AU2008202217A9 - Nucleic acids and corresponding proteins entitled 191PAD12(b) useful in treatment and detection of cancer - Google Patents

Description

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AUSTRALIA

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

COMPLETE SPECIFICATION STANDARD PATENT Invention Title.

Nucelic acids and corresponding proteins entitled 191PAD12(b) 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:-

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NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 191 o00 0 P4D12(b) USEFUL IN TREATMENT AND DETECTION OF CANCER This is a divisional of AU 2003228717, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The invention described herein relates to genes and their encoded proteins, termed 191P4D 12(b), expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 191 SP4D12(b).

BACKGROUND OF THE INVENTION 00 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 patents 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 30,000 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 cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities.

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

On the diagnostic front, the lack of a prostate tumor marker that can accurately 00 detect early-stage, localized tumors remains a significant limitation in the diagnosis and N, 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.

00 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 00 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 al., 1997, Nat. Med. 3:402). More recently Identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl.

Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 1445- 51), STEAP (Hubert, et al., Proc Natl Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) S (Reiter et 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 cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma C0 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 C"1 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 00 cancer.

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

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

SLung 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 c.i 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, 00 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

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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, ovan'an 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.

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

SThere 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 S may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease In mortality rates among men (about-0.9% per year) while L| rates have increased slightly among women.

SSurgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment 00 options can extend survival and/or relieve symptoms In many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION The present invention relates to a gene, designated 191P4D12(b), that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 191P4D12(b) 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 191P4D12(b) are provided. The tissue-related profile of 191P4D12(b) in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 191P4D12(b) 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 191P4D12(b) genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 191P4D12(b)-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 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 191P4D12(b)-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 191 P4D12(b) genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 191P4D12(b) genes, mRNAs, or to 191P4D12(b)-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 191P4D12(b). Recombinant DNA molecules containing 191P4D12(b) polynudeotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 191P4D12(b) gene products are also provided.

The invention further provides antibodies that bind to 191P4D12(b) 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 embodimenls, 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 191 P4D12(b) polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 191P4D12(b). A typical embodiment of this invention provides methods for monitoring 191P4012(b) gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

0 0 The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 191P4D12(b) such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 191P4D12(b) as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 191P4D12(b) 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 191P4D12(b). Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent Is a moiety that is immunoreactive with 191P4D12(b) 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 S antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

00 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 191P4D12(b) and/or one or more ri than 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 191P4D12(b) as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 191P4D12(b). Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 191P4D12(b) antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 191P4D12(b) production) or a ribozyme effective to lyse 191P4D12(b) 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 oligonucleotide 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 00 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 0 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, c 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) 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 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 00 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.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The 191P4D12(b) SSH sequence of 223 nucleotides.

Figure 2. A) The cDNA and amino acid sequence of 191P4D12(b) variant 1 (also called "191P4012(b) v.1" or "191P4D12(b) variant is shown in Figure 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

B) The cDNA and amino acid sequence of 191P4D12(b) variant 2 (also called "191P4D12(b) is shown in Figure 28. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

C) The cDNA and amino acid sequence of 191P4D12(b) variant 3 (also called "191P4D12(b) is shown in Figure 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

D) The cDNA and amino acid sequence of 191P4D12(b) variant 4 (also called "191P4D12(b) is shown in Figure 2D. The codon for the start methionine Is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

E) The cDNA and amino acid sequence of 191P4D12(b) variant5 (also called "191P4D12(b) is shown in Figure 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

F) The cDNA and amino acid sequence of 191P4D12(b) variant 6 (also called "191P4D12(b) is shown in Figure 2F. The codon for the start methlonine is underlined. The open reading frame extends from nucleic acid 789-1676 including the stop codon.

G) The cDNA and amino acid sequence of 191P4D12(b) variant 7 (also called "191P4D12(b) is shown in Figure 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1721 including the stop codon.

H) The cDNA and amino acid sequence of 191P4D12(b) variant8 (also called "191P4D12(b) Is shown in Figure 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

I) The cDNA and amino acid sequence of 191P4D12(b) variant 9 (also called "191P4D12(b) is shown in Figure 21. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 708-1121 including the stop codon.

O J) The cDNA and amino acid sequence of 191P4D12(b) variant 10 (also called "191P4D12(b) v.10") is shown in Figure 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

K) The cDNA and amino acid sequence of 191P4012(b) variant 11 (also called "191P4D12(b) v.11") is shown in Figure 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

L) The cDNA and amino acid sequence of 191P4D12(b) variant 12 (also called "191P4D12(b) v.12") is shown in Figure 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1796 including the stop codon.

M) The cDNA and amino acid sequence of 191P4D12(b) variant 13 (also called "191P4D12(b) v.13") is shown in 00 Figure 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 264-1799 including the stop codon.

0i N) The cDNA and amino acid sequence of 191P4D12(b) variant 14 (also called "191P4D12(b) v.14") is shown in Figure 2N. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 708-1121 including the stop codon.

Figure 3.

A) The amino acid sequence of 191P4D12(b) v.1 is shown in Figure 3A; it has 510 amino acids.

B) The amino acid sequence of 191P4D12(b) v.2 is shown in Figure 3B; it has 510 amino acids.

C) The amino acid sequence of 191P4D12(b) v.6 is shown in Figure 3C; it has 295 amino acids.

D) The amino acid sequence of 191P4D12(b) v.7 is shown in Figure 3D; it has 485 amino acids.

E) The amino acid sequence of 191P4D12(b) v.10 is shown in Figure 3E; it has 510 amino acids.

F) The amino acid sequence of 191P4D12(b) v.11 is shown in Figure 3F; it has 510 amino acids.

G) The amino acid sequence of 191P4D12(b) v.12 is shown in Figure 3G; it has 510 amino acids.

H) The amino acid sequence of 191P4D12(b) v.13 is shown in Figure 3H; it has 511 amino acids.

I) The amino acid sequence of 191P4D12(b) v.9 is shown in Figure 31; it has 137 amino acids.

J) The amino acid sequence of 191P4D12(b) v.14 is shown in Figure 3J; it has 137 amino acids.

As used herein, a reference to 191P4D12(b) includes all variants thereof, including those shown in Figures 2, 3, and 11, unless the context clearly indicates otherwise.

Figure 4. Alignment of 191P4D12(b) with known homologs. Figure 4(A) Alignment of 191P4D12(b)with human Ig superfamily receptor LNIR (gi 14714574). Figure 4(B) Alignment of 191P4D12(b) with mouse nectin 4 (gi 18874521).

Figure 5. Hydrophilicity amino acid profile of 191P4012(b)v.1, v.7, and v.9 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp Woods 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 191P4D12(b)v.1, v.7, and v.9 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 acid profile of 191P4D12(b)v.1, v.7, and v.9 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-bln/protscale.pl) through the ExPasy molecular biology server.

Figure 8. Average flexibility amino acid profile of 191P4012(b)v.1, v.7, and v.9 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/cgibin/protscale.pl) through the ExPasy molecular biology server.

Figure 9. Beta-turn amino acid profile of 191P4D12(b)v.1, v.7, and v.9 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, Roux B. 1987 Protein Engineering 1:289-294) S 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 10. Schematic alignment of SNP variants of 191P4D12(b). Variants 191P4D12(b) v.2 through v.5 and C(1 through v.12 are variants with single nucleotlde differences. Compared with v.1, v.13 had an insertion of three bases (GCA) 00 between 1262 and1263 and added one amino acid to the protein. Variant v.14 was a SNP variant of transcript variant v.9, corresponding to the SNP at 2688 of v.1. Though these SNP variants were shown separately, they could also occur in any combinations and in any transcript variants, as shown in Fig. 12, that contained the base pairs. Numbers correspond to those of 191P4D12(b) v.1. Black box shows the same sequence as 191P4D12(b) v.1. SNPs are indicated above the box.

Figure 11. Schematic alignment of protein variants of 191P4D12(b). Protein variants correspond to nucleotide variants. Nucleotide variants 191P4D12(b) v.3, v.4, v.5 and v.8 coded for the same protein as v.1. Nucleotide variants 191P4D12(b) v.6, v.7, v.8 and v.9 were splice variants of v.1, as shown in Figure 12. Variant v.9 translated to a totally different protein than other variants, with two isoforms that different from each other by one amino acid at 64: A or D. Variant v.13 had an insertion of one amino acid at 334. Single amino acid differences were indicated above the boxes. Black boxes represent the same sequence as 191P4D12(b) v.1. Numbers underneath the box correspond to 191P4D12(b) v.1.

Figure 12. Exon compositions of transcript variants of 191P4D12(b). Variant 191P4D12(b) v.6, v.7, v.8 and v.9 are transcript variants of v.1. Variants v.6, v.7 and v.8 spliced out 202-321, 1497-1571 and 2951-3013 of v.1, respectively.

Variant v.9 was part of the last exon of v.1. The order of the potential exons on the human genome is shown at the bottom.

Poly A tails were not shown in the figure. Ends of exons are shown above the boxes. Numbers in undemeath the boxes correspond to those of 191P4D12(b) v.1. Lengths of introns and exons are not proportional.

Figure 13. Secondary structure and transmembrane domains prediction for 191P4D12(b) protein variants.

The secondary structure of 191P4D12(b) protein variants 1 (SEQ ID NO: 127), v6 (SEQ ID NO: 128), v7 (SEQ ID NO: 129), and v9 (SEQ ID NO: 130) (Figures 13A-D respectively) were predicted using the HNN Hierarchical Neural Network method (Guermeur, 1997, http:l/pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa n.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.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. Figures 13E, 13G, 131,13K: Schematic representations of the probability of existence of transmembrane regions and orientation of 191P4D12(b) variants 1, 6, 7, and 9, respectively, 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). Figures 13F, 13H, 13J, 13L. Schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 191P4D12(b) variants 1, 6, 7, and 9, respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L.L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markbv 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 00 0 the World Wide Web at (.expasy.ch/tools/).

Figure 14. 191P4D12(b) Expression by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal kidney, prostate cancer pool, bladder cancer pool, colon cancer pool, lung cancer pool, breast cancer pool and cancer metastasis pool; prostate cancer metastasis to lymph node, 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, and LAPC prostate xenograft pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 191P4D12(b), was S performed at 26 and 30 cycles of amplification. In results show strong expression of 191P4D12(b) in bladder cancer pool. Expression of 191P4D12(b) was also detected in prostate cancer pool, colon cancer pool, lung cancer pool, breast CN cancer pool and cancer metastasis pool but very weakly in vital pool 1 and vital pool 2. In results show strong expression of 191P4D12(b) in prostate, bladder, kidney, colon, lung, ovary, breast, cancer metastasis, and pancreas cancer specimens.

0 0 Figure 15. Expression of 191P4D12(b) in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/Iane were probed with the 191P4D12(b) sequence. Size standards in kilobases (kb) are indicated on the side.

Results show expression of an approximately 4kb transcript In placenta and very weakly in prostate but not in any other normal tissue tested. A smaller 191P4D12(b) transcript of approximately 2.5kb was detected in heart and skeletal muscle.

Figure 16. Expression of 191P4D12(b) In Patient Cancer Specimens and Normal Tissues. RNA was extracted from a pool of 3 bladder cancer patient specimens, as well as from normal prostate normal bladder normal kidney normal colon normal lung normal breast (NBr), normal ovary and normal pancreas (NPa). Northern blot with 10 ug of total RNA/lane was probed with 191P4D12(b) SSH sequence. Size standards in kilobases (kb) are indicated on the side. The 191P4D12(b) transcript was detected in the bladder cancer specimens, but not in the normal tissues tested.

Figure 17. Expression of 191P4D12(b) in Bladder Cancer Patient Specimens. RNA was extracted from bladder cancer cell lines normal bladder and bladder cancer patient tumors Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4012(b) transcript in the bladder tumor tissues but not in normal bladder. A smaller transcript was detected in the HT1197 cell line but not in the other cancer cell lines tested.

Figure 18. Expression of 191P4D12(b) in Prostate Cancer Xenografts. RNA was extracted from normal prostate, and from the prostate cancer xenografts LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in all the LAPC xenograft tissues but not in normal prostate.

Figure 19. Expression of 191P4D12(b) in Cervical Cancer Patient Specimens. RNA was extracted from normal cervix, Hela cancer cell line, and 3 cervix cancer patient tumors Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in 2 out of 3 cervix tumors but not in normal cervix nor in the Hela cell line.

Figure 20. Expression of 191P4D12(b) in Lung Cancer Patient Specimens. RNA was extracted from lung cancer cell lines normal lung bladder cancer patient tumors and normal adjacent tissue (Nat). Northern blots with ug of total RNA were probed with the 191P4D12(b). Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in the lung tumor tissues but not in normal lung nor in the cell lines tested.

Figure 21. Figure 21A. 191P4D12(b) Expression In Lung Cancer. First strand cDNA was prepared from a panel of lung cancer specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show 00 C expression of 191P4D12(b) in 97% of the 31 lung cancer patient specimens tested. Figure 21B. 191P4D12(b) Expression in Bladder Cancer. First strand cDNA was prepared from a panel of bladder cancer specimens. Normalization was Sperformed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 94% of the 18 Sbladder cancer patient specimens tested. Figure 21C. 191P4012(b) Expression in Prostate Cancer. First strand cDNA was prepared from a panel of prostate cancer specimens, and four LAPC prostate cancer xenografts. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak CK1 expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 100% of the S'prostate cancer patient specimens tested, and in all 4 prostate cancer xenografts. Figure 21D. 191P4D12(b) Expression in 00 Colon Cancer. First strand cDNA was prepared from a panel of colon cancer specimens. Normalization was performed by 0 PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 100% of the 22 colon cancer patient specimens tested. Figure 21E. 191P4D12(b) Expression in Uterus Cancer. First strand cDNA was prepared from a panel of uterus cancer specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 100% of the 12 uterus cancer patient specimens tested. Figure 21F. 191P4D12(b) Expression in Cervical Cancer. First strand cDNA was prepared from a panel of cervix cancer specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 100% of the 14 cervix cancer patient specimens tested.

Figure 22. Transient Expression of 191P4D12(b) in Transfected 293T Cells. 293T cells were transfected with either 191P4D12(b) .pTag5, 191P4D12(b).pcDNA3.1/mychis or pcDNA3.1/mychis vector control. Forty hours later, cell lysates and supernatant were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with antihis antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression from 191P4D12(b).pTag5 plasmid of 191P4D12(b) extracellular domain in the lysate (Lane 2) and secretion in the culture supernatant (Lane Also, expression of 191P4D12(b) was detected from in the lysates of 191P4D12(b).pcDNA3.1/mychis transfected cells (Lane but not from the control pcDNA3.1/mychis (Lane 4).

Figure 23. Expression of 191P4012(b) in Transduced Cells Following Retroviral Gene Transfer. 3T3 cells were transduced with the pSRa retroviral vector encoding the 191P4D12(b) gene. Following selection with neomycin, the cells were expanded and RNA was extracted. Northern blot with 10 ug of total RNA/lane was probed with the 191P4D12(b) SSH sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of the 191P4D12(b) transcript driven from the retroviral LTR, which migrates slower than the endogenous 4 kb 191P4D12(b) transcript detected in the positive control LAPC-4AD.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections Definitions II.) 191P4D12(b) Polynucleotides 00 II.A.) Uses of 191P4D12(b) Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities ll.A.2.) Antisense Embodiments II.A.3.) Primers and Primer Pairs II.A.4.) Isolation of 191P4D12(b)-Encoding Nucleic Acid Molecules Recombinant Nucleic Acid Molecules and Host-Vector Systems II1.) 191P4D12(b)-related Proteins III.A.) Motif-bearing Protein Embodiments III.B.) Expression of 191P4D12(b)-related Proteins SIII.C.) Modifications of 191P4D12(b)-related Proteins III.D.) Uses of 191P4D12(b)-related Proteins 00 IV.) 191P4D12(b) Antibodies 191P4D12(b) Cellular Immune Responses C VI.) 191P4D12(b) Transgenic Animals VII.) Methods for the Detection of 191P4D12(b) VIII.) Methods for Monitoring the Status of 191P4D12(b)-related Genes and Their Products IX.) Identification of Molecules That Interact With 191P4D12(b) Therapeutic Methods and Compositions Anti-Cancer Vaccines XB.) 191P4D12(b) as a Target for Antibody-Based Therapy XC.) 191P4D12(b) as a Target for Cellular Immune Responses X.C.1. Minigene 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 and/or HTL Peptides Adoptive Immunotherapy Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 191P4D12(b).

XII.) Inhibition of 191P4D12(b) Protein Function XII.A.) Inhibition of 191P4D12(b) With Intracellular Antibodies XII.B.) Inhibition of 191P4D12(b) with Recombinant Proteins XII.C.) Inhibition of 191P4D12(b) Transcription or Translation XII.D.) General Considerations for Therapeutic Strategies XIII.) Identification, Characterization and Use of Modulators of 191P4D12(b) 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 0 0 commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized 0 molecular cloning methodologies described in Sambrook et Molecular Cloning: A Laboratory Manual 2nd. edition (1989) SCold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of S commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

S

The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to Include stage C disease under the American Urological Association (AUA) system, stage C1 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 recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes 0 compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically 00 identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or Induration above the Sprostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 191P4D12(b) (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 191P4D12(b). 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 191 P4D12(b)-related protein). For example, an analog of a 191 P4D12(b) protein can be specifically bound by an antibody or T cell that specifically binds to 191 P4D12(b).

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-191P4D12(b) 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-191P4D12(b) antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-191 P4D12(b) 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 chemical libraries include, but are not limited to, peptide libraries (see, U.S. Patent No. 5,010,175, Furka, Pept. Prot.

Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptolds (PCT Publication No WO 91/19735), encoded t peptides (PCT Publication WO 93/20242), random bio- 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. Sci.

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)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem.

59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, Stratagene, Corp.), peptide nucleic acid libraries (see, U.S. Patent 5,539,083), antibody libraries (see, Vaughn et al., Nature 00 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); isoprenoids, U.S. Patent No. 5,569,588; thlazolidinones 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, maytansinolds, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposlde, 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, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At 211 1131, 1125, Y90, Re' 86 Re' 8 8 Sm' 53 Bi212o213, P32 and radioactive isotopes of Lu including Lu' 7 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 00 encoded by the nucleic acids of Figure 2. In one embodiment, a cancer amino acid sequence is used to determine 0 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 ct herein.

"High throughput screening" assays for the presence, absence, quantification, or other properties of particular nucleic 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 proteins; 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.

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, 00 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 m 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 pig/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1% SDS are above 55 degrees C.

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 polynuceotides that correspond or are complementary to genes other than the 191P4D12(b) genes or that encode polypeptides other than 191P4D12(b) gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 191P4D12(b) polynucleotide. A protein is said to be "solated," for example, when physical, mechanical or chemical methods are employed to remove the 191P4D12(b) proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 191P4D12(b) protein.

Altematively, 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 0C metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation.

0 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 C^ prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term "modulator" or "test compound" or "drug candidate" or grammatical equivalents as used herein describe any molecule, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the C=K capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, a nucleic acid or Sprotein sequences, or effects of cancer sequences signaling, gene expression, protein interaction, etc.) In one aspect, 00 a modulator will neutralize the effect of a cancer protein of the Invention. By "neutralize" is meant that an activity of a protein 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 andlor 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 adds, 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 peplides 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 "monodonal antibody" refers to an antibody obtained from a population of substantially homogeneous 00 antibodies, the antibodies comprising the population are identical except for possible naturally occuning mutations that are O present in minor amounts.

CK1 A "motif", as in biological motif of a 191P4D12(b)-related protein, refers to any pattern of amino acids forming part Sof the primary sequence of a protein, that is associated with a particular function protein-protein interaction, protein-DNA S 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 ri 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 S 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 acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor CK residues.

00 0A "pharmaceutical excipient" comprises a material such as an adjuvant, a carrer, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

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

"Radioisotopes" include, but are not limited to the following (non-limiting exemplary uses are also set forth): Examples of Medical Isotopes: Isotope Description of use Actinium-225 See Thorium-229 (Th-229) (AC-225) 7 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the S Actinium-227 skeleton resulting from cancer breast and prostate cancers), and cancer (AC-227) radioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (81-212) Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cadm1 09 Cancer detection (Cd-109) Radiation source for radiotherapy of cancer, for food Irradiators, and for sterilization of medical supplies \Copper-64 Copper-64 A positron emitter used for cancer therapy and SPECT imaging cN (Cu-64) Copper-67 Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies breast 1 (Cu-67) and colon cancers, and lymphoma) 00 Dysprosium-166 Cancer radioimmunotherapy (Dy-166) S Erbium-169 Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and (Er-169) toes Europium-152 Radiation source for food irradiation and for sterilization of medical supplies (Eu-152) Europium-154 Radiation source for food irradiation and for sterilization of medical supplies (Eu-154) Gadolinium-153 Osteoporosis detection and nuclear medical quality assurance devices (Gd-153) Gold-198 Implant and Intracavity therapy of ovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone (Ho-166) marrow ablation, and rheumatoid arthritis treatment Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, lodine-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 radiolmmunotherapy 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 radiolmmunotherapy 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 Cancer radioimmunotherapy (Os-194) Palladium-103 Prostate cancer treatment (Pd-103) Platinum-195m Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug (Pt-195m) Phosphorus-32 Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer 00 (P-32) 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 Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of (P-33) blocked arteries arteriosclerosis and restenosis) Radium-223 (Ra-223) Rhenium-186 (Re-186) See Actinium-227 (Ac-227) Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, (Re-188) treatment of rheumatoid arthritis, and treatment of prostate cancer Rhodium-105 (Rh-105) Samarium-145 (Sm-145) Samarium-153 (Sm-153) Cancer radioimmunotherapy Ocular cancer treatment Cancer radioimmunotherapy and bone cancer pain relief Scandium-47 Cancer radioimmunotherapy and bone cancer pain relief (Sc-47) Strontium-89 (Sr-89) 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 Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy Technetium-99mSee Molybdenum-99 (Mo-99) (Tc-99m) Thorium-228 (Th-228) Thorium-229 (Th-229) Thulium-170 (Tm-170) Tin-117m (Sn-117m) Tungsten-188 (W-188) Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimmunotherapy Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha emitters used in cancer radioimmunotherapy Gamma source for blood irradiators, energy source for implanted medical devices Cancer immunotherapy and bone cancer pain relief Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries arteriosclerosis and restenosis) Xenon-127 Neurolmaging of brain disorders, high resolution SPECT studies, pulmonary function tests, (Xe-127) and cerebral blood flow studies Ytterblum-175 (Yb-175) Yttrium-91 (Y-91) Cancer radioimmunotherapy 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 0 (or nucleic acids, discussed herein) can incorporate any nudeotide 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 C agents.

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

SA "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation 00 in vitro.

SNon-limiting examples of small molecules include compounds that bind or interact with 191P4D12(b), ligands C( including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 191P4D12(b) protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 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, 191P4D12(b) 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 50°C; employ during hybridization a denaturing agent, such as formamide, for example, 50% (vfv) 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 p.g/ml), 0.1% SDS, and 10% dextran sulfate at 42 oC, with washes at 42oC in 0.2 x SSC (sodium chloride/sodium, citrate) and 50%.formamide at 55 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 Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the 00 filters in 1 x SSC at about 37-500C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as 0 necessary to accommodate factors such as probe length and the like.

