcttttcatctcaaatacccagtagtgtaaataagt 876 v.1 3351 ctatgaactctagaagatctctggcttctaggaggtctcttattaatatg 3400 v.11 877 ctatgaactctagaagatctctggcttctaggaggtctcttattaatatg 926 v.1 3401 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 3450 v.11 927 gttttagaccacgtggaggacatggaggaaagacttgacgacagcagtga 976 v.1 3451 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg 3500 v.11 977 agcaaagggtcctgaagattatccagaagaaggggtggaggaaagcagtg 1026 v.1 3501 gcgaagcctccaagtatacagaagaggatccttccggagaaacactgtct 3550 v.11 1027 gcgaagcctccaagtatacagaagaggatccttccgg aaaacatgtct 1076 v.1 3551 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 3600 v.11 1077 tcagaaaacaagtccagctggttaatgacgtctaagcctagtgctctagc 1126 v.1 3601 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 3650 v.11 1127 tcaagagacctctcttggtgcccctgagcctttgtctggtgaacagttgg 1176 v.1 3651 ttggttctccccaggataaggcggcagaggctacaaatgactatgagact 3700 v.11 1177 ttggttctccccaggataaggcggcagaggctacaaatgactatgagact 1226 v.1 3701 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 3750 v.11 1227 cttgtaaagcgtggaaaagaactaaaagagtgtggaaaaatccaggaggc 1276 v.1 3751 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag, 3800 v.11 1277 cctaaactgcttagttaaagcgcttgacataaaaagtgcagatcctgaag, 1326 v.1 3801 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 3850 v.11 1327 ttatgctcttgactttaagtttgtataagcaacttaataacaattgagaa 1376 V.1 3851 tgtaacctgtttattgtattttaaagtgaaactgaatatgagggaatttt 3900 v. 11 1377 tgt aacc tgtt tat tgt attt taaagtgaaactgaatatgagggaatttt 1426 v.1. 3901 tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc 3950 v.1 127tgttcccataattggattctttgggaacatgaagcattcaggcttaaggc 1476 v.1 3951 aagaaagatctcaaaagcaacttctgccctgcaacgccccccactccat 4000 v.11 1477 aagaaagatctcaaaagcaacttctgccctgcaacgccccccactccat 1526 v.1 4001 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt 4050 v.11 1527 agtctggtattctgagcactagcttaatatttcttcacttgaatattctt 1576 v.1 4051 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt 4100 0 v.11 1577 atattttaggcatattctataaatttaactgtgttgtttcttggaaagtt 1626 (i v.1 4101 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 4150 v.11 1627 ttgtaaaattattctggtcattcttaattttactctgaaagtgatcatct 1676 v.1 4151 ttgtatataacagttcagataagaaaattaaagttacttttctc 4194 v.11 1677 ttgtatataacagttcagataagaaaattaaagttacttttctc 1720 Table LIV(d). Peptide sequences of protein coded by 273P4137 v.11 (SEQ ID NO: 131) MEKSFATKNE AVQKETLQEG PKQEALQEDP LESFNYVLSK STKADIGPNLi DQLKDDEILR HCNPWPIISI TNESQNAESN VSIIEIADDL SASHSALjQDA QASEAKEJEEE PSASSPQYAC 120 DFNTJFLEDSA DNRQNFSSQS LEHVEKE1NSL CGSAPNSRAG FVHSKTCLSW EFSEKDDEPE 180 EVVVXAKIRS KARRIVSDGE DEDDSFKDTS SINPFNTSLF QFSSVKQFDA STP1K'1fISPP 240 GRFFSSQIPS SVNKSMNSRR SLASRRSLIN MVLDHVEDME ERLDDSSEAK GPEDYPEEGV 300 EESSGEASKY TEEDPSGETL SSENKSSWLM TSKPSALAQE TSLGAPEPLS GEQLVGSPQD 360 KAAEATNDYE TLVKRGKELaK ECGKIQEALN CLVKALDIKS ADPEVMLTLTL SLYKQIMNNN 419 Table LV(d). Amino acid sequence alignment of 273134137 v.11 (SEQ ID NO: 132) and 273P4B7 M. (SEQ ID NO: 133) V. 1 801 DGKGTGSADSIATLPKGFGSVEELCTNSSLjGMEKSFATKNBAVQKETLQE 850 v.11 1 MEKSFATKNEAVQKETLQE 19 v. 1 851 GPKQEALjQEDPILESFNYVLSKSTKAflIGPNLDQLKDDEILRHCNPWPIIS 900 v.11 20 GPKQEALQEDPLESFNYVLSKSTKAI3IGP:NILDQLKDDEILaRHCNPWPIIS 69 v.1 901 ITNESQNAESNVSIIEIADDLSASHSALQDAQASEAKLEEEPSASSPQYA 950 v.11 70 ITNESQNAESNVSIIEIADDLSASHSALQDAQASEAKLEEEPSASSPQYA 119 v.1 951 CDFNLFLEDSAflNRQNFSSQSLEHVEKENSLCGSAPNSRAGFVHSKTCLS 1000 v.11 120 CDFNLFLEDSAflNRQN~FSSQSLEHVEKENSLCGSAPNSRAGFVHSKTCLS 169 v.1 1001 WEFSEKDDEPEEVVVKAKIRSKARRIVSDGEDEDDSFKDTSSINPFNTSL 1050 v. 11 170 WEFSEKDDEPEEVVVKAJIRSKAJRIVSDGEDEDDSFKDTSSINPFNTSL 219 v.1 1051 FQFSSVKQFDASTPKNDISPPGRFFSSQIPSSVNKSMSRRSLASRRSLI 1100 v.11 220 FQFSSVKQFDASTPKINDISPPGRFFSSQIPSSVNKSMSRRSLASRRSLI 269 v.1 '1101 NMVLDRVEDMEERLDDSSEAKGPEDYPEEGVEESSGEASKYTEEDPSGET 1150 v.11 270 NMVLDHVEDMEERLDDSSEAKGPEDYPEEGVEESSGEASKYTEEDPSGET 319 v.1 1151 LSS ENKSSWLMTSKPSALAQETSLGAPEPLSGEQLVGSPQDKAAEATNDY 1200 ci v.11 320 LSSENKSSWLMTSKPSALAQETSLGAPEPLSGEQLVGSPQDKAA..ATNDY 369 v.1 1201 ETLVKRGKELKECGKIQEAIJNCLVKALDIKSADPEVMLLTLSLYKQLM 1250 v.11 370 BTVRKLEGIELCVALISDEMLLLKLN 419 00 Table LVI: SNP and codon.'changes in 273P4B7 v.1 and v.2. .*AA :amino acid; deletion of the corresponding base.