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

Overall phenotypic frequences of HLA-supertypes in different ethnic populations are set forth in Table IV The nonlimiting 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 SA3 A3, All, A31, A*3301, A*6801, A*0301, A*1101, A'3101 B7: B7, 8*3501-03, B*51, 8*5301, 8*5401, 8*5501, B*5502, B'5601, B'6701, B*7801, B*0702, B*5101, B*5602 I B44: B*3701, B*4402, 8*4403, B*60 (B*4001), B61 (B*4006) C Al: A*0102, A*2604, A*3601, A*4301, A*8001 C A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003 C 827: B*1401-02, 8*1503, B*1509, 8*1510, 8*1518, B*3801-02, B*3901, 8*3902, B*3903-04, 8*4801-02, B*7301, 00 0 B'2701-08 858: B*1516, B*1517, 8*5701, B*5702, B58 B62: 8*4601, 852, B*1501 (B62), B*1502 (B75), B*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, an embryonic stage. A "transgene" is a DNA 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 191P4D12(b) 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 "191P4D12(b)-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 Sdifferent 191P4D12(b) proteins or fragments thereof, as well as fusion proteins of a 191P4012(b) protein and a heterologous polypeptide are also included. Such 191P4D12(b) proteins are collectively referred to as the 191P4D12(b)-related proteins, the 00 proteins of the invention, or 191P4D12(b). The term "191P4D12(b)-related protein" refers to a polypeptide fragment or a 0 191P4D12(b) protein sequence 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 C-i amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70,80, 85, 90, 95,100,105, 110,115,120,125,130,135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.

II.) 191P4D12(b) Polynucleotides One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 191P4D12(b) gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 191P4D12(b)-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or C ollgonucleotides complementary to a 191P4D12(b) gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 191P4D12(b) gene, mRNA, or to a 191P4012(b) encoding polynudeotide (collectively, 0 191P4D12(b) polynucleotides"). In all instances when referred to in this section, T can also be U in Figure 2.

SEmbodiments of a 191P4D12(b) polynucleotide include: a 191P4012(b) polynucleotide having the sequence C= shown in Figure 2, the nucleotide sequence of 191P4D12(b) as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nudeotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 191P4D12(b) 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 nudeotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2C, from nucleotide residue number 264 through nucleotide residue number 1796, including the a stop codon, wherein T can also be U; a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D, from nucleotide residue number 264 through nuceotide residue number 1796, including the stop codon, wherein T can also be U; (VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2E, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; (VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2F, from nucleotide residue number 789 through nucleotide residue number 1676, including the stop codon, wherein T can also be U; (VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 0 2G, from nucleotide residue number 264 through nucleotide residue number 1721, including the stop codon, 0 wherein T can also be U; [c (IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2H, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 21, from nucleotide residue number 708 through nucleotide residue number 1121, including the stop codon, wherein T can also be U; Ci (XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure C 2J, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, C] wherein T can also be U; 00 S(XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure CK1 2K, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; (XIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2L, from nucleotide residue number 264 through nucleotide residue number 1796, including the stop codon, wherein T can also be U; (XIV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2M, from nucleotide residue number 264 through nucleotide residue number 1799, including the stop codon, wherein T can also be U; (XV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2N, from nucleotide residue number 708 through nucleotide residue number 1121, including the stop codon, wherein T can also be U; (XVI) a polynucleotide that encodes a 191P4D12(b)-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-N; (XVII) a polynucleotide that encodes a 191P4D12(b)-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-N; (XVIII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX; (XIX) 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-B and 3E-G in any whole number increment up to 510 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 (XX) 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-B and 3E-G In any whole number increment up to 510 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 less than in the Hydropathicity profile of Figure 6; 00 O (XXI) a polynudeotide 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-B and 3E-G in any whole number increment up to 510 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 in the Percent Accessible Residues profile of Figure 7; c (XXII) 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-B and 3E-G in any whole number increment up to 510 that includes 1, 2,3,4, 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 acid position(s) having a value greater than in the Average Flexibility profile of Figure 8; C0 (XXIII) a polynudeotde that encodes a peptide region of at least 5, 6,7, 8, 9, 10,11, 12,13,14,15,16,17,18, 0 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-B and 3E-G C N I in any whole number increment up to 510 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 in the Beta-turn profile of Figure 9; (XXIV) a polynucleoude 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 3C in any whole number increment up to 295 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 Hydrophilidty profile of Figure (XXV) a polynudeotide that encodes a peptide region of at least 5,6, 7, 8, 9,10,11,12,13,14,15,16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a peptide of Figure 3C in any whole number increment up to 295 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 less than 0.5 in the Hydropathicity profile of Figure 6; (XXVI) a polynudeotide that encodes apeptide 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 3C in any whole number incrementup to 295 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; (XXVII) 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 3C in any whole number increment up to 295 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; (XXVIII) a polynucleotide that encodes a peptide region of at least 5,6, 7, 8, 9,10,11,12,13,14,15,16,17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3C in any whole number incrementup to 295 thatincludes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18,19, 20,21,22, 00 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 amino acid positon(s) having a value greater than 0.5 in the Beta- O turn profile of Figure 9 cl (XXIX) 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 ofa peptide of Figure 3D in any whole number increment up to 485 that includes 1, 2,3,4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, S23, 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 (XXX) 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 3D in any whole C number increment up to 485 that includes 1,2, 3,4, 5, 6,7, 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, S23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35 amino acid position(s) having a value less than 0.5 in the 00 Hydropathicity profile of Figure 6; S(XXXI) 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 3D in any whole number increment up to 485 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; (XXXII) 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 3D in any whole number increment up to 485 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; (XXXIII) 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 3D in any whole number increment up to 485 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 Betaturn profile of Figure 9 (XXXIV) 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 3H in any whole number Increment up to 511 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 (XXXV) 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 3H in any whole number increment up to 511 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 less than 0.5 in the Hydropathicity profile of Figure 6; (XXXVI) a polynucleotide that encodes a peptide region of at least 5,6, 7 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 3H in any whole 00 numberincrementupto511 thatincludes 1,2,3,4,5,6,7, 8, 9 ,10,11,12,13,14,15,16,17, 18,19,20,21, 22, S23, 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; (XXXVII) 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 3H in any whole number increment up to 511 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; (XXXVIII) 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 3H in any whole (N number increment up to 511 that includes 1,2, 3,4, 5, 6,7, 8, 9,10,11,12,13,14,15,16,17,18,19,20, 21, 22, 00 S23, 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 Betaq turn profile of Figure 9 (XXXIX) 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 31-J in any whole number increment up to 137 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 (XL) 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 31-J in any whole number increment up to 137 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 less than 0.5 in the Hydropathicity profile of Figure 6; (XLI) a polynudeotide that encodes a peptide region of at least 5, 6,7,8, 9,10,11,12,13,14,15,16,17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 31-J in any whole number increment up to 137 that Indudes 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; (XLII) 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 31-J in any whole number increment up to 137 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; (XLIII) 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 31-J in any whole number increment up to 137 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-turn profile of Figure 9 (XLIV) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XLIII).

00 (XLV) a peptide that is encoded by any of to (XLIV); and

O

S(XLVI) a composition comprising a polynucleotide of any of (I)-(XLIII) or peptide of (XLV) together with a pharmaceutical excipient and/or in a human unit dose form.

(XLVII) a method of using a polynucleotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a method to modulate a cell expressing 191P4D12(b),

L

c (XLVIII) a method of using a polynuceotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 191P4D12(b) (XLIX) a method of using a polynucleotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 191P4D12(b), said cell from a cancer of a tissue listed In Table I; <00 00(L) a method of using a polynucleotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a Smethod to diagnose, prophylax, prognose, or treat a a cancer; (LI) a method of using a polynucleotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and, (LII) a method of using a polynucleotide of any (I)-(XLIV) or peptide of (XLV) or a composition of (XLVI) in a method to identify or characterize a modulator of a cell expressing 191P4D12(b).

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

Typical embodiments of the invention disclosed herein include 191P4D12(b) polynucleotides that encode specific portions of 191P4D12(b) mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: 4, 5, 6, 7, 8, 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, 505 or 510 more contiguous amino acids of 191P4D12(b) variant 1; the maximal lengths relevant for other variants are: variant 2, 510 amino acids; variant 6, 295 amino acids, variant 7, 485 amino acids, variant 10, 510 amino acids, variant 11,510 amoni adds, variant 12, 510 amoni acids, variant 13, 511 amino acids, variant 9, 137 amino acids, and variant 14, 137 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 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 191P4D12(b) protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid to about amino acid 100 of the 191P4D12(b) protein shown in 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 00 of the 191P4D12(b) protein are embodiments of the invention. Wherein it is understood that each particular amino acid 0 position discloses that position plus or minus five amino acid residues.

l Polynucleotides encoding relatively long portions of a 191P4D12(b) protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid (or 30, or 40 or 50 etc.) of the 191P4012(b) protein "or variant shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynudeotide fragments can include any portion of the 191P4D12(b) sequence r as shown in Figure 2.

Additional illustrative embodiments of the invention disclosed herein include 191P4D12(b) polynucleotide fragments encoding one or more of the biological motifs contained within a 191P4D12(b) protein "or variant" sequence, C- including one or more of the motif-bearing subsequences of a 191P4D12(b) protein "or varianf' 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 ri of 191P4D12(b) protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, 00 typical polynucleotide fragments can encode one or more of the 191P4D12(b) 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 191P4D12(b) Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 191P4D12(b) gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 191P4D12(b)." For example, because the 191P4D12(b) gene maps to this chromosome, polynucleotides that encode different regions of the 191P4D12(b) 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 al., 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 191P4D12(b) proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 191P4D12(b) 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 (1994)).

Furthermore, as 191P4D12(b) was shown to be highly expressed in prostate and other cancers, 191P4D12(b) polynucleotides are used in methods assessing the status of 191P4D12(b) gene products in normal versus cancerous 0 tissues. Typically, polynucleotides that encode specific regions of the 191P4D12(b) proteins are used to assess the S presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 191P4D12(b) 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 C regions within the protein.

II.A.2.) Antisense Embodiments Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including C alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or 00 C protein expression of 191P4D12(b). 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 0 in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 191P4D12(b) 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, 191P4D12(b). See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 191P4D12(b) 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 O-oligos with 3H-1,2benzodithiol-3-one-1,1-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 al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 191P4D12(b) antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, Partridge et al., 1996, Antisense Nucleic Acid Drug Development 6: 169-175).

The 191P4D12(b) 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 191P4D12(b) genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonudeotide complementary to this region allows for the selective hybridization to 191 P4D12(b) mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 191P4D12(b) antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 191P4D12(b) mRNA. Optionally, 191P4D12(b) 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 191P4012(b). Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 191P4D12(b) 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 radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 00 191P4D12(b) polynucleotide in a sample and as a means for detecting a cell expressing a 191P4D12(b) protein.

O

SExamples of such probes include polypeptides comprising all or part of the human 191P4D12(b) cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 191P4D12(b) mRNAs are also described in the ct Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 191P4D12(b) mRNA.

0 The 191P4D12(b) polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification andlor detection of the 191P4D12(b) gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 191P4D12(b) polypeptides; as tools for modulating or inhibiting the expression of the 191P4D12(b) gene(s) and/or translation of the 191P4D12(b) transcript(s); and as therapeutic agents.

0 The present invention includes the use of any probe as described herein to identify and isolate a 191P4D12(b) or 00 191P4D12(b) related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence perse, which would comprise all or most of the sequences found in the probe used.

ll.A.4.) Isolation of 191P4D12(b)-Encoding Nucleic Acid Molecules The 191P4D12(b) cDNA sequences described herein enable the isolation of other polynucleotides encoding 191P4D12(b) gene product(s), as well as the isolation of polynucleotides encoding 191P4D12(b) gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 191P4D12(b) gene product as well as polynucleotides that encode analogs of 191P4D12(b)-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 191P4D12(b) 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 191P4D12(b) gene cDNAs can be identified by probing with a labeled 191P4D12(b) cDNA or a fragment thereof. For example, in one embodiment, a 191P4D12(b) 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 191P4D12(b) gene. A 191P4D12(b) gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 191P4D12(b) DNA probes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems The invention also provides recombinant DNA or RNA molecules containing a 191P4D12(b) polynudeotide, 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 191P4D12(b) 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 191P4D12(b) or a fragment, analog or homolog thereof can be used to generate 191P4D12(b) proteins or fragments thereof using any number of Shost-vector systems routinely used and widely known In the art.

O A wide range of host-vector systems suitable for the expression of 191P4D12(b) proteins or fragments thereof are C= available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors S for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRatkneo (Muller etal., 1991, MCB 11:1785). Using these expression vectors, 191P4D12(b) 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 C' systems of the invention are useful for the production of a 191P4D12(b) protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 191P4D12(b) and 191P4D12(b) mutations or analogs.

Recombinant human 191P4D12(b) protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 191P4D12(b)-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 191P4D12(b) or fragment, analog or homolog thereof, a 191P4D12(b)- 1 related protein is expressed in the 293T cells, and the recombinant 191P4D12(b) protein is isolated using standard 00 C purification methods affinity purification using anti-191P4D12(b) antibodies). In another embodiment, a 191P4D12(b) o 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 191P4D12(b) 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 191P4D12(b) coding sequence can be used for the generation of a secreted form of recombinant 191P4D12(b) protein.

As discussed herein, redundancy in the genetic code permits variation in 191P4D12(b) 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.jpl-nakamura/codon.html.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonlintron 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)).

III.) 191P4D12(b)-related Proteins Another aspect of the present invention provides 191 P4D12(b)-related proteins. Specific embodiments of 191P4D12(b) proteins comprise a polypeptide having all or part of the amino acid sequence of human 191P4D12(b) as shown in Figure 2 or Figure 3. Alternatively, embodiments of 191P4D12(b) proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 191 P4D12(b) shown in Figure 2 or Figure 3.

Embodiments of a 191P4D12(b) polypeptide include: a 191P4D12(b) polypeptide having a sequence shown in Figure 2, a peptide sequence of a 191P4D12(b) 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 191P4D12(b) peptides comprise, without limitation: 00 a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in Figure 2A-N or Figure 3A-J; (II) a 191P4D12(b)-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-N or 3A-J; (III) a 191P4D12(b)-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-N or 3A-J; (IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso C that it is not an entire protein of Figure 2; 00 00 a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is 0also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of Figure 2; C (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-B or 3E-G, in any whole number increment up to 510 respectively that includes at least 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 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-B or 3E-G, in any whole number increment up to 510 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 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-B or 3E-G, in any whole number increment up to 510 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-B or 3E-G, in any whole number increment up to 510 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 OO than 0.5 in the Average Flexibility profile of Figure 8;

O

S(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-B or 3E-G in any whole number c increment up to 510 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 Sthan 0.5 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3C, in any whole number increment up to 295 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, C 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 00 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, C 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3C, in any whole number increment up to 295 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, 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 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 3C, in any whole number increment up to 295 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, 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 3C, in any whole number increment up to 295 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 3C in any whole number increment up to 295 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-turn profile of Figure 9; (XIX) 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 3D, In any whole number increment up to 485 respectively that indudes 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 (XX) 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 3D, in any whole number increment up to 485respectively 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, 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 0 (XXI) 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 31), in any whole number Increment up to 485 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 c-K1 the Percent Accessible Residues profile of Figure 7; (XXII) 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 3D, in any whole number increment up to 485 respectively that includes atleast at least1, 2 3 4 6 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in 00 the Average Flexibility profile of Figure 8; (XXIII) 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 3D in any whole number increment up to 485 respecively that includes at least at leastl1, 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-turn profile of Figure 9; (XXIV) 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 3H, in any whole number increment up to 611 respectively that includes at leasti1, 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 (XXV) 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 3H, in any whole number increment up to 511 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, 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; (XXVI) 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 3H, in any whole number increment up to 511 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, 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; (XXVII) 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 3H, in any whole number increment up to 511 respectively that includes atleast atleast, 2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XXVIII) 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, 00 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of Figure 3H in any whole number increment up to O 511 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, C22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9; (XXIX) 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 31-J, in any whole number increment up to 137 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 0.5 in the Hydrophilicity profile of Figure S(XXX) 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 31-J, in any whole number increment 0 0 up to 137 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, 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; (XXXI) 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 31-J, in any whole number Increment up to 137 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, 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; (XXXII) 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 31-J, in any whole number increment up to 137 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, 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; (XXXIII) 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 31-J in any whole number increment up to 137 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-turn profile of Figure 9; (XXXIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively; (XXXV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively; (XXXVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively; (XXXVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively; (XXXVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XXXIX) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX; (XL) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XLI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX; (XLII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide: 0 i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino 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 Hydrophilicity profile of Figure

C

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 Sthan 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; 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 RFigure 7; iv) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment 00 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, C 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; (XLIII) a composition comprising a peptide of (I)-(XLII) or an antibody or binding region thereof together with a pharmaceutical excipient andlor in a human unit dose form.

(XLIV) a method of using a peptide of or an antibody or binding region thereof or a composition of (XLIII) in a method to modulate a cell expressing 191P4D12(b), (XLV) a method of using a peptide of (I)-(XLII) or an antibody or binding region thereof or a composition of (XLIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 191P4D12(b) (XLVI) a method of using a peptide of (I)-(XLII) or an antibody or binding region thereof or a composition (XIIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 191P4D12(b), said cell from a cancer of a tissue listed in Table I; (XLVII) a method of using a peptide of (I)-(XLII) or an antibody or binding region thereof or a composition of (XLIII) in a method to diagnose, prophylax, prognose, or treat a a cancer; (XLVIII) a method of using a peptide of (I)-(XLII) or an antibody or binding region thereof or a composition of (XLIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and, (XLIX) a method of using a a peptide of (I)-(XLII) or an antibody or binding region thereof or a composition (XLIII) in a method to identify or characterize a modulator of a cell expressing 191P4D12(b).

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

Typical embodiments of the invention disclosed herein include 191P4D12(b) polynucleotides that encode specific portions of 191P4D12(b) mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: 4, 5, 6, 7, 8, 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, 00 75, 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, O 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 505, or 510 or more contiguous amino acids of 191P4D12(b) C1 variant 1; the maximal lengths relevant for other variants are: variant 2, 510 amino acids; variant 6, 295 amino acids, variant 7, 485 amino acids, variant 10, 510 amino acids, variant 11, 510 amino acids, variant 12, 510 amino acids, variant 13, 511 amino acids, variant 9, 137 amino acids, and variant 14, 137 amino acids..

In general, naturally occurring allelic variants of human 191P4D12(b) share a high degree of structural identity and C homology 90% or more homology). Typically, allelic variants of a 191P4D12(b) protein contain conservative amino acid substitutions within the 191P4D12(b) sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 191P4D12(b). One class of 191P4D12(b) allelic variants are proteins that share a high degree of homology with at least a small region of a particular 191P4D12(b) 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 CN sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics.

00 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); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei 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 191P4D12(b) proteins such as polypeptides having amino acid insertions, deletions and substitutions. 191P4D12(b) variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.

Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10.6487 (1987)), cassette mutagenesis (Wells et al., 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 191P4D12(b) 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.

00 As defined herein, 191P4D12(b) variants, analogs or homologs, have the distinguishing attribute of having at least 0 0 one epitope that is "cross reactive" with a 191P4D12(b) protein having an amino acid sequence of Figure 3. As used in this sentence, "cross reactive" means that an antibody or T cell that specifically binds to a 191P4D12(b) variant also specifically c binds to a 191P4D12(b) protein having an amino acid sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 191P4D12(b) protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded 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 al., J Immunol (1985) 135(4):2598-608.

L

C Other classes of 191P4D12(b)-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 191P4D12(b) protein variants or 00 analogs comprises one or more of the 191P4D12(b) biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 191P4D12(b) fragments (nucleic or amino acid) that have altered Cl functional 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 191P4D12(b) 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 191P4D12(b) 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 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 191P4D12(b) protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 191P4D12(b) 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 191P4D12(b) protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

191P4D12(b)-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 191P4D12(b)-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 191P4D12(b) protein (or variants, homologs or analogs thereof).

III.A.) Motif-bearing Protein Embodiments OO Additional illustrative embodiments of the invention disclosed herein include 191P4D12(b) polypeptides comprising S the amino acid residues of one or more of the biological motifs contained within a 191P4D12(b) polypeptide sequence set Cr 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 Internet sites (see, URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edulseq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; O ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsitl .html; EpimatrixTM and Eplmer

T

Brown University, S brown.edulResearch/TB-HIVLab/epimatrixepimatx.html; and BIMAS, bimas.dcrtnih.gov/.).

Motif bearing subsequences of all 191P4D12(b) variant proteins are set forth and identified in Tables VIII-XXI and

XXII-XLIX.

C 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 C family, motif name or description and most common function; location information is included if the motif is relevant for 00 0 location.

S Polypeptides comprising one or more of the 191P4D12(b) motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 191P4D12(b) motifs discussed above are associated with growth dysregulation and because 191P4D12(b) is overexpressed in certain cancers (See, Table I).

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 etal., 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 al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., 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. 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in 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 191P4D12(b) protein that are capable of optimally binding to specified HLA alleles Table IV; Epimatrix M and Epimer

TM

Brown University, URL brown.edulResearch/TB- HIV_Lablepimabix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have 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 epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

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

A variety of references reflect the art regarding the identification and generation of epitopes In a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201- 212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney etal., J. Immunol. 1996 157(8): 3480-90; and Falk et Nature 351: 290-6 00 (1991); Hunt et al., Science 255:1261-3(1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker etal., J. Immunol.

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

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

Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred S embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of Cr 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 00 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.

C 191P4D12(b)-related proteins are embodied in many forms, preferably in isolated form. A purified 191P4D12(b) protein molecule will be substantially free of other proteins or molecules that impair the binding of 191P4012(b) to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 191P4D12(b)-related proteins include purified 191P4D12(b)-related proteins and functional, soluble 191P4D12(b)-related proteins. In one embodiment, a functional, soluble 191P4D12(b) protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 191P4D12(b) proteins comprising biologically active fragments of a 191P4D12(b) amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 191P4D12(b) protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 191P4D12(b) 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 bind to the starting protein.

191P4D12(b)-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, Gamier-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-191P4D12(b) antibodies or T cells or in identifying cellular factors that bind to 191P4D12(b). 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.

Hydropathidty profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, 1982, J. Mol. Biol. 157:105-132. Percent Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran Ponnuswamy 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, Roux 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a 191P4D12(b) 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 Epimatrix T M and Epimer T M Brown University, URL (brown.edu/ResearchTfB- HIVLab/epimatrixepimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 191P4D12(b) that are presented in the context of human MHC Class I molecules, HLA-A1, A2, A3, All, A24, 87 and B35 were 0 predicted (see, Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 191P4D12(b) protein 0 and relevant portions of other variants, for HLA Class I predictions 9 flanking residues on either side of a point mutation C- or exon Juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction Scorresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatlcs and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmiheidelberg.com/.

CN 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 al., Nature 351: 290-6 (1991); r- Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 LC (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 peplides are 8- 1 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine or methionine at 00 C position 2 and a valine or leucine at the C-terminus (see, Parker et at., J. Immunol. 149:3580-7 (1992)). Selected S results of 191P4D12(b) 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, 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 37oC 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 EpimatrixTM 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.bml-heidelberg.com/, or BIMAS, bimas.dcrtnih.gov/) are to be "applied" to a 191P4D12(b) protein in accordance with the invention. As used in this context "applied" means that a 191P4D12(b) protein is evaluated, visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 191P4D12(b) protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 191P4D12(b)-related Proteins In an embodiment described in the examples that follow, 191P4D12(b) 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 191P4D12(b) 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 191P4D12(b) protein in transfected cells. The secreted HIS-tagged 191P4D12(b) In the culture media can be purified, using a nickel column using standard techniques.

III.C.) Modifications of 191P4D12(b)-related Proteins 00 Modifications of 191P4D12(b)-related proteins such as covalent modifications are included within the scope of this 0 invention. One type of covalent modification includes reacting targeted amino acid residues of a 191P4D12(b) polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a t 191P4D12(b) protein. Another type of covalent modification of a 191P4D12(b) polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent 0 modification of 191P4D12(b) comprises linking a 191P4D12(b) 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 191P4D12(b)-related proteins of the present invention can also be modified to form a chimeric molecule comprising 191P4D12(b) 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 tumor- 00 associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of S fragments of a 191P4D12(b) sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, CK 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 191P4D12(b). A chimeric molecule can comprise a fusion of a 191P4D12(b)-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 carboxylterminus of a 191P4D12(b) protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 191P4D12(b)-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 191P4012(b) 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, U.S. Patent No. 5,428,130 issued June 27, 1995.

III.D.) Uses of 191P4D12(b)-related Proteins The proteins of the invention have a number of different specific uses. As 191P4D12(b) is highly expressed in prostate and other cancers, 191P4D12(b)-related proteins are used in methods that assess the status of 191P4D12(b) gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 191P4D12(b) protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 191P4D12(b)-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 191P4D12(b) polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 191P4D12(b)-related proteins that contain the amino acid residues of one or more of the biological motifs in a 191P4D12(b) protein are used to screen for factors that interact with that region of 191P4D12(b).

191P4D12(b) protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies antibodies recognizing an extracellular or intracellular epitope of a 191P4D12(b) protein), for identifying agents or cellular factors that bind to 191P4D12(b) 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 191P4D12(b) genes, or by analogs, homologs or fragments thereof, have a variety of 00 uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular O constituents that bind to a 191P4D12(b) gene product. Antibodies raised against a 191P4D12(b) protein or fragment thereof CN are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 191P4D12(b) protein, such as those listed in Table 1. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 191P4D12(b)-related nucleic acids or proteins are also used in generating HTL or CTL responses.

C Various immunological assays useful for the detection of 191P4D12(b) 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 si capable of detecting 191P4D12(b)-expressing cells in radioscintigraphic imaging methods). 191P4D12(b) proteins are also c particularly useful in generating cancer vaccines, as further described herein.

00 0 IV.) 191P4D12(b) Antibodies Another aspect of the invention provides antibodies that bind to 191P4D12(b)-related proteins. Preferred antibodies specifically bind to a 191P4D12(b)-related protein and do not bind (or bind weakly) to peptides or proteins that are not 191P4D12(b)-related 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 altematively in a range of pH 7.0 to 8.0, or altematively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4°C to 37 0 C. For example, antibodies that bind 191P4D12(b) can bind 191P4D12(b)-related proteins such as the homologs or analogs thereof.

191P4D12(b) 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 other cancers, to the extent 191P4D12(b) is also expressed or overexpressed in these other cancers.

Moreover, intracellularly expressed antibodies single chain antibodies) are therapeutically useful in treating cancers in which the expression of 191P4D12(b) is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 191P4D12(b) and mutant 191P4D12(b)-related proteins. Such assays can comprise one or more 191P4D12(b) antibodies capable of recognizing and binding a 191P4012(b)-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, enzymelinked immunosorbent assays (ELSA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

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

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 191P4012(b) are also provided by the invention, including but not limited to radioscinligraphic imaging methods using labeled 191P4D12(b) antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 191P4D12(b) expressing cancers such as prostate cancer.

191P4D12(b) antibodies are also used in methods for purifying a 191P4D12(b)-related protein and for isolating 191P4D12(b) homologues and related molecules. For example, a method of purifying a 191P4D12(b)-related protein comprises incubating a 191P4D12(b) antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 191P4D12(b)-related protein under conditions that permit the 191P4D12(b) antibody to bind to the 191P4D12(b)-related protein; washing the solid matrix to eliminate impurities; and eluting the 191P4D12(b)-related protein from the coupled antibody. Other uses of 191P4D12(b) antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 00 191P4D12(b) protein.

SVarious 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 191P4D12(b)-related protein, peptide, or fragment, in isolated or c 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 191P4D12(b) can also be used, such as a 191P4D12(b) GST- 0 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 191P4012(b)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 191P4D12(b)related protein or 191P4D12(b) expressing cells) to generate an immune response to the encoded immunogen (for review, see 0 Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

00 The amino acid sequence of a 191P4D12(b) protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 191P4D12(b) protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a

C

N 191P4D12(b) amino acid sequence are used to identify hydrophilic regions in the 191P4D12(b) structure. Regions of a 191P4D12(b) 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-Schultz 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. Hydropathlcity profiles can be generated using the method of Kyte, J. and Doolittle, 1982, J. Mol. Biol. 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 191P4D12(b) 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 methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 191P4D12(b) immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

191P4D12(b) 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 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 191P4D12(b)-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.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 191P4D12(b) protein can also be produced in the context of chimeric or complementaritydetermining region (CDR) grafted antibodies of multiple species origin. Humanized or human 191P4D12(b) 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 00 for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et 1993, Proc. Natl. Acad. Sd. USA 89: 4285 and Sims et al., 1993, J. Immunol.

CN 151:2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 191P4D12(b) monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries phage display) (Griffiths and 1 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); I> Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 191P4D12(b) monoclonal K antibodies can also be produced using transgenic mice engineered to contain human Immunoglobulin gene loci as described in PCT Patent Application W098124893, Kucherlapati and Jakobovits et al., published December 3, 1997 (see also, Jakobovits, CN 1998, Exp. Opin. Invest. Drugs 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 00 0 2000; and, 6,114598 issued 5 September 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 191P4D12(b) antibodies with a 191P4D12(b)-related protein can be established by a number of well known means, including Western blot, Immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 191P4D12(b)related proteins, 191P4D12(b)-expressing cells or extracts thereof. A 191P4D12(b) 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 radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 191P4D12(b) 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).

191P4D12(b) 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 worldwide 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 (Buus, S. etal., Cell 47:1071, 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 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 al., J. Immunol. 160:3363,1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scrpts.hlaserver.dll/home.htm); Sette, A.

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. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunof. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 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 cleft/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 Immunity 4:203, 1996; Fremont et al., Immunity 8:305,1998; Stem et 00 Structure 2:245, 1994; Jones, E.Y. Curr. Opin. Immunol. 9:75,1997; Brown, J. H. et Nature 364:33, 1993; Guo, H. C.

O

et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, S1992; Matsumura, M. et Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science c 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 0 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, S amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or 0 immunogenicity.

00 Various strategies can be utilized to evaluate cellular immunogeniclty, including: S1) Evaluation of primary T cell cultures from normal individuals (see, Wentworth, P. A. et al., Mol. Immunol.

CN" 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796,1997; Kawashima, I. et al., 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 J. Immunol. 26:97, 1996; Wentworth, P.

A. et Int. Immunol. 8:651, 1996; Alexander, J. et 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 51Cr-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 and/or from chronically ill patients (see, Rehermann, B. etal., J. Exp. Med. 181:1047, 1995; Doolan, D. L. e al., Immunity 7:97, 1997; Bertoni, R. et J. Clin. Invest. 100:503,1997; Threlkeld, S. C. et J. Immunol. 159:1648, 1997; Diepolder, H. M. et J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "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 targets, T cell proliferation, or lymphokine release.

VI.) 191P4D12(b) Transgenic Animals Nucleic acids that encode a 191P4D12(b)-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 191P4D12(b) can be used to clone genomic DNA that encodes 191P4D12(b). The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 191P4D12(b). 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 S 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 191P4D12(b) transgene 0 incorporation with tissue-specific enhancers.

CK Transgenic animals that include a copy of a transgene encoding 191P4D12(b) can be used to examine the effect of increased expression of DNA that encodes 191P4D12(b). 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, CN compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 191P4D12(b) can be used to construct a 191P4D12(b) "knock out" animal that has a defective or altered gene encoding 191P4D12(b) as a result of homologous recombination between the C endogenous gene encoding 191P4D12(b) and altered genomic DNA encoding 191P4D12(b) introduced into an embryonic CK cell of the animal. For example, cDNA that encodes 191P4D12(b) can be used to clone genomic DNA encoding 00 191P4012(b) in accordance with established techniques. A portion of the genomic DNA encoding 191P4D12(b) 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, e.g., 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, 69:915 (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 191 P4D12(b) polypeptide.

VII.) Methods for the Detection of 191P4D12(b) Another aspect of the present invention relates to methods for detecting 191P4D12(b) polynucleotides and 191P4D12(b)-related proteins, as well as methods for identifying a cell that expresses 191P4D12(b). The expression profile of 191P4D12(b) makes it a diagnostic marker for metastasized disease. Accordingly, the status of 191P4D12(b) gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 191P4D12(b) gene products in patient samples can be analyzed by a variety protocols that are well known In the art including immunohistochemical analysis, the variety of Northem blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of 191P4D12(b) polynudeotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 191P4D12(b) polynucleotides include, for example, a 191P4D12(b) gene or fragment thereof, 191P4D12(b) mRNA, alternative splice variant 191P4D12(b) mRNAs, and recombinant DNA or RNA molecules that contain a 191P4D12(b) polynucleotide. A number of methods for amplifying andlor detecting the presence of 191P4D12(b) 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 191P4D12(b) 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 191P4D12(b) 00 polynucleotides as sense and antisense primers to amplify 191P4D12(b) cDNAs therein; and detecting the presence of the amplified 191P4D12(b) cDNA. Optionally, the sequence of the amplified 191P4D12(b) cDNA can be determined.

In another embodiment, a method of detecting a 191P4D12(b) gene in a biological sample comprises first isolating c genomic DNA from the sample; amplifying the Isolated genomic DNA using 191P4D12(b) polynucleotides as sense and antisense primers; and detecting the presence of the amplified 191P4D12(b) gene. Any number of appropriate sense and S antisense probe combinations can be designed from a 191P4D12(b) nucleolide sequence (see, Figure 2) and used for this purpose.

The invention also provides assays for detecting the presence of a 191P4D12(b) protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 191P4D12(b)- CKl related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot 0 analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 00 191P4D12(b)-related protein in a biological sample comprises first contacting the sample with a 191P4D12(b) antibody, a S 191P4D12(b)-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 191P4D12(b) C1 antibody; and then detecting the binding of 191P4D12(b)-related protein in the sample.

Methods for Identifying a cell that expresses 191P4D12(b) are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 191P4D12(b) gene comprises detecting the presence of 191P4D12(b) 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 191 P4D12(b) riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 191P4D12(b), 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 191P4D12(b) gene comprises detecting the presence of 191P4D12(b)-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 191P4D12(b)-related proteins and cells that express 191P4D12(b)-related proteins.

191P4D12(b) expression analysis is also useful as a tool for identifying and evaluating agents that modulate 191P4D12(b) gene expression. For example, 191P4D12(b) 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 191P4D12(b) expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 191P4D12(b) expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 191P4D12(b)-related Genes and Their Products Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, Alers et al., Lab 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 191P4D12(b) 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 191P4D12(b) in a biological sample of interest can be compared, for example, to the status of 191P4D12(b) 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 191P4D12(b) 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 00 predetermined normative value such as a predetermined normal level of mRNA expression (see, Grever et al., J. Comp.

S Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 191P4D12(b) status in a sample.

CK1 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 191P4D12(b) O expressing cells) as well as the level, and biological activity of expressed gene products (such as 191P4012(b) mRNA, CI polynucleotides and polypeptides). Typically, an alteration in the status of 191P4D12(b) comprises a change in the location of 191P4D12(b) and/or 191P4D12(b) expressing cells and/or an increase in 191P4D12(b) mRNA and/or protein expression.

191P4D12(b) status in a sample can be analyzed by a number of means well known in the art, including without aC 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 191P4D12(b) gene and gene rC products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern 00 0 Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 191P4D12(b) in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 191P4D12(b) gene), Northern analysis and/or PCR analysis of 191P4D12(b) mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 191P4D12(b) 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 191P4D12(b) proteins and/or associations of 191P4D12(b) proteins with polypeptide binding partners). Detectable 191P4D12(b) polynucleotides include, for example, a 191P4D12(b) gene or fragment thereof, 191P4D12(b) mRNA, alternative splice variants, 191P4D12(b) mRNAs, and recombinant DNA or RNA molecules containing a 191P4D12(b) polynucleotide.

The expression profile of 191P4D12(b) 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 191P4D12(b) provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 191P4D12(b) status and diagnosing cancers that express 191P4D12(b), such as cancers of the tissues listed in Table I. For example, because 191P4D12(b) mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 191P4D12(b) mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 191P4D12(b) dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 191P4D12(b) provides information including the presence, stage and location of dysplastic, precancerous 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.

Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 191P4D12(b) in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 191P4D12(b) in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 191P4012(b) in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 191P4D12(b) expressing cells those that express 191P4D12(b) mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 191P4D12(b)-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 191P4D12(b) 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, 00 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 S disease progression (see, Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., 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 191P4D12(b) gene products by determining the 0 status of 191P4D12(b) 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 191P4D12(b) gene products in a corresponding normal sample. The presence of aberrant 191P4012(b) gene products in S 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, 00 comprising detecting a significant increase in 191P4D12(b) 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 191P4D12(b) mRNA can, for Cl example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 191P4D12(b).

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 191P4D12(b) mRNA or express it at lower levels.

In a related embodiment, 191P4D12(b) status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 191P4D12(b) protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 191P4D12(b) expressed in a corresponding normal sample. In one embodiment, the presence of 191P4D12(b) protein is evaluated, for example, using immunohistochemical methods.

191P4D12(b) antibodies or binding partners capable of detecting 191P4D12(b) 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 191P4D12(b) nudeotide 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 observed in a large number of proteins associated with a growth dysregulated phenotype (see, Marrogi et at., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 191P4D12(b) may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 191P4D12(b) indicates a potential 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 nuceic acid or amino acd sequences of 191P4D12(b) gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing 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).

Additionally, one can examine the methylation status of a 191P4D12(b) gene in a biological sample. Aberrant demethylation andlor hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the 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 al., 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 ;0 but is expressed in 25-50% of prostate cancers) is Induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art For example, one can utilize, in Southern hybridization S 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 blsulfite (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.

C Gene amplification is an additional method for assessing the status of 191P4D12(b). Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the 00 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. Alternatively, antibodies are CK" 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 191P4D12(b) expression. The presence of RT-PCR amplifiable 191P4D12(b) 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 etal., 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 191P4D12(b) mRNA or 191P4D12(b) protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 191P4D12(b) mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 191P4D12(b) in prostate or other tissue is examined, with the presence of 191P4D12(b) 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 191P4D12(b) nuceotide 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 in 191P4D12(b) gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 191P4D12(b) mRNA or 191P4D12(b) protein expressed by tumor cells, comparing the level so determined to the level of 191P4D12(b) mRNA or 191P4D12(b) protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 191P4D12(b) mRNA or 191 P4D12(b) 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 191P4D12(b) is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 191P4D12(b) nudeotide 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.

C0 Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an S individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 191P4D12(b) mRNA or 191P4D12(b) protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 191P4D12(b) mRNA or 191P4D12(b) protein expressed in an equivalent tissue S sample taken from the same individual at a different time, wherein the degree of 191P4D12(b) mRNA or 191P4D12(b) protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the S progression of a cancer is evaluated by determining 191P4D12(b) expression in the tumor cells over time, where Increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 191P4D12(b) nudeotide 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.

CK1 The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic 00 protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 191P4D12(b) gene and 191P4D12(b) gene products (or perturbations in 191P4D12(b) gene and 191P4D12(b) 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, Bocking et al., 1984, Anal. Quant Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 191P4D12(b) gene and 191P4D12(b) gene products (or perturbations in 191P4012(b) gene and 191P4D12(b) 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 191P4D12(b) gene and 191P4D12(b) gene products (or perturbations in 191P4D12(b) gene and 191P4D12(b) gene products) and another factor associated with malignancy entails detecting the overexpression of 191P4D12(b) mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 191P4D12(b) mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment the expression of 191P4D12(b) and PSA mRNA in prostate tissue is examined, where the coincidence of 191P4D12(b) 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 191P4D12(b) mRNA or protein are described herein, and standard nudeic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 191P4D12(b) mRNA include in situ hybridization using labeled 191P4D12(b) riboprobes, Northern blot and related techniques using 191P4D12(b) polynucleotide probes, RT-PCR analysis using primers specific for 191P4D12(b), and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 191P4D12(b) mRNA expression. Any number of primers capable of amplifying 191P4D12(b) 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 191P4D12(b) protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 191P4D12(b) 00 0 The 191P4012(b) protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, 01 small molecules and other agents that interact with 191P4D12(b), as well as pathways activated by 191P4D12(b) 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 L-i 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 al., Nature 402: 4 November 1999, 83-86).

Cl Alternatively one can screen peptide libraries to identify molecules that interact with 191P4012(b) protein L) sequences. In such methods, peptides that bind to 191P4D12(b) are identified by screening libraries that encode a random S or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage 0 coat proteins, the bacteriophage particles are then screened against the 191P4D12(b) 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 191P4D12(b) 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 191 P4D12(b) are used to identify protein-protein interactions mediated by 191P4D12(b). Such interactions can be examined using immunoprecipitation techniques (see, Hamilton et al.

Biochem. Biophys. Res. Commun. 1999, 261:646-51). 191P4D12(b) protein can be immunoprecipitated from 191P4D12(b)expressing cell lines using anti-191P4D12(b) antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 191P4D12(b) and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 5 S-methlonine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 191P4D12(b) 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 191P4D12(b)'s ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 191P4D12(b)-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 191P4D12(b) (see, Hille, Ionic Channels of Excitable Membranes 2 nd Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate 191P4D12(b) function can be identified based on their ability to bind 191P4D12(b) 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 ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 191P4D12(b) and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 191P4D12(b).

An embodiment of this invention comprises a method of screening for a molecule that interacts with a 191P4D12(b) amino acid sequence shown In Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 0 0 191P4D12(b) amino acid sequence, allowing the population of molecules and the 191P4D12(b) amino acid sequence to 0 interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 191P4D12(b) amino acid sequence, and then separating molecules that do not interact with the 191P4D12(b) 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 191P4D12(b) amino acid sequence. The identified molecule can be used to modulate a function performed by 191P4D12(b). In a preferred embodiment, the 191P4D12(b) amino acid sequence is contacted with a library of peptides.

X) Therapeutic Methods and Compositions The identification of 191P4D12(b) 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 00 such cancers.

SOf 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 HER2/neu 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 been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very 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.

Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth 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.

Accordingly, therapeutic approaches that inhibit the activity of a 191P4D12(b) protein are useful for patients suffering from a cancer that expresses 191P4D12(b). These therapeutic approaches generally fall into two classes. One

O

O class comprises various methods for inhibiting the binding or association of a 191P4D12(b) protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 191P4D12(b) gene or translation of 191P4D12(b) mRNA.

Anti-Cancer Vaccines SThe invention provides cancer vaccines comprising a 191P4D12(b)-related protein or 191P4D12(b)-related nucleic acid.

In view of the expression of 191P4D12(b), cancer vaccines prevent and/or treat 191P4D12(b)-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 C rodent PAP immunogens (Hodge et al., 1995, Int J. Cancer 63:231-237; Fong et al, 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 191 P4D12(b)-related protein, or a 191P4D12(b)-encoding 0 0 nucleic acid molecule and recombinant vectors capable of expressing and presenting the 191P4D12(b) immunogen (which 0 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, Heryln et al., 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 humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope an epitope present in a 191P4D12(b) 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 191P4D12(b) immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 191P4D12(b) indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.

The entire 191P4D12(b) 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 al., In: Concepts in vaccine development, Kaufmann, S. H. ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537,1986; Kieny, et al., AIDS Bio/Technology4:790, 1986; Top, F.

H. etal., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. 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; Gupta, R. K. et al., Vaccine 11:293,1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585,1992; Rock, K. Immunol.

Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, 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 Eldridge, J. H. etal., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

In patients with 191P4D12(b)-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, surgery, chemotherapy, drug therapies, radiation therapies, etc.

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

Cellular Vaccines: K CTL epitopes can be determined using specific algorithms to identify peptides within 191P4D12(b) protein that bind S corresponding HLA alleles (see Table IV; EpimerM and EpimatrixT

M

Brown University (URL brown.edulResearch/TB- HIV.Lab/epimalrix/epimatrix.html); and, BIMAS, (URL bimas.dcrtnih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/).

0 In a preferred embodiment, a 191P4D12(b) 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 and/or a peptide S 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 S 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, 00 the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound 0 by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are C1 length specific, 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 191P4D12(b) protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 191P4D12(b) in a host, by contacting the host with a sufficient amount of at least one 191P4D12(b) B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 191P4D12(b) B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 191P4D12(b)-related protein or a man-made multiepitopic peptide comprising: administering 191P4D12(b) immunogen (e.g.

a 191P4D12(b) protein or a peptide fragment thereof, a 191P4D12(b) 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, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 191P4D12(b) 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 191P4D12(b) immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, U.S. Patent No.

5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 191P4D12(b), in order to generate a response to the target antigen.

Nucleic Acid Vaccines: Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate

OO

0 prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 191P4D12(b).

S Constructs comprising DNA encoding a 191P4D12(b)-related proteinlimmunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 191P4D12(b) protein/immunogen. Alternatively, a vaccine comprises a 191P4D12(b)-related protein.

Expression of the 191P4D12(b)-related protein immunogen results in the generation of prophylactic or therapeutic humoral LC and cellular immunity against cells that bear a 191P4D12(b) protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet 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 98/04720. Examples of C-l DNA-based 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.

S 5,922,687).

SFor therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the 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 191P4D12(b)-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 191P4D12(b)-related nucleic acid molecule. In one embodiment, the full-length human 191P4D12(b) cDNA is employed. In another embodiment 191P4D12(b) nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) andlor 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 191P4D12(b) antigen to a patient's immune system.

Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 191P4D12(b) peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 191P4D12(b) peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 191P4D12(b) protein. Yet another embodiment involves engineering the overexpression of a 191P4D12(b) gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivlrus, adeno-associated virus, DNA transfection (Ribas et 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 191P4D12(b) can also be engineered to 0 express immune modulators, such as GM-CSF, and used as immunizing agents.

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C 191P4D12(b) as a Target for Antibody-based Therapy 191P4D12(b) 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 191 P4D12(b) is expressed by cancer cells of various lineages relative to C1 corresponding normal cells, systemic administration of 191P4D12(b)-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 191P4D12(b) are useful to C=K treat 191P4D12(b)-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

ri 191P4D12(b) antibodies can be introduced into a patient such that the antibody binds to 191P4D12(b) and 00 modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells S 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 191P4D12(b), 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 191P4D12(b) 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 191P4D12(b)), 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- 191P4D12(b) antibody) that binds to a marker 191P4D12(b)) 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 191P4D12(b), comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 191P4D12(b) 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 pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent Cancer immunotherapy using anti-191P4D12(b) antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et 1996, Leuk. Res.

20:581-589), colorectal cancer (Moun etal., 1994, Cancer Res. 54:6160-6166; Velders et 1995, Cancer Res. 55:4398- 4403), and breast cancer (Shepard et 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies 00 ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarM, Coulter Pharmaceuticals), while others involve co-administration of 0 antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.). The C<1 antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 191P4D12(b) antibodies can be S administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin S such as calicheamicin Mylotarg

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Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid taxane-based Tumor-Activated Prodrug, TAP, Cl platform, ImmunoGen, Cambridge, MA, also see US Patent 5,416,064).