SNP SP inv. ISNP in v.2 SP Alleles SNP PFo-sitioni AA* AA Position -AA AA N.vratchange position change position a/ 159 -7-11- 36 2 alg v4 608 00 17-2 748 RG 4 3 a/g v.5 1185 -KR 364 1T325 K/k) 241 4 a/Wg v.-6 2759 I/V 889 2899 I/V 766 7 T -8 1188 -3798 1065 6- a/g v.8 3j8-50 -3990 00

Claims (12)

1. 3. A recombinant expression vector comprising a polynucleotide of claims 1 or 2.
2. 4. A host cell that contains an expression vector of claim 3. ci 5. An isolated protein comprising the amino acid sequence selected from the 0j groups consisting of SEQ ID NO: 13, 14, 15, 16, 17, 18, and 19.
3. 6. A process for producing the protein according to claim 5, comprising culturing C, a host cell according to claim 4 under conditions sufficient for the production of 00 the protein.
4. 7. An antibody or fragment thereof that immunospecifically binds to an epitope on Sthe protein according to claim
5. 8. The antibody or fragment thereof according to claim 7, which is monoclonal.
6. 9. The antibody or fragment thereof according to claim 7 or claim 8, which is conjugated with a cytotoxic agent. A hybridoma that produces an antibody according to claim 8.
7. 11. A method for detecting the presence of a protein or a polynucleotide in a test sample comprising: contacting the sample with an antibody or a probe, respectively, that specifically binds to the protein according to claim 5 or the polynucleotide according to claim 1, respectively; and detecting binding of protein or polynucleotide, respectively, in the sample thereto.
8. 12. The method according to claim 11, wherein the detecting step comprises comparing an amount of binding of the antibody or the probe that specifically binds to the protein or the polynucleotide to the presence of the protein or the ,I polynucleotide in a corresponding normal sample.
9. 13. The method according to claim 12, wherein the presence of elevated polynucleotide or protein in the test sample relative to the normal tissue sample provides an indication of the presence of cancer. 00 14. The method according to claim 13, wherein the cancer is selected from the 00 group consisting of prostate cancer, bladder cancer, kidney, colon, lung, ovary, breast, pancreas, bone, skin, cervix, lymph node, stomach and uterus. A method of inhibiting growth of a cell expressing the protein according to claim 5, comprising providing an effective amount of an antibody according to any one of claims 7 to 9 to the cell, whereby the growth of the cell is inhibited.
10. 16. A method of delivering a cytotoxic agent to a cell expressing the protein according to claim 5, comprising providing an effective amount of an antibody according to any one of claims 7 to 9 to the cell.
11. 17. A method of inducing an immune response to the protein according to claim the method comprising: providing a protein epitope; and Contacting the epitope with an immune system T cell or B cell, whereby the immune system T cell or B cell is induced.
12. 18. Use of an epitope from the protein according to claim 5 for the preparation of a medicament to induce a T cell or B cell immune response in a subject.