Although 191P4D12(b) antibody therapy is useful for all stages of cancer, antibody therapy can be particularly S appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients S who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a ri chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, Cl antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not 00 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 191P4D12(b) 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 191P4D12(b) expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 191P4D12(b) imaging, or other techniques that reliably indicate the presence and degree of 191P4D12(b) 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-191P4D12(b) 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-191P4D12(b) 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-191P4D12(b) mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 191P4D12(b). 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-191P4D12(b) mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from 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 specifically to the target 191P4D12(b) 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-191P4D12(b) mAbs as well as 0 combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain SmAbs 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- 191P4D12(b) mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various O chemotherapeutic agents, androgen-blockers, immune modulators IL-2, GM-CSF), surgery or radiation. The anti- 191P4D12(b) mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-191P4D12(b) antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, 0 intradermal, and the like. Treatment generally involves repeated administration of the anti-191P4D12(b) antibody OO preparation, via an acceptable route of administration such as intravenous injection typically at a dose in the range of O about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the C1 range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the HerceptinTM 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- 191P4012(b) 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 191P4D12(b) expression in the patient, the extent of circulating shed 191P4D12(b) 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 191P4012(b) in a given sample the levels of circulating 191P4D12(b) antigen and/or 191P4D12(b) 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-idiotypic anti-191P4D12(b) antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 191P4D12(b)-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-191P4D12(b) antibodies that mimic an epitope on a 191P4D12(b)-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 be used in cancer vaccine strategies.

191P4D12(b) as a Target for Cellular Immune Responses Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the 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 peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with

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0 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 S synthesis.

Carriers 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, polyamino acids such as poly L-lyslne, poly L-glutamic acid, (C 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 CK the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P 3 CSS). Moreover, an adjuvant such as a Ni synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL 00 responses 10- to 100-fold. (see, e.g. Davila and Cells, J. Immunol. 165:539-547 (2000)) SUpon 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 191 P4D12(b) 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 PADRET (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 polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic 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.

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 aL, Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

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

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

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

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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 S HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per C sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal Q epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, C such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it 00 0 does not have pathological or other deleterious biological properties.

S6.) 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 concern 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.

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.

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

67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotifand/or motif-bearing epitopes derived 191P4D12(b), the PADRE® universal helper T cell epitope or multiple HTL epitopes from 191P4D12(b) (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 0 CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be 0 correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these C-l experiments can show that the minigene serves to both: generate a CTL response and that the induced CTLs S recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression In human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the CKl 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 andlor immunogenicity, additional S elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated S and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a S ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and CKl HTL epitopes may be improved by including synthetic poly-alanine) or naturally-occurring flanking sequences adjacent 00 0 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. coil 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. coli 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.

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

(PADRE

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M, Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II 00 pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune

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S response by co-expression of immunosuppressive molecules TGF-P) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E, coli, followed by ct purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation 0 technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized 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 S minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods 00 have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can O also be used in the formulation (see, as described by WO 93/24640; Mannino Gould-Fogerite, BioTechniques 6(7): C1 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et al., Proc. Nat'l Acad. Scl. 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 5 sCr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 5 sCr release, indicates both production of, and HLA presentation of, mlnigene-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, 5 'Cr-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.

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

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

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

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

X.C.2. Combinations of CTL Peptides with Helper Peptides

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Vaccine compositions comprising CTL peptides of the invention can be modified, analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.

00 O For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, Ala, Gly, or other S 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 S 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 CK terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be C acylated.

00 In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a 0 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: 44), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNWNS; SEQ ID NO: 45), and Streptococcus 18kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 46). 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: XKXVAAWTLKAAX (SEQ ID NO: 47), where is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either o-alanine or L-alanine, 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 altemative 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 0-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 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 e- and a- amino groups of Lys, which is attached via linkage, 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-Sglycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate 0 0 peptide (see, Deres, et at., Nature 342:561, 1989), Peptides of the invention can be coupled to P3CSS, for example, 0 and the lipopeptide administered to an Individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with 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 O 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 patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Pharmacia-Monsanto, St Louis, MO) or GM-CSFIIL-4.

After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In C= this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

00 The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 191P4D12(b).

0 Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class 11 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 191P4D12(b).

X.D. Adoptive Immunotherapy Antigenic 191P4D12(b)-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 withithe invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, 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 191P4D12(b). 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 the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 191P4D12(b). The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 191 P4D1 2(b)-associated cancer.

00 This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The 0 embodiment of the vaccine composition including, but not limited to embodiments such as peptide cocktails, C=K 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 t 191P4D12(b), a vaccine comprising 191P4D12(b)-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

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

L\i The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is

L

C 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 CKl human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient. Boosting dosages of between about 00 0 1.0 pg to about 50,000 pg of peptide pursuant to a boosting regimen over weeks to months may be administered depending Ci 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 pg to about 50,000 jig 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% glyclne, 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 combined with a sterile solution prior to administration.

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

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

c A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity 0 that is known by those of skill in the art to be used for administration of such compositions to humans (see, Remington's Pharmaceutical Sciences, 17 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 pig, generally 100-5,000 Rpg, 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 Ig) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then 00 administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x10 9 pfu.

For antibodies, a treatment generally involves repeated administration of the anti-191P4D12(b) 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- 191P4D12(b) 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 191P4D12(b) expression in the patient, the extent of circulating shed 191P4D12(b) 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, 500g 1mg, 1mg 50mg, 50mg 100mg, 100mg 200mg, 200mg 300mg, 400mg 500mg, 500mg 600mg, 600mg 700mg, 700mg 800mg, 800mg 900mg, 900mg ig, 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 mglkg; 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 polynuceotides 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 polynucleotide 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 independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 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 00 direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

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

CN 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 Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, S 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. Liposomes include emulsions, foams, micelles, S insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the CK1 peptide to be delivered is incorporated as part of a liposome, alone or In conjunction with a molecule which binds to a 00 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 of, 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, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition Is ordinarily propellant. A carrier can also be included, as desired, as with, lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 191P4D12(b).

As disclosed herein, 191P4D12(b) 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, both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in 00 the Example entitled "Expression analysis of 191P4D12(b) in normal tissues, and patient specimens").

O

191P4D12(b) can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to Identify and monitor the presence of prostate cancer (see, Merrill et al., J. Urol.

t 163(2): 503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., S Tulchinsky et al., Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 191 P4D12(b) polynucleotides and polypeptides (as well as 191 P4D12(b) polynucleotide probes and anti- 191P4D12(b) 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.

STypical embodiments of diagnostic methods which utilize the 191 P4D12(b) polynucleotides, polypeptides, reactive 00 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 CK (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 191P4D12(b) polynucleotides described herein can be utilized in the same way to detect 191P4D12(b) 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 al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, Alanen et Pathol. Res. Pract. 192(3):233-7 (1996)), the 191P4D12(b) polypeptides described herein can be utilized to generate antibodies for use in detecting 191P4D12(b) 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 191P4D12(b) 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 191P4D12(b)-expressing cells (lymph node) is found to contain 191P4D12(b)-expressing cells such as the 191P4D12(b) expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 191 P4D12(b) 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 191P4D12(b) or express 191P4D12(b) at a different level are found to express 191P4D12(b) or have an increased expression of 191P4D12(b) (see, the 191P4D12(b) 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 191P4D12(b)) such as PSA, PSCA etc. (see, Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a 191P4D12(b) polypeptide within a tissue section can 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 00 the cancer cells, as compared to corresponding non-malignant tissue.

O The 191P4D12(b) polypeptide and immunogenic compositions are also useful in view of the phenomena of altered C subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and Is often associated with changes in subcellular protein localizationldistribution. For example, cell membrane S 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.

CK1 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 S of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an S apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 r C (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45:1547-1557 (1997)). In addition, normal breast rC epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express 00 the protein over the whole cell surface (De Potter, et al, 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 concerning 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 191P4D12(b), the 191P4D12(b) 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 191P4D12(b) protein and immune responses related thereto very useful. Use of the 191P4D12(b) compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.

Immunohistochemical reagents specific to 191P4D12(b) are also useful to detect metastases of tumors expressing 191P4D12(b) when the polypeptide appears in tissues where 191P4D12(b) is not normally produced.

Thus, 191P4D12(b) 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 polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 191P4D12(b) 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 al., 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 191P4D12(b) in normal tissues, and patient specimens," where a 191P4D12(b) polynucleotide fragment is used as a probe to show the expression of 191P4D12(b) RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for 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 polynucleotide sequence a 191P4D12(b) polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.

00 Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that O specifically binds to that epitope are used in methods of monitoring PSA. 191P4D12(b) polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems'such as fusion proteins being used by practitioners (see, Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick 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 order to generate immune responses specific for different portions of a polypeptide of interest (see, U.S.

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 CN one of the 191P4D12(b) 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 0c context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence a 191P4D12(b) polypeptide shown in Figure 3).

C As shown herein, the 191P4D12(b) polynudeotides and polypeptides (as well as the 191P4D12(b) polynucleotide probes and anti-191P4D12(b) 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 191P4D12(b) 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 al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 191P4D12(b) polynucleotides and polypeptides (as well as the 191P4D12(b) polynucleotide probes and anti-191P4D12(b) 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 191P4D12(b) 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 191P4D12(b) gene maps (see the Example entitled "Chromosomal Mapping of 191P4D12(b)" below). Moreover, in addition to their use in diagnostic assays, the 191P4D12(b)-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 Sc Int 1996 Jun 28;80(1-2): 63-9).

Additionally, 191P4D12(b)-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 191P4D12(b). 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 191P4D12(b) antigen.

Antibodies or other molecules that react with 191P4D12(b) can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 191P4D12(b) Protein Function The invention includes various methods and compositions for inhibiting the binding of 191P4D12(b) to its binding partner or its association with other protein(s) as well as methods for inhibiting 191P4D12(b) function.

XII.A.) Inhibition of 191P4D12(b) With Intracellular Antibodies OO In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 191P4D12(b) 0 are introduced into 191P4D12(b) expressing cells via gene transfer technologies. Accordingly, the encoded single chain CN anti-191P4D12(b) antibody Is expressed intracellularly, binds to 191P4D12(b) protein, and thereby inhibits its function.

Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as "intrabodies", are specifically targeted to a particular compartment within the cell, providing control over where the s inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see C Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, Richardson et al., 1995, Proc. Natl. Acad. Scd. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

iSingle 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 C- variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered 00 0 into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to O 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 191P4D12(b) in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 191P4D12(b) intrabodies in order to achieve the desired targeting. Such 191P4D12(b) intrabodies are designed to bind specifically to a particular 191P4D12(b) domain. In another embodiment, cytosolic intrabodies that specifically bind to a 191P4D12(b) protein are used to prevent 191P4D12(b) from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus preventing 191P4D12(b) 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 andlor 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 191P4D12(b) with Recombinant Proteins In another approach, recombinant molecules bind to 191P4D12(b) and thereby inhibit 191P4D12(b) function. For example, these recombinant molecules prevent or inhibit 191P4D12(b) 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 191P4D12(b) specific antibody molecule. In a particular embodiment, the 191P4D12(b) binding domain of a 191P4D12(b) binding partner Is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 191P4D12(b) 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 CH 3 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 191P4D12(b), whereby the dimeric fusion protein specifically binds to 191P4D12(b) and blocks 191P4D12(b) interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of 191P4D12(b) Transcription or Translation 00 The present invention also comprises various methods and compositions for inhibiting the transcription of the S 191P4D12(b) gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 191P4D12(b) mRNA into protein.

SIn one approach, a method of inhibiting the transcription of the 191P4D12(b) gene comprises contacting the 191P4D12(b) gene with a 191P4D12(b) antisense polynucleotide. In another approach, a method of inhibiting 191P4D12(b) mRNA translation comprises contacting a 191P4D12(b) mRNA with an antisense polynucleotide. In another approach, a 191P4D12(b) specific ribozyme is used to cleave a 191P4D12(b) message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 191P4D12(b) gene, such as 191P4D12(b) promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 191P4D12(b) gene transcription factor are C used to inhibit 191P4D12(b) 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 00 and translation is well known in the art.

SOther factors that inhibit the transcription of 191P4D12(b) by interfering with 191P4D12(b) transcriptional activation C are also useful to treat cancers expressing 191P4D12(b). Similarly, factors that interfere with 191P4D12(b) processing are useful to treat cancers that express 191P4D12(b). 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 191P4D12(b) antisense, ribozyme, polynucleotides encoding intrabodies and other 191P4D12(b) inhibitory molecules). A number of gene therapy approaches are known in the art Recombinant vectors encoding 191P4D12(b) antisense polynucleotides, ribozymes, factors capable of interfering with 191P4D12(b) 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 191P4D12(b) to a binding partner, etc.

In vivo, the effect of a 191P4D12(b) 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 W098/16628 and U.S. Patent 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In 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 fod are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used In the practice of the foregoing methods can be formulated into pharmaceutical LC- 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 i non-reactive with the patient's Immune system. Examples Include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, C-K Remington's Pharmaceutical Sciences 16(h Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, Lq parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation Cl for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile LC- unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for 00 SInjection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, Sand 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 191 P4D12(b) Methods to Identify and Use Modulators In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to 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 and/or modulate the biological activity of the gene product.

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

Modulator-related Identification and Screening Assays: Gene Expression-related Assays Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a "gene expression profile," expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high 00 throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-

L

c 94,1996).

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

rli A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds 00 are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. "Modulation" in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the Cl 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.

In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a 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 00 0 throughput screening (HTS) methods are employed for such an analysis.

SAs noted above, gene expression monitoring is conveniently used to test candidate modulators protein, S 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.

If required, the target sequence is prepared using known techniques. For example, a sample Is treated to lyse the C' 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 nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or The target sequence can be labeled with, a fluorescent, a chemiluminescent, a chemical, or a radioactive C1 signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, Ssuch as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a 00 product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that S 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 streptavldin 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.

Biological Activity-related Assays The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a 00 cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention.

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

CK1 In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of S physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells cell-cell contacts). In 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 critical structural features of the compound.

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

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

As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, 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, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being 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 Aaar 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.

Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of 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 0 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, similarly an MTT 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.

In this assay, labeling index with 3 H)-thymidine at saturation density is a preferred method of measuring density 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 C= incorporated cpm.

SContact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular 00 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 et al., 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 Matrinel to Idenlify 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.

Briefly, 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 filler, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125s and counting the radioactivity on the distal side of the filter or bottom of the dish. See, Freshney (1984), supra.

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

Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in 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 carcinogens.

To prepare transgenlc chimeric animals, mice, a DNA construct is introduced into the nuclei of embryonic CK1 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 00 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 NC 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 thymectornized mouse, or an irradiated mouse (see, Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J.

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

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

Opin. Biotechnol. 1998: 9:624).

00 As outlined above, in vitro screens are done on individual genes and gene products. That Is, having identified a Sparticular 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.

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 expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better 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 00 used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the Samount andfor 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

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

00 A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the S invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, by

C

attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound Ct a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.

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

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

Competitive Binding to Identify and Characterize Modulators In one embodiment, the binding of the "test compound" is determined by competitive binding assay with a C00 "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, C<1 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 400C.

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

In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.

In an 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 drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins.

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

0 Positive controls and negative controls can be used In the assays. Preferably control and test samples are CN performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to S 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.

LC= 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 or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease Inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides Cl for the requisite binding.

00 SUse of Polynucleotldes to Down-regulate or Inhibit a Protein of the Invention.

O Polynucleotide modulators of cancer can be introduced into a cell containing the target nuceotide 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 phosphorothioate 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 Biosystems. 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 oligonudeotides. 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 00 &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

Ribozvmes c In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancerassociated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different 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 properties of different ribozymes).

The general features of hairpin ribozymes are described, in Hampel et al., Nucl. 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 CN 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 00 Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

0 CN 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 Peotides 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.

00 0 O Methods 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 S variant cancer genes, determining the presence of, all or part, the sequence of at least one endogenous cancer gene in Sa 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 CK1 sequence of the sequenced gene to a known cancer gene, a wild-type gene to determine the presence of family Smembers, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the 00 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.) KitslArticles of Manufacture For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a Figure 2-related protein or a Figure 2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers comprising 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 the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit.

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

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

The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or 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.

c l The container can alternatively hold a composition which is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or S a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an r antibody capable of specifically binding 191P4D12(b) and modulating the function of 191P4D12(b).

The label can be on or associated with the container. A label a can be on a container when letters, numbers or c0 other characters forming the label are molded or etched into the container itself; a label 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 CN indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose 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 are intended to limit the scope of the invention.

Example 1: SSH-Generated Isolation of cDNA Fragment of the 191P4D12(b) Gene To isolate genes that are over-expressed in prostate cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from prostate cancer tissues. The 191P4D12(b) SSH cDNA sequence was derived from bladder tumor minus cDNAs derived from a pool of 9 normal tissues. The 191P4D12(b) cDNA was identified as highly expressed in the bladder cancer.

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

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

RNA Isolation: Tissues were homogenized in Trizol reagent (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 spectrophotometric analysis 260/280 nm) and analyzed by gel electrophoresis.

Oliqonucleotides: The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): 5'TTTTGATCAAGCTT3o3' (SEQ ID NO: 48) Adaptor 1: S5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 49) (SEQ ID NO: Adaptor 2: 5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 51) (SEQ ID NO: 52) PCR primer 1: 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: 53) Nested primer (NP)1: N 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 54) SNested primer (NP)2: 00 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: Suppression Subtractive Hybridization: Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.

The gene 191P4D12(b) sequence was derived from bladder cancer minus normal tissue cDNA subtraction. The SSH DNA sequence (Figure 1) was identified.

The cDNA derived from of pool of normal tissues was used as the source of the "driver" cDNA, while the cDNA from bladder cancer was used as the source of the "tester" cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 pig of poly(A)* RNA isolated from the relevant xenograft 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. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 370C. Digested cDNA was extracted with phenol/chloroform and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) 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 pd 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 pl at 160C overnight, using 400 u ofT4 DNA ligase (CLONTECH). Ugation was terminated with 1 pl of 0.2 M EDTA and heating at 72oC for 5 min.

The first hybridization was performed by adding 1.5 pi (600 ng) of driver cDNA to each of two tubes containing 1.5 l ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 pl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 980C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 680C. The two hybridizations were then mixed together with an additional 1 pi of fresh denatured driver cDNA and were allowed to hybridize overnight at 68C0. The second hybridization was then diluted in 200 pl of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 70C0 for 7 min. and stored at -200C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH: To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 pl of the diluted final hybridization mix was added to 1 Ip of PCR primer 1 (10 pM), 0.5 pl dNTP mix (10 pM), 2.5 pi 10 x 00 reaction buffer (CLONTECH) and 0.5 pi 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 pi. PCR 1

O

was conducted using the following conditions: 75oC for 5 min., 940C for 25 sec., then 27 cycles of 94°C for 10 sec, 660C for 30 sec, 72oC 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 pl 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 94oC for 10 sec, 680C 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 overnight. 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.

00 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.

SRT-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 420C with reverse transcriptase followed by RNAse H treatment at 370C for 20 min. After completing the reaction, the volume can be increased to 200 pl 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: 56) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 57) to amplify p-actin. First strand cDNA (5 pi) were amplified In a total volume of 50 pC containing 0.4 pM primers, 0.2 pM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCI2, 50 mM KCI, pH8.3) and 1X Klentaq DNA polymerase (Clontech). Five pl 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 94oC for 15, 650C for 2 min, 720C 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 191P4D12(b) gene, 5 pl of normalized first strand cDNA were analyzed by PCR using 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 were designed using the 191P4D12(b) SSH sequence and are listed below: 191P4D12(b).1 GGCTGGAGTTCAATGAGGTTTATTT 3' (SEQ ID NO: 58) 191P4D12(b).2 TCCAGCAGATTTCAGACTAAAAGAAGA- 3' (SEQ ID NO: 59) A typical RT-PCR expression analysis is shown in Figure 14. First strand cDNA was prepared from vital pool 1 (liver, lung 00 and kidney), vital pool 2 (pancreas, colon and stomach), normal kidney, prostate cancer pool, bladder cancer pool, colon cancer pool, lung cancer pool, breast cancer pool and cancer metastasis pool. Normalization was performed by PCR using primers to actin and ri GAPDH. Semi-quantitative PCR, using primers to 191P4D12(b), was performed at 26 and 30 cycles of amplification. Results show strong expression of 191P4D12(b) in bladder cancer pool. Expression of 191P4D12(b) was also detected in prostate cancer pool, colon cancer pool, lung cancer pool, breast cancer pool and cancer metastasis pool but very weakly in vital pool 1 and vital pool 2.

Example 2: Isolation of Full Length 191P4D12(b) Encoding cDNA The 191P4D12(b) SSH cDNA sequence was derived from a subtraction consisting of bladder cancer minus a mixture of 9 normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine and heart. The SSH cDNA C sequence of 223 bp (Figure 1) was designated 191P4D12(b).

O 191P4D12(b) v.1 (clone 1Al) of 3464 bp was cloned from bladder cancer cDNA library, revealing an ORF of 510 amino 00 acids (Figure 2 and Figure Other variants of 191P4D12(b) were also identified and these are listed in Figures 2 and 3, 191P4D12(b) v.1, v.2, v.10, v.11, and v.12 proteins are 510 amino acids in length and differ from each other by one amino acid as shown in Figure 11. 191P4D12(b) v.3, v.4, v.5, and v.8 code for the same protein as 191P4D12(b) v.1.

191P4D12(b) v.6 and v.7 are splice variants and code for proteins of 295 and 485 amino acids, respectively. 191P4D12(b) v.13 clone 9C was cloned from bladder cancer cDNA and has one amino acid insertion at position 334 compared to 191P4D12(b) v.1.

191P4D12(b) v.9 clone BCP1 is a splice variant of 191P4D12(b) v.1 and was cloned from a bladder cancer cDNA library.

191P4D12(b) v.14 is a SNP variant and differs from 191P4D12(b) v.9 by one amino acid as shown in Figure 2.

191P4D12(b) v.1 shows 99% identity over 2744 to the Ig superfamily receptor LNIR (nectin-4), accession number NM_030916. 191P4D12(b) v.9 protein is 100% identical to clone AF218028 with function of inhibiting cancer cell growth.

Example 3: Chromosomal Mapping of 191P4D12(b) 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 Comell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).

191P4D12(b) maps to chromosome 1q22-q23.2 using 191P4D12(b) sequence and the NCBI BLAST tool located on the World Wide Web at (.ncbi.nlm.nih.gov/genomelseq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4: Expression Analysis of 191P4D12(b) In Normal Tissues and Patient Specimens Expression analysis by RT-PCR demonstrated that 191P4D12(b) is strongly expressed in bladder cancer patient specimens (Figure 14). First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal kidney, prostate cancer pool, bladder cancer pool, colon cancer pool, lung cancer pool, breast cancer pool and cancer metastasis pool; prostate cancer metastasis to lymph node, 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, and LAPC prostate xenograft pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 191P4D12(b), was performed at 26 and 30 cycles of amplification. In results show strong expression of 191P4D12(b) in bladder cancer pool. Expression of 191P4D12(b) was also detected in prostate cancer pool, colon cancer pool, lung cancer pool, breast cancer pool and cancer metastasis pool but very weakly In vital pool 1 and vital pool 2. In results show strong expression of 191P4D12(b) in prostate, bladder, kidney, colon, lung, ovary, breast, cancer metastasis, and pancreas cancer specimens.

00 Northern blot analysis of 251 P5G2 is a technique known to those skilled in the art to detect 251 P5G2 protein C production. Northern blotting detects relative levels of mRNA expressed from a 251 P5G2 gene. Specific mRNA is Smeasured using a nucleic acid hybridization technique and the signal is detected on an autoradiogram. The stronger the C signal, the more abundant is the mRNA. For 251 P5G2 genes that produce mRNA that contains an open reading frame S flanked by a good Kozak translation initiation site and a stop codon, in the vast majority of cases the synthesized mRNA is expressed as a protein.

The level of expression of the 251P5G2 gene is determined in various normal tissues and in various tumor tissues and tumor cell lines using the technique of Northern blotting, which detects production of messenger RNA. It is well known in 1 the art that the production of messenger RNA, that encodes the protein, is a necessary step in the production of the protein ri itself. Thus, detection of high levels of messenger RNA by, for example, Northern blot, is a way of determining that the protein itself is produced. The Northern blot technique is used as a routine procedure because it does not require the time 00 delays (as compared to Western blotting, immunoblotting or immunohistochemistry) involved in isolating or synthesizing the Sprotein, preparing an immunological composition of the protein, eliciting a humoral immune response, harvesting the Cr antibodies, and verifying the specificity thereof.

The Kozak consensus sequence for translation initiation CCACCATGG, where the ATG start codon is noted, is the sequence with the highest established probability of initiating translation. This was confirmed by Peri and Pandey Trends in Genetics (2001) 17: 685-687. The conclusion is consistent with the general knowledge in the art that, with rare exceptions, expression of an mRNA is predictive of expression of its encoded protein. This is particularly true for mRNA with an open reading frame and a Kozak consensus sequence for translation initiation.

It is understood in the art that the absolute levels of messenger RNA present and the amounts of protein produced do not always provide a 1:1 correlation. In those instances where the Northern blot has shown mRNA to be present, it is almost always possible to detect the presence of the corresponding protein in the tissue which provided a positive result in the Northern blot. The levels of the protein compared to the levels of the mRNA may be differential, but generally, cells that exhibit detectable mRNA also exhibit detectable corresponding protein and vice versa. This is particularly true where the mRNA has an open reading frame and a good Kozak sequence (See, Peri and Pandey, supra.).

Occasionally those skilled in the art encounter a rare occurrence where there is no detectable protein in the presence of corresponding mRNA. (See, Fu, L, et al., Embo. Journal, 15:4392-4401 (1996)). In many cases, a reported lack of protein expression is due to technical limitations of the protein detection assay. These limitations are readily known to those skilled in the art. These limitations include but are not limited to, available antibodies that only detect denatured protein and not native protein present in a cell and unstable proteins with very short half-life. Short-lived proteins are still functional and have been previously described to induce tumor formation. (See, Reinstein, et al., Oncogene, 19: 5944- 5950). In such situations, when more sensitive detection techniques are performed and/or other antibodies are generated, protein expression Is detected. When studies fail to take these principles into account, they are likely to report artifactually lowered correlations of mRNA to protein. Outside of these rare exceptions the use of Northern blot analysis is recognized to those skilled in the art to be predictive and indicative of the detection of 251 P5G2 protein production.

Extensive expression of 191P4D12(b) in normal tissues is shown in Figure 15. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNAnane were probed with the 191P4D12(b) sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 4kb transcript in placenta and very weakly in prostate but not in any other normal tissue tested. A smaller 191P4D12(b) transcript of approximately 2.5kb was detected in heart and skeletal muscle.

Expression of 191P4D12(b) in bladder cancer patient specimens and human normal tissues is shown In Figure 16.

RNA was extracted from a pool of 3 bladder cancer patent specimens, as well as from normal prostate normal bladder O normal kidney normal colon normal lung normal breast (NBr), normal ovary and normal 0 pancreas (NPa). Northern blot with 10 ug of total RNAlane was probed with 191P4D12(b) SSH sequence. Size standards in kilobases (kb) are indicated on the side. The 191P4D12(b) transcript was detected in the bladder cancer specimens, but not in the normal tissues tested.

Analysis of individual bladder cancer patient specimens Is depicted in Figure 17. RNA was extracted from bladder cancer cell lines normal bladder and bladder cancer patient tumors Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in the bladder tumor tissues but not in normal bladder. A smaller transcript was detected in the HT1197 cell line but not in the other cancer cell lines tested.

t^ Expression of 191P4D12(b) was also detected in prostate cancer xenograft tissues (Figure 18). RNA was extracted from normal prostate, and from the prostate cancer xenografts LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI.

00 Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards In kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in all the LAPC xenograft tissues but not in normal prostate.

Figure 19 shows expression of 191P4D12(b) In cervical cancer patient specimens. RNA was extracted from normal cervix, Hela cancer cell line, and 3 cervix cancer patient tumors Northern blots with 10 ug of total RNA were probed with the 191P4D12(b) SSH fragment. Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in 2 out of 3 cervix tumors tested but not in normal cervix nor in the Hela cell line.

191P4D12(b) was also expressed in lung cancer patient specimens (Figure 20). RNA was extracted from lung cancer cell lines normal lung bladder cancer patient tumors and normal adjacent tissue (Nat). Northem blots with 10 ug of total RNA were probed with the 191P4D12(b). Size standards in kilobases are on the side. Results show expression of the approximately 4kb 191P4D12(b) transcript in the lung tumor tissues but not in normal lung nor in the cell lines tested.

191P4D12(b) expression was tested in a panel of individual patient cancer specimens (Figure 21). First strand cDNA was prepared from a panel of lung cancer specimens bladder cancer specimens prostate cancer specimens colon cancer specimens uterus cancer specimens and cervix cancer specimens Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 191P4D12(b) SSH fragment, was performed at 26 and 30 cycles of amplification. Expression level was recorded as 0 no expression detected; 1 weak expression, 2 moderate expression; 3 strong expression. Results show expression of 191P4D12(b) in 97% of the 31 lung cancer patient specimens tested, 94% of 18 bladder cancer patient specimens, 100% of 20 prostate cancer patient specimens, 100% of 22 colon cancer patient specimens, 100% of 12 uterus cancer patient specimens, and 100% of 14 cervix cancer patient specimens tested.

The restricted expression of 191P4D12(b) in normal tissues and the expression detected in cancer patient specimens suggest that 191P4D12(b) is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5: Transcript Variants of 191P4D12(b) Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or altemative 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 00 coding and/or non-coding or 3' end) portions, from the original transcript. Transcript variants can code for similar or S different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in CN 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 cellular or S extracellular localizations, secreted versus Intracellular.

STranscript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, 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 C1 compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful C for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

00 SMoreover, computer programs are available 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/EmptyGrailForm) 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 al., 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 multiple forms of mature caprine alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR-based Validation: Wellmann S, et 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 betadefensins 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 Biophys Acta. 1997 Aug 7; 1353(2): 191-8).

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

Disclosed herein is that 191P4D12(b) has a particular expression profile related to cancer. Alternative transcripts and splice variants of 191P4D12(b) may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.

Using the full-length gene and EST sequences, four additional transcript variants were identified, designated as 191P4D12(b) v.6, v.7, v.8 and v.9 as shown in Figure 12. The boundaries of exons in the original transcript, 191P4D12(b) v.1 were shown in Table LI. Compared with 191P4D12(b) v.1, variant v.6 spliced out 202-321 from the first exon of v.1 while variant v.8 spliced out 63 bases from the last exon of v.1. Variant v.7 spliced out exon 8 of v.1. Variant 9 was part of the last exon of v.1. Theoretically, each different combination of exons in spatial order, e.g. exons 2, 3, 5, 7 and 9 of v.1, is a potential splice variant.

Tables LlI through LV are set forth on a variant-by-variant bases. Tables LII shows nucleotide sequence of the transcript variants. Tables LIII shows the alignment of the transcript variant with nucleic acid

OO

0 sequence of 191P4D12(b) v.l. Tables LIV lays out amino acid translation of the transcript variant for the identified i reading frame orientation. Tables LV displays alignments of the amino acid sequence encoded by the splice variant with that of 191P4D12(b) v.1.

Example 6: Single Nucleotide Polymorphisms of 191P4D12(b) C 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), CN 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 o SNP cause inherited diseases; others contribute to quantitative variations In phenotype and reactions to environmental S 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):15-26).

SNP are identified by a variety of art-accepted methods Bean, "The promising voyage of SNP target 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 sequences from different DNA samples. With the rapid accumulation of sequence data in public and private 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 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 polymorphisms," Annu. 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, seven SNP and one insertion/deletion of three bases were identified in the original transcript, 191P4D12(b) v.1, at positions 420 2184 2341 2688 367 699 1590 and insertion of GCA in between 1262 and 12631. The transcripts or proteins with alternative allele were designated as variant 191P4D12(b) v.2 through v.5 and v.10 through v.13, as shown in Figure 10. Figure 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in Figure 11. 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 191P4D12(b) v.9) that contains the site of the SNP. The SNP at 2688 of v.1 occurs also in transcript variant v.9 and contributed to one codon change of v.9 at amino acid 64 from Ala to Asp (Figure 11).

00 0 Example 7: Production of Recombinant 191P4D12(b) in Prokarvotic Systems C To express recombinant 191P4D12(b) and 191P4D12(b) variants in prokaryotic cells, the full or partial length 191P4D12(b) and 191 P4D12(b) 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 191P4D12(b) 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 Cr contiguous amino acids from 191P4D12(b), variants, or analogs thereof.

A. In vitro transcription and translation constructs: pDRII: To generate 191P4D12(b) sense and anti-sense RNA probes for RNA in situ investigations, pCRII Ci constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 191P4D12(b) cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 191P4D12(b) RNA for use as probes in RNA C in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 191 P4D12(b) at the 00 RNA level. Transcribed 191P4D12(b) RNA representing the cDNA amino acid coding region of the 191P4012(b) gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize 191 P4D12(b) protein.

B. Bacterial Constructs: pGEX Constructs: To generate recombinant 191P4D12(b) proteins in bacteria that are fused to the Glutathione Stransferase (GST) protein, all or parts of the 191P4D12(b) 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 191P4D12(b) protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-lerminus. 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 PreScission T M recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 191P4D12(b)-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 191P4D12(b) proteins that are fused to maltose-binding protein (MBP), all or parts of the 191P4D12(b) 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 191P4D12(b) protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl-terminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6X His epitope tag is generated by adding 6 histidine codons to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 191P4D12(b). The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs: To express 191P4D12(b) in bacterial cells, all or parts of the 191P4D12(b) cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 191P4D12(b) 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 TM 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 191P4D12(b) protein are expressed as amino-terminal fusions to NusA.

00 C. Yeast Constructs: pESC Constructs: To express 191P4D12(b) in the yeast species Saccharomyces cerevlsiae for generation of recombinant protein and functional studies, all or parts of the 191P4D12(b) 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 Flag T M or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 191P4D12(b). In addition, expression in yeast yields similar post-translational modifications, such as S glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 191P4D12(b) in the yeast species Saccharomyces pombe, all or parts of the 191P4D12(b) cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 191P4D12(b) protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST 00 which aids purification of the recombinant protein. A FlagT epitope tag allows detection of the recombinant protein with anti- SFlag T M antibody.

Example 8: Production of Recombinant 191P4D12(b) in Higher Eukaryotic Systems A. Mammalian Constructs: To express recombinant 191P4D12(b) in eukaryotic cells, the full or partial length 191P4012(b) cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of S191P4D12(b) are expressed in these constructs, amino adds 1 to 510, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 191P4D12(b) v.1, v.2, v.10, v.11, v.12; amino acids 1 to 511, 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 191P4D12(b) v.13, 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-191P4D12(b) polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 191P4D12(b) in mammalian cells, a 191P4D12(b) ORF, or portions thereof, of 191P4D12(b) were cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 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.

pcDNA3.11MycHis Constructs: To express 191P4D12(b) in mammalian cells, a 191P4D12(b) ORF, or portions thereof, of 191 P4D12(b) with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovlrus (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 episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Figure 22 shows expression of 191P4D12(b).pcDNA3.1/MycHis following vector transfection into 293T cells. 293T cells were transfected with either 191P4D12(b).pcDNA3.1/mychis or pcDNA3.11mychis vector control. Forty hours later cell lysates 00 were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression of 191P4D12(b) in the lysates of 191P4D12(b).pcDNA3.1/mychis transfected cells (Lane but not from the control pcDNA3.1/mychis (Lane 4).

pcDNA3.11CT-GFP-TOPO Construct: To express 191P4D12(b) in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 191P4D12(b) 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 r 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 00 T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the Sampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional N constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 191P4D12(b) protein.

PAPta: A 191P4D12(b) 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 191P4D12(b) protein while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of a 191P4D12(b) protein. The resulting recombinant 191P4D12(b) 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 191 P4D12(b) 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 coli.

A 191P4D12(b) v.1 extracellular domain was cloned into pTag-5 plasmid. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 191P4D12(b) protein with an amino-terminal IgGK signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification.

The resulting recombinant 191P4D12(b) 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 191P4D12(b) proteins.

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

Figure 22 shows expression and secretion of the extracellular domain of 191P4D12(b) following 191P4D12(b).pTag5 vector transfection into 293T cells. 293T cells were transfected with 191P4D12(b) .pTag5. Forty hours later, cell lysate and supematant were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody.

The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression from 191P4D12(b).pTag5 plasmid of 191P4D12(b) extracellular domain in the lysate (Lane 2) and secretion in the culture supematant (Lane 1).

191P4D12(b) ORF, or portions thereof, is also cloned into pTag-5 plasmid.

PsecFc: A 191P4D12(b) ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 191P4D12(b) proteins, while fusing the IgGK signal sequence to N-terminus. 191P4D12(b) fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 0 o 191P4D12(b) proteins are optimized for secretion Into the media of transfected mammalian cells, and can be used as 0 immunogens or to identify proteins such as ligands or receptors that interact with 191P4D12(b) protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRa Constructs: To generate mammalian cell lines that express 191P4D12(b) constitutively, 191P4D12(b) ORF, or portions thereof, of 191P4D12(b) 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 (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 191P4D12(b), into the host cell-lines. Protein C expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for p selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection 0 0 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.

Figure 23 shows stable expression of 191P4D12(b) following 191P4D12(b).pSRa transduction into 3T3 cells. 3T3 cells were transduced with the pSRa retroviral vector encoding the 191P4D12(b) gene. Following selection with neomycin, the cells were expanded and RNA was extracted. Northern blot with 10 ug of total RNAlane was probed with the 191P4D12(b) SSH sequence. Size standards In kilobases (kb) are indicated on the side. Results show expression of the 191P4D12(b) transcript driven from the retroviral LTR, which migrates slower than the endogenous 4 kb 191P4D12(b) transcript detected in the positive control LAPC-4AD.

Additional pSRa constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 191P4D12(b) 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: 60) 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 191P4D12(b) proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 191P4012(b). High virus titer leading to high level expression of 191P4D12(b) is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 191P4D12(b) coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 191P4D12(b) 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 191P4D12(b) in mammalian cells, coding sequences of 191P4D12(b), 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 191P4D12(b). These vectors are thereafter used to control expression of 191P4D12(b) in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems To generate recombinant 191P4D12(b) proteins in a baculovirus expression system, 191P4D12(b) ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the Nterminus. Specifically, pBlueBac-191P4D12(b) 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.

00 Recombinant 191P4D12(b) protein is then generated by infection of HighFive insect cells (Invitrogen) with purified S baculovirus. Recombinant 191P4D12(b) protein can be detected using anti-191P4D12(b) or anti-His-tag antibody.

Cr 191P4012(b) protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 191P4D12(b).

Example 9: Antigenicity Profiles and Secondary Structure c Figure Figure Figure Figure and Figure 9(A-C) depict graphically five amino acid profiles of 191P4D12(b) variants 1, 7, and 9, each assessment available by accessing the ProtScale website located on the S World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.

C1 These profiles: Figure 5, Hydrophilicity, (Hopp Woods 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828); Figure 6, Hydropathicity, (Kyte Doolittle 1982. J. Mol. Biol. 157:105-132); Figure 7, Percentage Accessible Residues (Janin 1979 Nature 277:491-492); Figure 8, Average Flexibility, (Bhaskaran and Ponnuswamy 1988.

00 Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, Roux B. 1987 Protein Engineering 1:289-294); and C optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 191P4D12(b) variant proteins. Each of the above amino acid profiles of 191P4D12(b) 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-tur 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 191P4D12(b) variant proteins indicated, by the profiles set forth in Figure Figure Figure Figure andlor Figure 9(A-C) are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-191P4D12(b) antibodies. The immunogen can be any 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 or more than contiguous amino acids, or the corresponding nucleic acids that encode them, from the 191P4D12(b) protein variants listed in Figures 2 and 3, of which the amino acid profiles are shown in Figure 9, or are identical to the variant sequences that are the same as a variant depicted in figure 9. 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 5; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the 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 3 in 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 0 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.

The secondary structure of 191P4D12(b) protein variants 1,7, and 9, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsann.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). The analysis indicates that 191P4D12(b) variant 1 is composed of 24.90% alpha helix, 18.63% extended strand, and 56.47% random coil (Figure 13A). Variant 6 is composed of 28.47% alpha helix, 19.32% extended strand, and 52.20% random coil (Figure 13B).

Variant 7 is composed of 26.19% alpha helix, 18.76% extended strand, and 55.05% random coil (Figure 13C). Variant 7 is C composed of 56.20% alpha helix, 8.76% extended strand, and 35.04% random coil (Figure 13D).

0 Analysis for the potential presence of transmembrane domains in the 191P4D12(b) variant proteins was carried out 0 using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Shown graphically in figure 13E and 13F are the results of analysis of variant 1 depicting the presence and location of 1 transmembrane domain using the TMpred program (Figure 13E) and 1 transmembrane domain using the TMHMM program (Figure 13F). Shown graphically In figure 13G and 13H are the results of analysis of variant 6 depicting the presence and location of 1 transmembrane domains using the TMpred program (Figure 13G) and 1 transmembrane domain using the TMHMM program (Figure 13H). Shown graphically in figure 131 and 13J are the results of analysis'of variant 7 depicting the presence and location of 1 transmembrane domain using the TMpred program (Figure 131) and 1 transmembrane domain using the TMHMM program (Figure 13J). Shown graphically in figure 13K and 13L are the results of analysis of variant 9 depicting the presence and location of 2 transmembrane domains using the TMpred program (Figure 1K) and 1 transmembrane domain using the TMHMM program (Figure 13L). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table VI and Table L.

Example 10: Generation of 191P4D12(b) Polyclonal 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 191P4D12(b) 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 Structures"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, Figure Figure 6(A C), Figure Figure 8(A or Figure 9(A-C) for amino acid profiles that indicate such regions of 191P4D12(b) protein variants).

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 191P4D12(b) protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 11. For example, in 191P4D12(b) variant 1, such regions include, but are not limited to, amino acids 27-39, amino acids 93-109, and amino acids 182-204. In sequence unique to variant 7, such regions include, but are not limited to, amino acids 400-420. In sequence specific for variant 9, such regions include, but are not limited to, amino acids 80-94. 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 00 encoding amino acids 52-63 of 191P4D12(b) variant 1 and amino acids 179-197 were each conjugated to KLH and used to S immunize separate rabbits. Alternatively the immunizing agent may include all or portions of the 191P4D12(b) variant C proteins, analogs or fusion proteins thereof. For example, the 191P4D12(b) 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 2-349 of C 191P4D12(b) variant 1 was 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, N thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 191P4D12(b) in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, Brady, Urnes, Grosmaire, Damle, and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).

00 SIn addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These c antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled "Production of Recombinant 191P4D12(b) in Eukaryotic Systems"), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 31-347 of variant 1, encoding the extracellular domain, was doned into the Tag5 mammalian secretion vector, and expressed in 293T cells resulting in a soluble secreted protein (Figure 22). The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 191P4D12(b) protein is then used as immunogen.

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

In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 pg, typically 100-200 pg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 pg, typically 100-200 pg, of the immunogen in incomplete Freund's adjuvant (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 -191P4D12(b) variant 1 protein, the full-length 191P4D12(b) variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled "Production of Recombinant 191P4D12(b) in Eukaryotic Systems").

After transfection of the constructs into 293T cells, cell lysates are probed with the anti-191P4D12(b) serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 191P4D12(b) protein using the Western blot technique. In addition, the Immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T (Figure 22) and other recombinant 191P4D12(b)-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 191P4D12(b) are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 191P4D12(b) 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- 191P4D12(b) variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel 1 matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP- 191P4D12(b) fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity S chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as 0 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 191P4D12(b) Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 191P4D12(b) 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 191P4D12(b) 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 191P4D12(b) protein S variant sequence, regions of the 191P4D12(b) protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, Figure Figure Figure Figure or Figure and the Example 00 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 C respective 191P4D12(b) variant, such as 293T-191P4D12(b) variant 1 or 300.19-191P4D12(b) variant Imurine Pre-B cells, are used to immunize mice.

To generate mAbs to a 191P4D12(b) variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 pg of protein immunogen or 107 191P4D12(b)-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 pg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cellbased immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 191P4D12(b) variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 31-347 was cloned into the Tag5 mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 191P4D12(b) variant 1 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus 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 191P4D12(b) 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 191P4D12(b) monoclonal antibodies, a Tag5-191P4D12(b) variant 1 antigen encoding amino acids 31-347, was expressed (Figure 22) and then purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 jg of the Tag5-191P4D12(b) variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 pig of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 191P4D12(b) variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 191P4D12(b) variant 1 cDNA (see the Example entitled "Production of Recombinant 191P4D12(b) in Eukaryotic Systems" and Figure 22). Other recombinant 191P4D12(b) variant 1-expressing cells or cells endogenously expressing 191P4D12(b) variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells 00 using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, S Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 191P4D12(b) specific antibody- Cl producing clones.

To generate monoclonal antibodies that are specific for each 191P4D12(b) variant protein, immunogens are S designed to encode sequences unique for each variant. In one embodiment, a GST-fusion antigen encoding the full sequence of 191P4D12(b) variant 9 (AA 1-137) is produced, purified, and used as immunogen to derive monoclonal LC antibodies specific to 191P4D12(b) variant 2. In another embodiment, an antigenic peptide composed of amino acids 400- 420 of 191P4D12(b) variant 7 is coupled to KLH and used as immunogen. Hybridoma supematants are then screened on the respective antigen and then further screened on cells expressing the specific variant and cross-screened on cells C<l expressing the other variants to derive variant-specific monoclonal antibodies.

The binding affinity of a 191 P4D 12(b) variant monoclonal antibody Is determined using standard technologies.

r1 Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 191P4D12(b) 00 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The S 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, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 12: HLA Class I and Class II Binding Assays HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125 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 presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and ICso_[HLA], the measured ICso 'values are reasonable approximations of the true Ko values. Peptide inhibitors are typically tested at concentrations ranging from 120 p.g/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 ICs0 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 0 0 confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of 0 population coverage is performed using the strategy described below.

Computer searches and algorithms for identification of supermotif and/or motif-bearing epitopes SThe searches performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles" and Tables VII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 191P4D12(b) set O 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 191P4D12(b) 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 S information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

SIdentified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity 00 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 S interactions can be approximated as a linear polynomial function of the type: "AG" aul x a2 x a x ani where aji is a coefficient which represents the effect of the presence of a given amino acid at a given position (i) 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 irrespective of the sequence of the rest of the peptide.

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

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

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 chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 supertype cross-reactive peptides Protein sequences from 191P4D12(b) are scanned utilizing motif identification software, to identify 9- 10- and 11-mer 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 191P4D12(b) protein sequence(s) scanned above is also examined for the presence of peptides with the HLAc0 A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized 0 and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3- C supertype 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.

C Selection of HLA-B7 supermotif bearing epitopes The 191P4D12(b) protein(s) scanned above is also analyzed for the presence of 9- 10-, or 11-mer peptides with I the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule C encoded by the most common 87-supertype allele the prototype B7 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- CK supertype molecules 8*3501, B*5101, B'5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype 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 191P4D12(b) protein can also be performed to identify HLA-A1- and A24-motif-containing 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 Immunogenicity 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: 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-restridted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 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 lpg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino adds, 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 supernatants. 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 200-250x10 6 PBMC are processed to obtain 0 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 S 20xl06cells/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°C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at 100x10 6 cells/ml (based on the original cell number) in PBS/AB serum containing C 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 1-2x10/ml in the presence of 3pg/ml 112- microglobulin for 4 hours at 20°C. The DC are then NC Irradiated (4,200 rads), washed 1 time with medium and counted again.

N Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1x10 s cells/ml) are co-cultured with 0.25ml of 00 CD8+ T-cells (at 2x10 6 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human 0 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 5x10 6 cells/ml and irradiated at -4200 rads. The PBMCs are plated at 2x10 6 in 0.5 ml complete medium per well and incubated for 2 hours at 370C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10pg/ml of peptide in the 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 cullures (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 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 1 Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNy ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL lytic activity by 5 'Cr release.

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

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200pCi of 1 Cr 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 3.3x10 6 /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 370C. 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 51 Cr release samplecpm of the spontaneous 51 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 00 higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

SIn situ Measurement of Human IFNy Production as an Indicator of Peptide-specific and Endogenous Recognition Cl 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 Mg 2 -free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 pl/well) and targets (100 pl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used l at a concentration of 1x10 6 cells/ml. The plates are incubated for 48 hours at 37°C with 5% CO2.

Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200pg/100 microliter/well and the plate incubated for two hours at 37°C. The plates are washed and 100 pl of biotinylated mouse anti-human IFN- S gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one 00 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 H 3 P0 4 Cl and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gammalwell 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, 5x10 4 CD8+ cells are added to a T25 flask containing the following: lx106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x10 5 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, sodium pyruvate, 25pM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours 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 lx10 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 1x10 6 /ml in the in situ IFNy assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3* as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5x104 CD8* cells are added to a T25 flask containing the following: 1x106 autologous PBMC per ml which have been peptide-pulsed with 10 pg/ml peptide for two hours at 37°C and 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.

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 191P4D12(b). 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 immunoqenicity 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 immunoqenicitv 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.

00 Peptides bearing other supermotifs/motifs, HLA-A1, HLA-A24 etc. are also confirmed using similar S methodology Example 15: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs SHLA 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 S 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.

00 Analoqing at Primary Anchor Residues Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For S example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, 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 binding affinity to any one (or more) of the supertype members to add population coverage.

The 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 A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have Increased immunogenicity and crossreactivity by T cells specific for the parent epitope (see, Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et a., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

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.

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

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

Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. 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 OO possible, targets that endogenously express the epitope.

O

CN Analoging at Secondary Anchor Residues Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides andlor 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 S 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.

C 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 r1 peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 191P4D12(b)- 00 expressing tumors.

SOther analoging strategies Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with ccamino 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 ct-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley Sons, England, 1999).

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

Example 16: Identification and confirmation of 191P4D12(b)-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 methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-supermotif-bearinq epitopes.

To identify 191P4D12(b)-derived, HLA class II HTL epitopes, a 191P4D12(b) antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DRsupermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

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

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

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

The 191P4D12(b)-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, DR6w19, 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 00 S 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. 191P4D12(b)-derived peptides found to S 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 LC 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 191P4D12(b) antigens are analyzed for sequences carrying CK1 one of the two DR3-specific binding motifs reported by Geluk et al. Immunol. 152:5742-5748, 1994). The corresponding LC peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 LM or better, less than 0 1 pM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

SDR3 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 for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 17: Immunogenicity of 191P4D12(b)-derived HTL epitopes This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

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 patients who have 191P4D12(b)-expressing tumors.

Example 18: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine breadth 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(1at)) (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 A'7401, 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, Sthe B7-like supertype-confirmed alleles are: B7, B83501-03, 851, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B7801 0 (potentially also 8*1401, B*3504-06, 8*4201, and B*5602).

CPopulation coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major S 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 C 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 at., J. Clin. Invest. 100:503,1997; Doolan et al., Immunity 7:97, c 1997; and Threlkeld et J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost CN always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in 00 0 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 used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred 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.

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 additional six days later, these cell lines are tested for cytotoxic activity on 51 Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 5'Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 191P4D12(b) expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 191P4D12(b) antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addilion to HLA-A*0201/Kb transgenic mice, several other transgenic 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 epitdpes.

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 191P4D12(b)-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 191P4D12(b)-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 S Immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide O 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 al., 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 C1 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 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 CK chimeric gene Vitiello et al,, J. Exp. Med. 173:1007, 1991) SIn vitro CTL activation: One week after priming, spleen cells (30x10 6 cells/flask) are co-cultured at 37*C with O 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.

Assay for cytotoxic activity: Target cells (1.0 to 1.5x106) are incubated at 37°C in the presence of 200 pl of s 5 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 slCr-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 gamma counter. The percent specific lysis is determined by the formula: percent specific release 100 x (experimental release spontaneous release)/(maximum release spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, s Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 51 Cr release assay. To obtain specific lytic units/10 6 the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide.

For example, if 30% 5'Cr release is obtained at the 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 and/or multiple HTL epitopes. In accordance with these procedures, It is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21: Selection of CTL and HTL epitopes for Inclusion in a 191P4D12(b)-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 191P4D12(b) clearance. The number of epitopes used depends on observations of patients who spontaneously clear 191P4D12(b). For 00 example, if it has been observed that patients who spontaneously clear 191P4D12(b)-expressing cells generate an immune response to at least three epitopes from 191P4D12(b) antigen, then at least three epitopes should be included for HLA class 1. 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 11, an ICso of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL 0 bimas.dcrt.nih.govl.

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 S 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.

O When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the 00 smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as O those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine Cl 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 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. 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. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system 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 analogs) directs the immune response to multiple peptide sequences that are actually present in 191P4D12(b), 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 191P4D12(b).

Example 22: Construction of "Minigene" Multi-Epltope DNA Plasmids 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, -B7 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 191P4D12(b), are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 191P4D12(b) 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 00 S 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 li 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-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein.

C-K The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

00 Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide 0 overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by 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°C for 15 sec, annealing temperature (50 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 pig 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 p1 reactions containing Pfu polymerase buffer (1x= 10 mM KCL, 10 mM (NH4)2S04, mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 gg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu 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 fulllength 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 Immunogenicity.

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of 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 al., J.

Immunol. 156:683-692, 1996; Demotz etal., 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 .g of 00 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 C 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 S 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.

C To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized 1 intramuscularly with 100 pg 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 Barnett at al., Aids Res. and Human Retroviruses 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 al, Vaccine 16:439- 445,1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177- 181, 1999; and Robinson etal., Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 jig 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 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 ig 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 and/or 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 191P4D12(b) expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid S comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are 0 also selected to target greater than 80% of the population, is administered to individuals at risk for a 191P4D12(b)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 Ci Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 pg, generally 100-5,000 pg, for a kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the S magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both C= safe and efficacious as a prophylaxis against 191P4D12(b)-associated disease.

SAlternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid- 00 based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 25: Polyepitopic Vaccine Compositions Derived from Native 191P4012(b) Sequences A native 191P4D12(b) polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify "relatively short" regions of the polyprotein that comprise multiple epitopes. The "relatively short" regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, "nested" epitopes can be used to generate a minigene construct.

The construct is engineered to express the peptide, which corresponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is 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 example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 191P4D12(b) antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity 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 191P4D12(b), 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: Polyepitopic Vaccine Compositions from Multiple Antigens The 191P4D12(b) 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 00 expresses 191P4D12(b) and such other antigens. For example, a vaccine composition can be provided as a single O polypeptide that incorporates multiple epitopes from 191P4D12(b) as well as tumor-associated antigens that are often C1 expressed with a target cancer associated with 191P4D12(b) expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene S construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

S Example 27: Use of peptides to evaluate an immune response Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, S CTL or HTL directed to 191P4D12(b). Such an analysis can be performed in a manner described by Ogg et al., Science CKl 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- 00 sectional analysis of, for example, 191P4D12(b) HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals c- at different stages of disease or following immunization comprising a 191P4D12(b) 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 (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-phycoerythrin.

For 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-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers 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 191P4D12(b) epitope, and thus the status of exposure to 191P4D12(b), 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 acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 191P4D12(b)-associated disease or who have been vaccinated with a 191P4D12(b) vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 191P4D12(b) 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) O supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 pg/ml), and Hepes (10mM) containing c heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising S an epitope of the invention is added at 10 .g/ml to each well and HBV core 128-140 epltope is added at 1 p.g/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4 x 10 5 PBMC are stimulated with peptide in 8 replicate cultures In 96-well round bottom r plate In 100 pl/well of complete RPMI. On days 3 and 10, 100 pl of complete RPMI and 20 U/ml final concentration of 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, r- rlL-2 and 10 s irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxlc activity on day 14. A N positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 61 Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med.

C 2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et a. J. Clin. Invest. 98:1432- 00 0 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 iCi of S 5 Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well s 5 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 x [(experimental release-spontaneous release)/maximum releasespontaneous release)]. Maximum release is determined by lysis of targets by detergent Triton X-100; Sigma Chemical Co., St. Louis, MO). Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 191P4D12(b) or a 191P4D12(b) vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5x10 5 cells/well and are stimulated with 10 ig/ml synthetic peptide of the Invention, whole 191P4D12(b) 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 pCi 3 H-thymldine 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 I: 3 subjects are injected with placebo and 6 subjects are injected with 5 pg of peptide composition; Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 pg peptide composition; Group III: 3 subjects are Injected with placebo and 6 subjects are injected with 500 pg of peptide composition.

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

O

0The 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 t 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.

SSafety: 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.

SThe vaccine is found to be both safe and efficacious.

00 Example 30: Phase II Trials In Patients Expressing 191P4D12(b) r Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 191P4D12(b). The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 191P4D12(b), 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: The 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.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and 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 191P4D12(b).

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 bdth safe and efficacious in the treatment of 191P4D12(b)associated 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 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 5-10 7 to 5x109 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 0 of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before S immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine.

S 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.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 191P4D12(b) 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 CN such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this C method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the 00 invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL 0 and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 191P4D12(b) protein from which the epitopes in the vaccine are derived.

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

A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin T 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 peptides.

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

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

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

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as ProgenipoietinTM 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 doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if ProgenipoletinTM 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 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex vivo activation of CTLHTL responses Altematively, ex vivo CTL or HTL responses to 191P4D12(b) 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. 191P4D12(b). Peptides produced 00 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 CK1 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 alternative 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 S nucleic acids that encode 191P4D12(b) to isolate peptides corresponding to 191P4D12(b) that have been presented on the CKl 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.

OC) As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele 00 and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize C=K that 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 Polynucleotldes Sequences complementary to the 191P4D12(b)-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 191P4D12(b). 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 of 191P4D12(b). 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 191P4D12(b)-encoding transcript.

Example 35: Purification of Naturally-occurring or Recombinant 191P4D12(b) Using 191P4D12(b)-Specific Antibodies Naturally occurring or recombinant 191P4D12(b) is substantially purified by immunoaffinity chromatography using antibodies specific for 191P4D12(b). An immunoaffinity column is constructed by covalently coupling anti-191P4D12(b) 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 manufacturers instructions.

Media containing 191P4D12(b) are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 191P4D12(b) high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/191P4D12(b) 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 191P4D12(b) 191P4D12(b), or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et at. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 191P4D12(b), washed, and any wells with labeled 191P4D12(b) complex are assayed. Data obtained using different concentrations of 191P4D12(b) are used to calculate values for the number, affinity, and association of 191P4D12(b) with the candidate molecules.

00 S Example 37: In Vivo Assay for 191P4D12(b) Tumor Growth Promotion S The effect of the 191P4D12(b) protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 191P4D12(b). For example, SCID mice are Injected subcutaneously on each flank with 1 x 106 of either 3T3, prostate PC3 cells), bladder UM-UC3 cells), kidney CaKI cells), or lung A427 cells) cancer cell lines containing tkNeo empty vector or 191P4D12(b). At least two strategies may be used: Constitutive 191P4D12(b) 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 2), 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 00 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 C 191P4D12(b)-expressing cells grow at a faster rate and whether tumors produced by 191P4D12(b)-expressing cells demonstrate characteristics of altered aggressiveness enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1 x 10 5 of the same cells orthotopically to determine if 191P4D12(b) has an effect on local growth in the prostate, and whether 191P4D12(b) affects the ability of the cells to metastasize, specifically to lymph nodes, and bone (Miki T et al, Oncol Res. 2001;12:209; Fu X et al, Int J Cancer. 1991, 49:938). The effect of 191P4D12(b) on bone tumor formation and growth may be assessed by injecting tumor cells intratiblally.

The assay is also useful to determine the 191P4D12(b) inhibitory effect of candidate therapeutic compositions, such as for example, 191P4D12(b) intrabodies, 191P4D12(b) antisense molecules and ribozymes.

Example 38: 191P4D12(b) Monoclonal Antibody-mediated Inhibition of Tumors In Vivo The significant expression of 191 P4D12(b) in cancer tissues and surface localization, together with its restrictive expression in normal tissues makes 191P4D12(b) a good target for antibody therapy. Similarly, 191P4D12(b) is a target for T cell-based immunotherapy. Thus, the therapeutc efficacy of anti-191P4D12(b) mAbs in human cancer xenograft mouse models, including prostate, lung, bladder, kidney and other -191P4D12(b)cancers listed in table 1, is evaluated by using recombinant cell lines such as PC3-191P4D12(b), UM-UC3-191P4D12(b), CaKi-191P4D12(b), A427-191P4D12(b) and 3T3-191P4D12(b) (see, Kaighn, et Invest Urol, 1979. 17(1): 16-23), as well as human prostate, kidney and bladder xenograft models such as LAPC 9AD, AGS-K3 and AGS-B1 (Saffran et al PNAS 1999,10:1073-1078).

Antibody efficacy on tumor growth and metastasis formation is studied, in a mouse orthotopic prostate, kidney, bladder, and lung cancer xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-191P4D12(b) mAbs inhibit formation of tumors in prostate kidney, bladder and lung xenografts. Anti-191P4012(b) mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-191P4D12(b) mAbs in the treatment of local and advanced stages several solid tumors. (See, Saffran, et al., PNAS 10:1073-1078 or world wide web URL pnas.org/cgildoi/10.1073/pnas.051624698).

Administration of the anti-191 P4D12(b) mAbs led to retardation of established orthotoplc tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 191P4D12(b) as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-191P4D12(b) mAbs for the treatment of local and metastatic prostate cancer. This example indicates that unconjugated 0 191P4D12(b) monodonal antibodies are effective to inhibit the growth of human prostate, kidney, bladder and lung tumor O xenografts grown In SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

CK Tumor Inhibition using multiple unconjugated 191P4D12(b) mAbs Materials and Methods 191P4D12(b) Monoclonal Antibodies: Monoclonal antibodies are raised against 191P4D12(b) as described in the Example entitled "Generation of 1 191P4D12(b) Monoclonal Antibodies (mAbs)." The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 191P4D12(b). Epitope mapping data for the anti-191P4D12(b) mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 191P4D12(b) protein. Immunohistochemical analysis C of prostate, kidney, bladder and lung cancer tissues and cells with these antibodies is performed.

SThe monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Cr Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are 00 performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of PC3, UM-UC3, CaKi and A427 tumor xenografts.

Cell Lines and Xenografts The cancer cell lines, PC3, UM-UC3, CaKi, and A427 cell line as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in RPMI (PC3) and DMEM (UM-UC3, CaKi, and A427, 3T3) respectively, supplemented with L-glutamine and 10% FBS.

PC3-191P4D12(b), UM-UC3-191P4D12(b), CaKi-191P4D12(b), A427-191P4D12(b) and 3T3-191P4D12(b) cell populations are generated by retroviral gene transfer as described in Hubert, et al., Proc Natl Acad Sci U S A, 1999. 96(25): 14523.

The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, et al., Nat Med. 1999, 5:280). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al. Similarly, kidney (AGS-K3) and bladder (AGS-B1) patient-derived xenografts are passaged in 6- to 8-week-old male ICR-SCID mice.

Xenograft Mouse Models.

Subcutaneous tumors are generated by injection of 2 x 10 6 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.e. 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 caliper measurements, and the tumor volume is calculated as length x width x height Mice with Subcutaneous tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdomen to expose the prostate and LAPC or PC3 tumor cells (5 x 105) mixed with Matrigel are injected Into the prostate capsule in a 10-pl volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure PSA levels. For kidney orthotopic models,.an incision is made through the abdominal muscles to expose the kidney. AGS-K3 cells mixed with Matrigel are injected under the kidney capsule. The mice are segregated into groups for the appropriate treatments, with anti-191P4D12(b) or control mAbs being injected i.p.

00 SAnti-191P4D12(b) mAbs Inhibit Growth of 191P4D12(b)-Expressing Xenoqraft-Cancer Tumors The effect of anti-191P4D12(b) mAbs on tumor formation is tested by using cell line PC3, UM-UC3, CaKI, A427, and 3T3) and patient-derived tumor LAPC9, AGS-K3, AGS-B1) orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse organ, such as prostate, bladder, kidney or S lung, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, et al., PNAS supra). The features make the orthotoplc model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

For example, tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and CK" treated with either: a) 200-500pg, of anti-191P4D12(b) Ab, or b) PBS three times per week for two to five weeks.

A major advantage of the orthotopic cancer models is the ability to study the development of metastases.

0 0 Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an 0 antibody against a tumor-specific cell-surface protein such as anti-CK20 for prostate cancer (Lin S et al, Cancer Detect Prev.

2001;25:202).

Another advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth is partly dependent on new blood vessel development. Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovascular is regulated by the xenograft tumor (Davidoff AM et al, Clin Cancer Res. 2001;7:2870; Solesvik O et al,, Eur J Cancer Clin Oncol. 1984, 20:1295). The effect of antibody and small molecule on neovascularization is studied in accordance with procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment.

Mice bearing established orthotopic tumors are administered 1000pg injections of either anti-191P4D12(b) mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-191P4D12(b) antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-191P4D12(b) antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-191P4D12(b) mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-191P4D12(b) mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39: Therapeutic and Diagnostic use of Anti-191P4D12(b) Antibodies In Humans.

Anti-191 P4D12(b) 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-191P4D12(b) mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 191P4D12(b) in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-191P4D12(b) 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-191P4D12(b) mAb specifically binds to carcinoma cells. Thus, anti- 191P4D12(b) 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 191P4D12(b). Shedding or release of an extracellular domain of 191P4D12(b) into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., 00 Hepatology 27:563-568 (1998)), allows diagnostic detection of 191P4D12(b) by anti-191P4D12(b) antibodies in serum and/or urine samples from suspect patients.

ri Anti-191P4D12(b) antibodies that specifically bind 191P4D12(b) are used in therapeutic applications for the treatment of cancers that express 191P4D12(b). Anti-191P4D12(b) 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-191P4D12(b) antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, the Example entitled "191P4D12(b) Monoclonal Antibody-mediated S Inhibition of Bladder and Lung Tumors In Vivo). Either conjugated and unconjugated anti-191P4D12(b) antibodies are used CN as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

00 Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human C Anti-191P4D12(b) Antibodies In vivo Antibodies are used in accordance with the present invention which recognize an epitope on 191P4D12(b), and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 191P4D12(b) expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

Adjunctive therapy: In adjunctive therapy, patients are treated with anti-191P4D12(b) antibodies in combination with a chemotherapeutic or antineoplastic agent andlor radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-191P4D12(b) antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-191P4D12(b) antibodies are utilized in several adjunctive 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).

II.) Monotherapy: In connection with the use of the anti-191P4D12(b) 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 radionuclide iodine or yttrium (1131, Y 9 0 to anti-191P4D12(b) 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 191P4D12(b). In connection with the use of the anti-191P4D12(b) antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (11' ln)-191P4D12(b) antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 191P4D12(b) (by analogy see, Divgi etal. J. Natl. Cancer nst. 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-191P4D12(b) antibodies can be administered with doses in the 00 range of 5 to 400 mg/m 2, with the lower doses used, In connection with safety studies. The affinity of anti- 191P4D12(b) 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-191P4D12(b) antibodies that are fully human antibodies, as c compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti- 191P4D12(b) antibodies can be lower, perhaps in the range of 50 to 300 mg/m 2 and still remain efficacious. Dosing in mg/m 2 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-191P4D12(b) antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the CK peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may 0 prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar 00 manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a O high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

NC Clinical Development Plan (CDP) Overview: The CDP follows and develops treatments of anti-191P4D12(b) 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-191P4D12(b) antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 191P4D12(b) expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to cytokine release syndrome, hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 191P4D12(b). Standard tests and follow-up are utilized to monitor each of these safety concerns.

Anti-191P4D12(b) antibodies are found to be safe upon human administration.

Example 41: Human Clinical Trial Adjunctive Therapy with Human Anti-191P4D12(b) Antibody and Chemotherapeutic Agent A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti- 191P4D12(b) 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-191P4D12(b) 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-191P4D12(b) antibody with dosage of antibody escalating from approximately about 25 mg/m 2 to about 275 mg/m 2 over the course of the treatment in accordance with the following schedule: Day 0 Day 7 Day 14 Day 21 Day 28 Day 00 mAb Dose 25 75 125 175 225 275 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m2 mg/m 2 SChemotherapy (standard dose) O Patients are closely followed for one-week following each administration of antibody and chemotherapy. In C 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 S antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that CK express 191P4D12(b). 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.

C The anti-191P4D12(b) antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy 0 and refine optimum dosing.

Example 42: Human Clinical Trial: Monotherapy with Human Antl-191P4D12(b) Antibody Anti-191P4D12(b) antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same 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-191P4D12(b) antibodies.

Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-191P4D12(b) Antibody Once 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-191P4D12(b) antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44: Homology Comparison of 191P4D12(b) to Known Sequences The human 191P4D12(b) protein exhibit a high degree of homology to a known human protein, namely Ig superfamily receptor LNIR (gi 14714574), also known as human nectin 4 (gi 16506807). Human LNIR shows 100% identity to 191P4D12(b) at the protein level. The mouse homolog of 191P4D12(b) has been Identified as murine nectin 4 (gi 18874521). It shows strong homology to 191P4D12(b), exhibiting 92% identity and 95% homology to 191P4D12(b). (See, Figure 4).

The prototype member of the 191P4D12(b) family, 191P4D12(b)v.1, Is a 510 amino acids protein, with the Nterminus located extracellulary and intracellular C-terminus. Initial bioinformatics analysis using topology prediction programs suggested that 191P2D14 may contain 2 transmembranes based on hydrophobicity profile. However, the first hydrophobic domain was identified as a signal sequence, rendering 191P2D12 a type I membrane protein, with an extracellular N-terminus.

The 191P4D12(b) gene has several variants, including one SNP represented in 191P4D12(b) v.2, an N-terminal deletion variant represented in 191P4D12(b) v.6 and 191P4D12(b) v.7 which lacks 25 amino acids between amino acids 411 and 412 of 191P4D12(b) v.1.

Motif analysis revealed the presence of several protein functional motifs in the 191P4D12(b) protein (Table L).

Two immunoglobulin domains have been identified at positions 45-129 and 263-317. In addition, 191P4D12(b) contains a 00 cadherin signature which includes and RGD sequence. Immunoglobulin domains are found in numerous proteins and participate in protein-protein such including protein-ligand interactions (Weismann et al, J Mol Med 2000, 78:247). In addition, Ig-domains function in cell adhesion, allowing the interaction of leukocytes and blood-born cells with the ct endothelium (Wang and Springer, Immunol Rev 1998, 163:197). Cadherins are single transmembrane proteins containing immunoglobulin like domains, and are involved in cell adhesion and sorting (Shan et al, Biophys Chem 1999, 82:157). They mediate tissue-specific cell adhesion, such as adhesion of lymphocytes to the surface of epithelial cells. Finally, the closest homolog to 191P4D12(b) is Nectin4, a known adhesion molecule that regulates epithelial and endothelial junctions, strongly suggesting that 191P4D12(b) participates in cell adhesion (Reymond N et al, J Biol Chem 2001, 276:43205).

The motifs found in 191P4D12(b) can participate In tumor growth and progression by enhancing the initial stages of tumorigenesis, such as tumor take or establishment of a tumor, by allowing adhesion to basement membranes and surrounding cells, by mediating cell communication and survival.

00 Accordingly, when 191P4D12(b) functions as a regulator of tumor establishment, tumor formation, tumor growth, cell signaling or as a modulator of transcription involved in activating genes associated with survival, invasion, tumorigenesis Cl or proliferation, 191P4D12(b) is used for therapeutic, diagnostic, prognostic and/or preventative purposes. In addition, when a molecule, such as a variant or SNP of 191P4D12(b) is expressed in cancerous tissues, such as those listed in Table I, they are used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Example 45: Regulation of Transcription The cell surface localization of 191P4D12(b) coupled to the presence of Ig-domains within its sequence indicate that 191P4D12(b) modulates signal transduction and the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, by studying gene expression in cells expressing or lacking 191P4D12(b). For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 191P4D12(b)-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, androgen or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001.11:41.).

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

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 positive and negative modulators of pathway activation.

Thus, 191P4D12(b) plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46: Identification and Confirmation of Potential 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). Immunoglobulin-like molecules in particular has been associated with several tyrpsine klnases including Lyc, Blk, syk the MAPK signaling cascade that control cell mitogenesis and calcium flux (Vilen J et al, J Immunol 1997, 159:231; Jiang F, Jia Y, Cohen I. Blood. 2002, 99:3579). In addition, the 191P4D12(b) protein contains several phosphorylation sites (see Table VI) indicating an association with specific signaling cascades.

Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 191P4D12(b) and 00 mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 191P4012(b), including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, C icatenin, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem.

1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). In order to determine whether expression of 191P4D12(b) is sufficient to regulate specific signaling pathways not otherwise active in resting PC3 cells, the effect of these genes on the activation of the p38 MAPK cascade was investigated in the prostate cancer cell line PC3 (Figure 21A-B).

Activation of the p38 kinase is dependent on its phosphorylation on tyrosine and serine residues. Phosphorylated p38 can be distinguished from the non-phosphorylated state by a Phospho-p38 mAb. This phospho-specific Ab was used to study the S phosphorylation state of p38 in engineered PC3 cell lines.

CK PC3 cells stably expressing 191P4D12(b) neo were grown overnight in either 1% or 10% FBS. Whole cell lysates were analyzed by western blotting. PC3 cells treated with the known p38 activators, NaSal or TNF, were used as a positive C control. The results show that while expression of the control neo gene has no effect on p38 phosphorylation, expression of 191P4D12(b) in PC3 cells is sufficient to induce the activation of the p38 pathway (Figure 21A). The results were verified K using western blotting with an anti-p38 Ab, which shows equal protein loading on the gels (Figure 21B).

In another set of experiments, the sufficiency of expression of 191P4D12(b) in the prostate cancer cell line PC3 to activate the mitogenic MAPK pathway, namely the ERK cascade, was examined (Figure 22A-B). Activation of ERK is dependent on its phosphorylation on tyrosine and serine residues. Phosphorylated ERK can be distinguished from the non-phosphorylated state by a Phospho-ERK mAb. This phospho-specific Ab was used to study the phosphorylation state of ERK in engineered PC3 cell lines. PC3 cells, expressing an activated form of Ras, were used as a positive control.

The results show that while expression of the control neo gene has no effect on ERK phosphorylation, expression of 191P4D12(b) in PC3 cells Is sufficient to induce an increase in ERK phosphorylation (Figure 22A). These results were verified using anti-ERK western blotting (Figure 22B) and confirm the activation of the ERK pathway by 191P4D12(b) and STEAP-2.

Since FBS contains several components that may contribute to receptor-mediated ERK activation, we examined the effect of 191P4D12(b) in low and optimal levels of FBS. PC3 cells expressing neo or 191P4D12(b) were grown in either 0.1% or 10% FBS overnight. The cells were analyzed by anti-Phospho-ERK western blotting. This experiment shows that 191P4D12(b) induces the phosphorylation of ERK in 0.1% FBS, and confirms that expression of 191P4D12(b) is sufficient to induce activation of the ERK signaling cascade in the absence of additional stimuli.

To confirm that 191P4D12(b) 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; growthlapoptosis/stress 2. SRE-luc, SRFfTCF/ELK1; MAPK/SAPK; growth/differentiation 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; 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; PKAlp38; growth/apoptosis/stress 7. TCF-luc, TCF/Lef; 0-catenin, Adhesion/invasion 0 0 Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be 0 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 C' luminometer.

Signaling pathways activated by 191P4D12(b) are mapped and used for the identification and validation of 0 therapeutic targets. When 191P4D12(b) is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

r"- Example 47: Involvement in Tumor Progression Based on the role of Ig-domains and cadherin motifs in cell growth and signal transduction, the 191P4D12(b) gene can contribute to the growth, invasion and transformation of cancer cells. The role of 191P4D12(b) in tumor growth is 00 confirmed in a variety of primary and transfected cell lines including prostate cell lines, as well as NIH 3T3 cells engineered to stably express 191P4D12(b). Parental cells lacking 191P4D12(b) and cells expressing 191P4D12(b) are evaluated for cell r C growth using a well-documented proliferation assay (Fraser SP, Grimes JA, Djamgoz MB. Prostate. 2000;44:61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7:288).

To confirm the role of 191P4D12(b) in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 191P4D12(b) are compared to NIH-3T3 cells expressing 191P4D12(b), using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000;60:6730).

To confirm the role of 191P4D12(b) 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 prostate, breast and kidney cell lines lacking 191P4D12(b) are compared to cells expressing 191P4D12(b). Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog.

Invasion Is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.

191P4D12(b) can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 191P4D12(b) are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol.

1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 191P4D12(b), including normal and tumor prostate cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, taxol, 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 191P4D12(b) can play a critical role in regulating tumor progression and tumor load.

When 191P4D12(b) plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 48: Involvement In Anglogenesis 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). Based on the effect of cadherins on tumor cell adhesion and their Interaction with endothelial cells, 191P4012(b) plays a role In angiogenesis (Mareel and Leroy: Physiol Rev, 83:337; DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have been developed to measure anglogenesis 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 role of 191P4D12(b) in angiogenesis, enhancement or inhibition, is confirmed.

00 O For example, endothelial cells engineered to express 191P4D12(b) are evaluated using tube formation and proliferation assays. The effect of 191P4D12(b) is also confirmed in animal models in vivo. For example, cells either expressing or lacking 191P4D12(b) are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 191P4D12(b) affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 49: Involvement in Protein-Protein Interactions Ig-domains and cadherin motifs have been shown to mediate interaction with other proteins, including cell surface protein. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate C- with 191P4D12(b). Immunoprecipitates from cells expressing 191P4D12(b) and cells lacking 191P4D12(b) are compared for CN specific protein-protein associations.

SStudies are performed to confirm the extent of association of 191P4D12(b) with effector molecules, such as O nuclear proteins, transcription factors, kinases, phosphates etc. Studies comparing 191P4D12(b) positive and 191P4D12(b) negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen 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 191P4D12(b)-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 191P4D12(b), 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 191P4D12(b).

Thus it is found that 191P4D12(b) associates with proteins and small molecules. Accordingly, 191P4D12(b) and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 50: Involvement of 191P4D12(b) in cell-cell communication.

Cell-cell communication is essential in maintaining organ Integrity and homeostasis, both of which become deregulated during tumor formation and progression. Based on the presence of a cadherin motif in 191P4D12(b), a motif known to be involved in cell interaction and cell-cell adhesion, 191P4D12(b) can regulate cell communication. Intercellular communications can be measured using two types of assays Biol. Chem. 2000, 275:25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing 191P4D12(b) are compared to controls that do not express 191P4D12(b), and it is found that 191P4D12(b) enhances cell communications.

Figure 19 and Figure 20 demonstrate that 191P4D12(b) mediates the transfer of the small molecule calcein between adjacent cells, and thereby regulates cell-cell communication in prostate cancer cells. In this experiment, recipient PC3 cells were labeled with dextran-Texas Red and donor PC3 cells were labeled with calcein AM (green). The donor (green) and recipient (red) cells were co-cultured at 37 0 C and analyzed by microscopy for the co-localization of Texas red and calcein.

The results demonstrated that while PC3 control cells (no detectable 191P4D12(b) protein expression) exhibit little calcein transfer, the expression of 191P4D12(b) allows the transfer of small molecules between cells (Figure 19), whereby the initially red recipient cells take on a brownish color, and co-localize the red and green molecules. Small molecules and/or 00 antibodies that modulate cell-cell communication mediated by 191P4D12(b) are used as therapeutics for cancers that S express 191P4D12(b). When 191P4D12(b) functions in cell-cell communication and small molecule transport, it Is used as a rCl target or marker for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 51: Modulation of 191P4D12(b) function.

Knock down of 191P4D12(b) expression rli Several techniques can be used to knock down or knock out 191 P4D12(b) expression in vitro and in-vivo, including RNA interference (RNAi) and other anti-sense technologies. RNAi makes use of sequence specific double stranded RNA to prevent gene expression. Small interfering RNA (siRNA) are transfected into mammalian cells and thereby mediate S sequence specific mRNA degradation. (Elbashir, et al, Nature, 2001; vol. 411: 494-498). Using this approach, 191P4D12(b)specific RNAi is introduced in 191P4D12(b)-expressing cells by transfection. The effect of knocking down the expression of CN 191P4D12(b) protein is evaluated using the biological assays mentioned in examples 44 to 50 above.

00 SReduction of 191P4D12(b) Protein expression is detected 24-48 hours after transfection by Immunostaining and S flow cytometry. The introduction of 191P4D12(b) specific RNAi reduced the expression of 191P4D12(b) positive cells and reduce the biological effect of 191P4D12(b) on tumor growth and progression.

Accordingly, the RNA oligonucleotide sequences are used in therapeutic and prophylactic applications. Moreover, the RNA oligonucleotide sequences are used to assess how modulating the expression of a 191P4D12(b) gene affects function of cancer cells and/or tissues.

Inhibition using small molecule and antibodies Using control cell lines and cell lines expressing 191P4D12(b), inhibitors of 191P4D12(b) function are identified.

For example, PC3 and PC3-191P4D12(b) cells can be incubated in the presence and absence of mAb or small molecule inhibitors. The effect of these mAb or small molecule inhibitors are investigated using the cell communication, proliferation and signaling assays described above.

Signal transduction and biological output mediated by cadherins can be modulated through various mechanisms, including inhibition of receptor binding, prevention of protein interactions, or affecting the expression of co-receptors and binding partners (Kamei et al, Oncogene 1999, 18:6776). Using control cell lines and cell lines expressing 191P4D12(b), modulators (inhibitors or enhancers) of 191P4D12(b) function are identified. For example, PC3 and PC3-191P4D12(b) cells are incubated in the presence and absence of mAb or small molecule modulators. When mAb and small molecules modulate, inhibit, the Iransport and tumorigenic function of 191P4D12(b), they are used for preventative, prognostic, diagnostic and/or 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 present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

TABLES:

00 TABLE 1: Tissues that Express 191NPDI 2(b): a. Malianant Tissues Prostate Bladder Kidney Colon Lung Pancreas Ovary Breast Uterus Cervix TABLE ii: Amino AcId Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine Ilie isoleucine M Met methionine T Thr threonine N Asn asparagine K LYS lysine V Val valine A Ala aianlne D Asp aspartic acid E Glu glutamic acid G Gly glycine TABLE III: Amino Acid Substitution Matrix 00 Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The S higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL S ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V W Y.

4 0 -2 -1 -2 0 -2 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C 6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F 6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G 8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 1 -2 -1 0 1 1 2 0 -1 -2 -3 -2 K CN14 2 -3-3 -2-2-2 -1 1 -2 -1 L -2 -2 0 -1 -1 -1 1 1 -1I M 00 6 -2 0 0 1 0 -3 -4-2 N 7 -1 -2 -1 -1 -2 -4 -3 P 1 0 -1 -2 -2 -1 Q 1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 0 -2 -2 T 4 -3 -1 V 1.1 2 W 7 Y TABLE IV: HLA Class 1111 Mo~f slSupermotifs 00 00 TABLE IV HLA Class I SupermotifslMotifs ISUPERMOTIF POSITION POSITION

POSITION

2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Al TIL VMS

FWY

A2 LIVMATQ

_____IVMATL

A3 VSMATLI

RK

A24 YFWIVLMT Fl YWLM B7 P

VILFMWYA

827 RHK B44 ED

________FWYLIMVA

B58 ATS 862 QLIVMP FWYMIVL A MOTIFS Al TSM

Y

Al IDEAS

Y

A2.1 LMVQIAT

VLIMAT

A3 LMVISATFCGD

KYRHFA

AllI VTMLISAGNCDF

KRYH

A24 YFWM

FLIW

A'3101 MVTAUIS A*3301 MVALFIST A*6801 AVTMSLI

RK

B'*0702 p LMFWVfAIV 8*3501 P

LMFWYIVA

B51 P _LIVF WYAM B*5301 P

IMFWYALV

B*5401 P

ATIVLMFWY

Bolded residues are preferred, italicized residues are less preferred: A peptide 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 11 Supermotif TABLE IV HLA Class 11 Motifs MOTIFS 1"a anchor 1 2 3 4 5 1 anchor 6 7 8 9 DR4 preferred FMYUIVW M T I VSTCPALIM MH MH deleterious W R WOE DR1 preferred MFLIVWY PAMQ VMATSPUIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GIRD N G DR3 MOTIFS 1 0anchor 1 2 3 1 *anchor 4 5 1 *anchor 6 Motif a preferred LIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR Supermotif MFLIVWY VMSTACPU/ 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 a Anchor I' Anchor TIL VMS FWY A2 10* Anchor 1 Anchor LIVMATQ LIVMAT A3 Preferred 1 0 Anchor YEW YFW YFW P 1 0 Anchor VSMATL/ RK deleterious DE DE P A24 1' Anchor 1 o Anchor YFV~1VLMT FlY WLM B7 Preferred FWY 10* Anchor FWY FWY 1 "Anchor LIVM P VILFMWYA deleterious DE DE G QN DE G(415); B27 1* Anchor I "Anchor RHK FYLWMIVA B41 *Anchor 1 0 Anchor ED FWYLIMVA 858 1 *Anchor 1" Anchor ATS FWYLIVMA B62 1 *Anchor 1" Anchor QUVMP FWYMIVLA Italicized residues indicate less preferred or "tolerated" residues TABLE IV HLA Class I Motifs POSITION 1 3 4 5 6 7 8 9 C- 00 00 terminus or C-terminus Al preferred GFYW 1 0 Anchor DEA YFW P DEQN YFW 1 *Anchor 9-mer STM Y deleterious DE RHKLIVMP A G A Al preferred GRHK ASTCLIVM 1 0 Anchor GSTC ASTC LIVMV DE 1 0 Anchor 9-mer DEAS

Y

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

Y

mer deleterious GP RHKGLIVM DE RHK ONA RHKYFW RHK A Al preferred YFW STCLIVMV 1 0 Anchor A YFW PG G YFW 1 0 Anchor IDEAS

Y

mer deleterious RHK RHKDEPYFW P G PRHK QN A2.1 preferred YEW l 0 Anchor YFW STO YFW A P l 0 Anchor 9-mer LMIVQAT

VLMAT

deleterious DEP DERKH RKH DERKH POSITION:l1 3 4 5 6 7 8 9 C- Terminus A2.1 preferred AYFW l 0 Anchor LVIm G G FYWL l 0 Anctior LMIVQAT vim VLMAT mer deleterious DEP DE RKHA P RKH DERKHRKH A3 preferred RHK l 0 Anctior YFW PRHKYF A YFW P 1*Anchor LMVISATFCGD W KXRHFA deleterious DEP DE AllI preferred A 1*Anchor YEW YEW A YEW YEW P l 0 Anchor VTLMISAGN CD

KRYH

F

deleterious DEP A G A24 preferred YFWRHK l 0 *Anchor STC YEW YFW l 0 Anchor 9-mer YFWM

FLIW

deleterious DEG DE G QNP DERHKG AQN A24 Preferred 1 0 Anchor P YFWP P 1 0 Anchor YFWM

FLIW

mer Deleterious GDE QN RHK DE A QN DEA A3101 Preferred RHK l 0 Anctior YFW P YFW YFW AP I Anchor MVTALIS

RK

Deleterious DEP DE ADE DE DE DE A3301 Preferred 1 0 Anctior YFW AYFW 1 0 Anchor MVALFIST

RK

Deleterious GP DE A6801 Preferred YFWSTC I *Anchor YFWLIV YEW P l 0 Anchor AVTMSLI M RK deleterious GP DEG RHK A B30702 Preferred RHKFWY' 1 0 Anchor RHK RHK RHK RHK PA 1 0 Anchor P LMFWYAI

V

deleterious DEQNP DEP DE DE GDE QN DE B3501 Preferred FWYLIVM 1 0 Anchor FWY FWY 1*Anchor P LMFWY/V POSITION I POION1 2 3 4 5 6 7 8 9 0- 00 00 terinIIus or Al peferedGFYW 1 Act~r DA YF P EON YFW C-terminus prf FW 8 Aco DA YWP DQ F Anchor 9-mer STM

Y

deleterious DE RHKLIVMP A G A Al preferred GRHK ASTCLIVM J 0 Anchor GSTC ASTC LIVM DE IcAnchor 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 AGPIDER IDE G IDEQN GDE

HKSTC

B5301 preferred LIVMFWY 1 *Anchor F5"Y STC FWY LI VMFWYF WY 1 Anctior P

IMFWYAL

V

deleterious AGPQN G RHKQN IDE 85401 preferred FWY 1 Anchor FWYLIVM LIVM ALIVM FWYA 1 *Anchor P P AT! VLMF

WY

deleterious GPQNDE GDESTC RHKDE IDE QNIDGE DE TABLE IV 00 0 Summary of HLA-supertypes Overall phenotypic frequencies of HLA-supertypes In different ethnic populations Specificity Phenotypic frequency Supertype Position 2 C-Terminus Caucasian N.A. Black Japanese Chinese Hispanic Average B7 P AILMVFWY43.2 55.1 57.1 43.0 49.3 49.5 ,3 ,LMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 24 F (WIVLMT) FI (YWLM) 23.9 38.9 58.6 0.1 8.3 0.0 B44_ E(D) .FWYLIMVA 3.0 21.2 42.9 39.1 39.0 37.0 1 I (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 27 HK YL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4 362 QL(IVMP) FWY (MIV) 12.6 .8 36.5 25.4 11.1 18.1 58 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 hinese Hispanic Average 83.0 86.1 87.5 88.4 36.3 86.2 2, A3 and 87 99.5 98.1 100.0 99.5 99.4 99.3 2, A3, B7, A24, B44 99.9 99.6 100.0 99.8 99.9 99.8 and Al A2, A3, B7, A24, B44, A1,B27, B62, nd B 58 otifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of ublished data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues aso predicted to be tolerated by multiple alleles within the supertype.

Fable V: Frequently Occurring Motifs Table V: Frequently Occurring Motifs

I

Name avrg.% Description Potential Function identity Nucleic acid-binding protein functions as transcription factor, nuclear location f-C2H2 34% Zinc finger, C2H2 type probable Cytochrome b(N- membrane bound oxidase, generate ;ytochrome_b_N 68% erminal)/b6/petB superoxide domains are one hundred amino acids long and include a conserved Ig 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 repeat protein interaction may function in targeting signaling PDZ 23% PDZ domain molecules to sub-membranous sites LRR 28% -eucine Rich Repeat short sequence motifs involved in protein-protein interactions conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP Pkinase 23% Protein kinase domain binding site and a catalytic site 00

O

0

O

pleckstrin homology involved in intracellular 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 Oxidored_qi 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 in formation of connective tissue. The Collagen triple helix repeat sequence consists of the G-X-Y and the Collagen 42% (20 copies) polypeptide 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 )onds seven hydrophobic transmembrane egions, with the N-terminus located 7 transmembrane receptor extracellularly while the C-terminus Is 7tm_1 19% (rhodopsin family) cytoplasmic. Signal through G proteins Table VI: Motifs and Post-translational Modifications of 191P4D12(b) Table VI: Post-translational modifications of 191P4D12(b) N-glycosylation site 281 284 NWTR (SEQ ID NO: 61) 430- 433 NSSC (SEQ ID NO: 62) 489- 492 NGTL (SEQ ID NO: 63) Tyrosine sulfatlon site 118- 132 VQADEGEYECRVSTF (SEQ ID NO: 64) Protein kinase C phosphorylation site 26- 28 TGR 192-194 SSR 195-197 SFK 249-251 SVR 322-324 SSR 339- 341 SGK 383-385 TQK 397-399 SIR 426-428 SLK 450-452 TVR 465-467 SGR 491 493 TLR Casein kinase II phosphorylation site 283 -286 TIRLD (SEQ ID NO: 322- 325 SSRD (SEQ ID NO: 66) 410-413 SQPE (SEQ ID NO: 67) 426 -429 SLKD (SEQ ID NO: 68) 00 450 453 TVRE (SEQ ID NO: 69) 456-459 TQTE (SEQ ID NO: N-myristoylaUon site.

135-140 GSFQAR (SEQ ID NO: 71) 162- 167 GQGLTL (SEQ ID NO: 72) 164-169 GLTLAA (SEQ ID NO: 73) 189-194 GTTSSR (SEQ ID NO: 74) c-i218-223 GQPLTC (SEQ ID NO: 311 -316 GIYVCH (SEQ ID NO; 76) 354 -359 GVIAAL (SEQ ID NO: 77) 464 -469 GSGRAE (SEQ ID NO: 78) 477 -482 GlKQAM (SEQ ID NO: 79) 490- 495 GTLRAK (SEQ ID NO: c-K1500- 505 WIING (SEQ ID NO: 81) c-1 RGD Cell attachment sequence 00 55 -57 RGD ci Table VII: Search Peptides 191 P412(b) v.1 aal-.510 9-mers, 10-mets and 15-mers (SEQ ID NO: 82) MPLSLGAEMW GPEAWLLLLL LLASFTGRCP AGELETSDVV TVVLGQDAKL PCFYRGDSGE QVGQVAWARV DAGEGAQELA LLHSKYGLHV SPAYEGRVEQ PPPPRNPLDG SVLLRNAVQA DEGEYECRVS TFPAGSFQAR LRLRVLVPPL PSLNPGPALE EGQGLTLMAS CTAEGSPAPS VTWDTEVKGT TSSRSFKHSR SAAVTSEFHL VPSRSMNGQP LTCWVSHPGL LQDQRITHIL HVSFLAEASV-RGLEDQNLWH IGREGAMLKC LSEGQPPPSY NWVTRLDGPLP SGVRVDGDTL GFPPLTTEHS GIYVCHVSNE FSSRDSQVTV DVLDPQEDSG KQVDLVSASV VVVGVIAALL FCLLVVVVVL MSRYHRRKAQ QMTQKYEEEL TLTRENSIRR LHSHHTDPRS QPEESVGLRA EGHPDSLKDN SSCSVMSEEP EGRSYSTLTT VREIETQTEL LSPGSGRAEE EEDQDEGIKQ AMNHFVQENG TLRAKPTGNG IYINGRGHLV v.2 aal -510 9-mets 45-6 1 GQDAKLPCLYRGDSGEQ (SEQ ID NO: 83) 44-62 LGQDAKLPCLYRGDSGEQV (SEQ ID NO: 84) 39-67 VVTVVLGQDAKLPCLYRGDSGEQVGQVAW (SEQ ID NO: v. ORFR 264..1721 Frame +3 9-mets 403-418 SHHTDPRSQSEEPEGR (SEQ ID NO: 86) 402-419 HSHHTDPRSQSEEPEGRS (SEQ ID NO: 87) 397-424 SIRRLHSHHTDPRSQSEEPEGRSYSTLT (SEQ ID NO: 88) V.9: AA 1-137; 9-mets, 1O-mers, 15-mets (SEQ ID NO: 89) MRRELLAGIL LRITFNFFLF FFLPFPLVVF FIYFYFYFFL EMESHYVAQA GLELLGSSNP PASASLVAGT LSVHHCACFE SFTKRKKKLK KAFRFIQCLL LGLLKVRPLQ HQGVNSCDCE RGYFQGIFMQ MAPWEGT SNP variant 9-mets 27-43 GRCPAGELGTSDVVTWV (SEQ ID NO: 1 0-mets 26-44 TGRCPAGELGTSDVVTWVL (SEQ ID NO: 91) 21-49 LLASFTGRCPAGELGTSDVVTVVLGQDAK (SEQ ID NO: 92) v11 SNP variant 9-mets 138-154 QARLRLRVMVPPLPSLN (SEQ ID NO: 93) 137-1 55 FQARLRLRVMVPPLPSLNP (SEQ ID NO: 94) 132-1 60 FPAGSFQARLRLRVMVPPLPSLNPGPALE (SEQ ID NO: 7v.12 SNP variant 9-mers 435-451 VMSEEPEGCSYSTLTTV (SEQ ID NO: 96) 1 0-mers 434-452 SVMSEEPEGCSYSTLTTVRE (SEQ ID NO: 97) 00 15-mers 429-457 DNSSCSVMSEEPEGCSYSTLTTVREIETQ (SEQ ID NO: 98) v.13 Insertion of one AA at 333-4 9-mers 426-442 SQVTVDVLADPQEDSGK (SEQ ID NO: 99) S 10-mers 425-443 DSQVTVDVLADPQEDSGKQ (SEQ ID NO: 100) 420-448 EFSSRDSQVTVDVLADPQEDSGKQVDLVS (SEQ ID NO: 101) S 191P4Dl2(b)v.14: AA56-72; 9-mers GSSNPPASASLVAGTLS (SEQ ID NO: 102) .191 P4DI 2(b) v.14: AA55-73; I 0-mers LGSSNPPASASLVAGTLSV (SEQ ID NO: 103) c~I 191 P4DI 2(b) v.1 4: AA5O-78; riAGLELLGSSNPPASASLVAGTLSVHHCAC (SEQ ID NO: 104) 00 Tables VillI- XXI: 00 00 1Table VIII-VI-H LA-Al -9mers- _191P4D126 Each peptide is a portion of SEQ ID NO: 3; each start Iposition Is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start positionJ [StartjSu-bsequence 1STrei ,F437 SEEPEGRSYF22500

I

EfLRV~fEPPPP]j 1.000] E7i LSGQPPPS! 21.700 107 PLDGq SVLLR -T I.FpjLEiELT LTR]L.3o F[41 qtEE§KyR I 2.2501 58FTE VKGTS~ 1.250] F7-67PidAEM PEAI i9I 45:'TSVVTWL] 1.5001 IL 3611 MSEEPEGRS[ 1.350] LU1 LTTH SGIY[1.5 I 405[ L!ZQPE T i- Lli.IIGPEWLLLL lLj.'zsi5 [84]1 RLDGLPSG f.00-0] l[hVD2 =.P0 158 I AL EGQ E0.0 li4 flL~~ G ooTJ7C L1RE HP I 0.900iIA TiEIE ELLJI 999_9, L486 IQNTR iiC9L 0.675 Lm.[ .I L. HLs Lff Jl AVQA5EGY f[150 Il EEDQDEGI _Kj [0.500 I31 ITILHVSF I 0.0 [Tble 111M Al9es 191 P401 2B 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 7rt] Subsequence ifScore F3675vvvWiR[ 0.500 L Y6 VLSIRYTTO500 1.71, LATL.LIKY IF-.67 F[7 87 QqM Tqll 0.500] Wf J[EYECRVSTF j 0.450 I~oi DEGEECR~0.450] 71 IL FPXCTj 0.450 I yGL( _HV SPYjIO.?50 1 318F§NEF SSDS 1 I_0.225_1 PT 1LAG AQE o [.225 L~E gEYCVI 0.225 LU75I LEEGQGLTLIL2I 1GLETSDVj =0.225 I 15ILPP_ LL I0200 [Ti][SVTWDTEVK II 92901 [41]TVLQDAKII 0.20 0 Li7jLLLLLLASF 0T QPEESVG T. 01 501 N_2I1 S L AGS F 15 7i K3.IDSK 0.1 2-5 7 I L2].ITRENSIRR. ff. 25 I 'F T51 GQVJ I.Z F I jTrSSRSFK Lo.1i25 j353. VGILFI 125j 313fI[VSNEfF_0.100 Table Vlll-VI-HLA-A1-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acdds, and the end position for each peptide is the start position plus eight.

Sia-rtl[utbseqencel -Score 6IQ-VGQVYAWAR iF0.100, F5 .ELLSPGSGR II 0100 Py?9 I TVDV-Y LDPQE [0.100 L20 LLLSFTGR L q I Y MNJ .0.100] 1467 iRA~f9P I0.090 I.1[x D q__qLIYH-q~ 0.075I F{j GS~_ FQARLRL1[_O.075 I FE77I DPQDqEGIKQA 00751 R[DGTLGFPP [o0762j L1L,6~vI FH =_0.0501 [folLvPsRsmNG -2oA650 1.W9 I .iL P i 0.0 5 01I U 1. _W LLLLL LLL50 I L~.iL~~LT L0.050 1 ITable VIII-V2-HL11A-A-9mers- 00 00 Each peptide is a portion of SEQ ID NO: 5; 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 ISubsequence i 17 [GQDAKLPCLj 0.1.50 FT AK-LPCLYRG IF 0--010 KF LqPCLRGDS Y IP003J [JJ PCYRGDG 000 VY1L P D L-qlF.oo1 D T-IEI CL YRGDSGE 0.000--oT §1r~Y~5GEQ. (000 Ta le il-V7-HLA-A1-9mers- Eah 191P4D128 j Eahpeptide is a p ortion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each pepplde is the start position plus eight.

I. I J ubsequence [Score 3[iiIHDPF q§j =.250 F--j SQSEEPEG-R .015 LI71 DP RSQ P P9:99T.

LJ [7DS QSE PEj 1009 Tiale Vill-V9-H LA-Al -9mers- 191P4D128 Each peptide is a portion of1 SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. [Strt jj ubsequencj ScorI I IDq~ F [5 q0 L2IJLIFNf -F L Ff 7I LFFWFIYYF lf76of [Table VI II-V9-HLA-A1-9mers- L- 191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position plus eight.

9--]FI GLLLGSSLa9J007o [TI. 17ELLGLR0.0 ~JZ..J~!.TfFFLF. 050 ~IJ18 [~EGYFQGj 0.450

ESFTRK

Fj77]I1 TEJL01 7 q F 991 LLGL vR 13 KJLGVNSCDCERI 0. 10 0 ACFESFTKR II0. 10] -qCLLLGL 1..GI_ GLLKV i 0.050 =6 JLWYYJ050] [~jfWAQGLEL] 0950 49_ IF2GLELLGS[01 [I I SPPASSL .050 '65. SLvAGTLSV 1=.5 56 GSNPS,03OJ I IGTLSV~cA -0.025 II 30 1L..FIXYFYF.0251 Table VIII-V9-HLA-A1-9mers- 191P4D12B Each pepUde Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position [par] SFubseq-uejco[(. E [7-21L FFLPFPLYY 0.025 Fj .iIFFLEMESH'URI 0.25 TIY_ LLSN6~(.020] fNFFL 1F6 i3 2= KVRqPWQJ 0.0201 LDD[ LALLRIT 11.0101 175- iFF HCAFFL'T!I0.1:3] [i Yl L.o =-1 128 D NMPEGI .005i 15-5 GSSNPPATJ0 K JI A1L_1 ILA Ij KKAFRI LP3 FUD21 YFQGIF9 0.010 DI]326ILL FF LEMS0.0f3 DEBJ.TE FFPP I0.=005

I

7 5][TLSVHHC I 0.9035 00 00 1Table Vil jHA-l9es Each peptide Is a portion of SEQ ID NO: 19; each startI position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position -plus ei1ght.

Stat Sbsqunc _03_ _93_FFFRFIQCLLL 1003 1[114 IVNSCDCERG!FO.003 QIFQ 0.003j I JF .A I I F F F L 0.0p 03j [107 RPLQHQGVN L0.003' :F 7 VH 9 91 E 0.003j 1 91RFIQCLLLG. 10P.003 'FI_ I.FLFFFLPFP 1[-0-002.

11081PLQHQVNS]0.002[ 61 I1E.AL-Gl I 17P9I -q~CLLLGLL1i021 L' iL 'AA 0.002j ~~10.00211 191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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 [.Subsequence rLj [7AGELGTSDV :FP7. 5I [7]GRCPAGELG J10.005 [771 LGSDvvT7V 0.05 ,able _VIII-V10-HLA-A-9mers-1 11412B I Each pepide Is a portion of1 SEQ ID NO: 21; 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. LStadr [-Subsequence1 LScorei [T CPAGELGTS F70003 I[117 -1 G EL GT SDVV F0 .0 0 E7I.PA _GGTS jFo.oooi, TbeVIll-VI 1-HLA-A1-9mers-: 191P4D12B Each peptide Is a portion of SEQ ID NO: 23; each start Iposition is specified, the length; of peptide is 9 amino acids, and theend position for each peptide Is the start position 1_____jpus eight_..

I

ILFrt j subsequence iscorel MV- P FP LL 11.1001 7 F R LVMVPPLPS 1 1:501 RLRV1VPL0.001.1 7T2 FLRLVMV 0.000 [Table VIII-Vi 2-HLA-A1 -9mers- L191P34D12B3 Each peptide is a portion of1 SEQ ID NO: 25; 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 et. f77 SEPEGC 1Y22.501 3 SEEEGCS 1301 7~ PEGOSYS O 04501 77] GCSYSTLTT 0.050; 17111 m_ EPEGC -0 _09 51 1Table AVII-V 2-HLA-A -9mers- 19P4D12BJ Each peptide is a portion of SEQ ID NO: 25; 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 eg t [Start .1 Sbsequee FS'ore [T GCSYSTILT IO 0 3] PEGSYSTL 1 Fo T~able ViW-V1 3-HLAA1-9rners-j 191P4D12B Each peptide is a porion of SEQ ID NO: 27; each start position is specified, the length of peptide is 9 amino acids,' and the end position for each Ipeptide is the start position 1- plus eight. L9 1 I2!L G~I 0.01e0 [11[LAP~qEDS 5.00 DillADPED ?GKII.00 TVnDVLADPii00] V77 KD~vVI: 0.005 [Table VIIIAVi4-H LA-Al .9mers-j Each peptide is a portion of SEQ ID NO: 29; 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.gj_ l ubsequence [core] 71 FASASLVAGT, IO391 G I E7 S ASLVAGTL 0 .010 WI NPPASASLVj FO 003 00 00 Table ViII.V14.HLA-A1-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 29; 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._ [T71 PASASLVAGi F50072 [aeIXi-Vl.HLAA1orers- Each peptide Is a portion of1 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 Scre I 71 j[ GffPSK i 135.0001 F33 2 VLDPQEDSG 100.0001 43 MSEEPEGRS 7

Q

L4 EGHPDSL1 18.000

QA~~**DEGEYEC

1 _.0 [453 J[ EIET-QTERLLS1[E 4.50 [306 TEHGI VC 300 j GQDAKLPCF 3.5 [86IQ ENGTLAI 2.70 [76 AELA7LHSKJ 2.700 250 E [57 1457! QT-ELSPGSG L2:.5 I 18j D3TEKGTTSSj2fi ,IRVEQPPPP .0 .t2 TfAE-GSPAPSVJ {T0-* I SD VT V GI1 ITable IX.V1-HLA-A1mers-I I 191 P40D12B 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.

[1 QPEESVGLR 1125 F11 T[GEAWLLLLL][ 151251 F6Ij[GEQNWHJ)A [09001

NQADEGE

1_61 0.L500] YTVGDK)~0 [~HLVPSRSMN O[00 G4IL~ 9 ILLL MS:jF.500 [12] EGEYECRVS 0.450 [47SEEPEGRSY 0.45 [1JRSQPEESVG 030 []RGDSGEQVG [0.250.

3181 ISNEFSSRDSI .2 F 1[ t LPEGRSYSTL2 7 1i6 FL LLLLLSF F. 2O I L Table I1X-V1-HLA-A1-lo0mers- 191P4D12B 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.____ [Strt I Subsequenqcoe 3 67]KVL

M

SRYHJL [0.2001 36~VLMSRYHRRI 0,2001 K I L2 251 VSHPDGLLQD 0.150 WL .GSFQARLRRI_ F §]RSYSTLTTVR L0.j 18) GTTSSRSFKH 0.12 L~qNS lL- 0.L92 05 LLTTEHSq2YYJ 0125) 1[400 RLHSHHTDPI 0.100o 11451 VLVPPLPSL iLO.100 E SVMSEEPEG 010 260] IHGEAj0100j I9jHVSPAYEGIR 0.0 E4 QDEGIKQAME00901 [4 7RAEEE)oDI 7 31Ir IKQA~f 0.075] RSNGQPLT~[0 357 MFAAUCLLWI 0.950 00 00 Table IXV1-1-L1A-Al1l0mers- 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 positionI plus nine. Itrt SubseqUence [Scre I F4 ILQDKPI 0.050 lL188Ji G TTSSRFK 0.050 217 NG~QP LCW 0.0501 29,4j RVDGDT7LGF 0.05 [i7fjq CTEGSPAPS1 0050 447Z LTLTTVREIET L0050- 221_ILTCWVSHPGLI 0.0sq0 S-S-R-DSQVTD 0 if 30D4WELTERSGjFY .050 2~ FEGQPPPS~YN 0050 1146 L- 4 1L 9511 1 485 WLLLQENGTLFL00 1 Table IX-V2-HLA-A10mers- ___191 P4D12B Each peptide is a portion of1 SEQ ID NO: 5; 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.

Is tartj Subsequence IS-core j,'T G~QDAKLPCy [3.750 17] KLCLRGI d .0-10q 'FI1 LGq9RKLPPL7Fq.005 Tabl IX-V2-HLA-A10mers- K 191 P4D12B Each peptide is a portion of SEQ ID NO: 5; eachstr position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position I.L PA.9J[S:9.

[7L LPCLYRGDSG 09-0O3 F' _CLYRGDSGEJL.00J 71LA ~S!~xGEQY10.000 ,[Table IX-V7-HLA-A1 -1 Omers- 191P4D128 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. j Fsi.a7.I __*Subsequence~ Fs9L~I FlHTDPRSQSEE~i [7ki R.SQSEEPEGR SHTDPRQ 0.01.5, ,I.§QSEEPEGIRS 10.02 f[ 7 (TDSQSEEP [0 000 ~Table IX-V9-LA-1-0mers.- 191 P4D1 2B Each peptide is a portion of1 SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for eachI peptide is the start position pusnie Subsequence ScorejI I 1 ITFNFFLFF 110 I 281 VVFHYYY-10 [Table IX-V9-H11A-A1-1 Omers- Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Start I Subsequence. JFr 117~ SCDCERGYFQ _5.00 I HOACFESFTK IF.00 L8_11 IA2f ILVVFHXYFYF .0.5o Lii U±RTFNF [ii~ LAURT 0.5001 SSNPPASASL 0.-3 0 9TiRRELLAGILL P]L.2251 12.j FLPffPLVVFF. 19:-2091q 101TLSVHHA9FiL.9 I 77J ACFESFKRK 112.290 L9U .Q GL b'5] NSCDCERGYF 1F0419I 1i!3 ILVNSCDCERGY L0:1 76~ CACFESFTKR F000 T I LLf 'FY.00] LTI LLGLKV L0A-0IFq. J1 E74T! I GLELLGN 10.0501 SV.HGT!fY [0.0750.

L31 IYFYYFFL 0.050 F 8 '1SNPPASASLV' 10.025] Fl RELLAGILLR 0.0251 [112! QGVNSCDCER-j9 110051 69 J~GTSVHHac. P7.251 LRITFNFFLF T9.025,, 00 00 Table IX-V9-HL11A-A-1-l0mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 19; 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.

I..821 -FKKKLKIK .2 291 VFFIYF F 025 [16 1[ NFFtLFFlPF_ 25~ YFFILEMEHY 005 LGSSNPPA _0.20I 1[ 62 ASASVAGT 10151 J1ESFTKRKKKL [IE175 FL .IL NPPSALV 7t13 [1'21 11 RGYFQGIFMQ 10.131 YARTLSYH FClaO~ 11051 KVRPLQHQGV jf0.0101 [1-ILLRITFNFFj 1oj I~FESFTKRKKK '10.010 I_49 1[QAGLELLSJ ~poi 14 iFYVqAGLELL [.olol I±!~1LVNSCDERG 0.1 I..LQCLL 0.010 Dl. FIFYFEYFF 0.1 [F SLAGT LvH 161 L1.90I LLLLK'RPL 0.oj [4811 AQAGLELLGS 1000 ffl GLLLKVRPLQH .005I I 5LGSSNPPASA LO.005] I LGLILKVRPILQ 905 T73, VHHCACFESF_]KooSI QGIFMQMPW. jo.

LLRITFNFFL I:2I 11767 R PLQ HQGVNS] FoKos iT'8ilFMQAAPWEGT .05 I 61 KKLKKAFRF q0031 F117,[ CDCERY FqGI0_3] [Table IXHAA1-l10mers- L191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptidle is 10 amino acids, and the end position for each peptide is the start position pLu nine.

lStartLubsqeneIsoe I93j FR FICLLILGF7:3 FLTiLYFYYFLEM 10. 00 120j1 ERGYfqGIFM 10.00 L22 YF QGI F6.7003 FT.3 YFFLEMES [O.7090 D[jI HYVAQAGLEL 000 [so I ALELLGSSN 110-003 I I 7 LSVHHCA F O.7002j [1 1 FN F1F L H .i I I LLVPFLHQ 10.001 FL[j ARFIQCLLGL o1 K..l GIKY 19 0.002 EachAI petd i ortono [fASE -DN:21 IahFtr I _rtI Susqec .661 F ll GRCP--IAE LGT10m 0251 EachpAetid s p pono ITable IX-V 0G-HLA-A1 1 Omers- 191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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.

M7LmLTSDVVT [EO1] L5ILGLTSPVE 10.0011 IK1TG-RCPALG 0.000 Table IX-Vi 1 -HLA-Al 1Omers- 191P4D12B Each peptidle is a portion of SEQ ID NO: 23; 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.

Jrt LJ&bseqyence Ls coreI MI.7 VMVPPLPSLN 1i.9JI _vL sL MI LY__rT-L1o~oo LE7 0RMPLS[.003j [DI1LW LLRVM lLp2j lifli RLRLRVMVP] 0.000I [IlLRLRVMVPPL 1[.7ooo L Tble IX-VI 12-HILA-Al-I Diers-i 191 P4D12B Each peptide is a portion of SEQ ID NO: 25; 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. 3 MSEEPEGCSYj j F SEEPEGCSYS_jo.450 F_7, [EPE GCSYSTL 0.22? 00 00 Table IX-V1 2-HLA-A1 -1 Omers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position p lu s n in e Lstartl Subsequence i[Screl Lull CgSYSTLTTVR]0.5 ITLE(CSSTIT_7 !EO 131 GCSY TLT j.010~ j*VMSEEPEGCSjO7.05J 1 Y! SYSTLTV 1EO [900~ PEGCYSTT 10.0001 Tbe IX-V1 3-HLA-A 1lmers- 191 P4D1 26 Each peptide Is a portion of SEQ ID NO: 27; 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.

[Sj Subsequenice jlre Lo-o1 F--1 DSqnTpy! J[ .03DO i. WLDVLADPQDS110.10 W37LQYTYVDJLAP 0.002 110 51LAPQEDSG-K9 b01 ablIXV1 4-HL-A-Al1l0mers-1 I191P4D128 Each peptide is a portion of SEQ ID NO: 29; each start Iposition is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine..

I..Start 11 Subsequence IS-core [ale IX-14-H-1-1 mer-10, Eahpeptide is a portion of SEQ ID NO: 29; 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. 1.§t.qj Subs,!quence _L[coreI [i SSPPASSLJ [0,3001 =07I ASLV AG TL S V 1T NPPAAs_ LV T.0251 [T]LAS aAGTL. 0~.015S [2 J[SNPASS0.-0105- P77! LGSSN~PPSA 10.00951 Table X- V1.-HLA-A201 -9mers-l 191P4D12B 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 ppde Is the start position 1_!plus eight. q]1 Subsequenice. iL 9 I ~LLL.F9!4 12.546~: [T8 L LSF T F2 I59? -80I [KT0_iIS~9L 1 [7ML0411 F1 EMWGPAWL L72§~ M)2~ VLLR NAV 11. Ii 742? WLGQDAKL 1111.7 I203[_, _TFHLV 111.5631 F341l SASM11 14101 SQPEESVGL j18.880 Table X- V1-HLA-A201-9mers-' 191 P4D12B_ 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._ lPti Su. bsequence j L~or S351j qVVIAAL i 7.309 LESVTJ .288 [10 jWGPEALLLJI4.471 1l32JELTDW 4.122 1± L SY STLTJ34 L24 I VLAAV _2.856 IILLAFT GII.1 1253]ELL PPNLWHI L.9 [i21GLLQDQRIT..2.261 [Ifl7iL v~y9yil__~..2?22 L~jNLDGSVL 21151 jGY~Rsj1 .775j E2I[DNGQPLTCV 1 [5~5j RIETTEL 1.7036] L87iGPLSGVRJI680 23 1 FLQDQ0TH I 3 Th L6 21EV 9AY1v Fl.32 I.1Z.LLLLLLA~L.I.P98 Ll iLLLLLAL J[1P78 I3fl F _ALLEo~ILJA.7 [i2611REGALCL [_2A.

00 00 Table X- V1 -HLA-A201-9mers- L 191P4D12B 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 li tart Subsequence JF sre I 390 1 LT L. 9k I j(M?8 FvQEN1LoF K I I2661 AmL cLSEG] 1 0.8 IF'!6lsLTTA Ii I__EAWLLLLL .425_I (11IDGYEGRvj .1 113061[ TT EISG IYV [0O.340 Jj~ AEEQGL 025j P~flI KQVDLVSAS F18JMT-QKY)-EELIOa2747 [1 3L iLnsyF-9,. FT2:4- I 14501 -T-!KV TT 123 71THILHVSF 088 P-1-71[ N9 9!!TpW 0.1861 1.20 j LLLA FTiO781 144811 LTTVEITJ 0111 12851 IDGq!PPSGV f( 0.164 Table X- V1-HLA-A201-9mers- I. 191 P4D1 2B 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 iI plus eight.

lq]aYtiusequencej score] 1.4 7 1 FDQ DEGI KQA1 0142 1L332JLSSRIDSQVTV][ 0.141] lR'Tfi 0LSRHR 1 41I 1z221TCWSHGL I0.139 1 P2 L -H G!EG-I014 JL~eII QLTLASC 0.120] I ?3 ii ASFTGRP II 0 ]0j [TbexV2-HLA-A201-9mers-} 191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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_____ jSaqtl Subsequence 11. Score FY]_QDALPC L93J t1I'- -GE~j 0.048 177 LPLYRG~qDSJL0-.T0 TJDAKLPC LYI 0.000 9 LyGDGQ] 0.90 Table X- W7-HLA-A201 -9mers- 191 P40128 B Each peptide is a portion of ISEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. stq [Subsequence scrj R-SQSEEPEG FP 796001 WTal -V-HLA-A201 -9mers-1 f~be 191 P4D12B Each peptide Is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.itl Fsubsequenej1 S FT1I TDPRSQSEI000 [LI SHTPRTSQ 0[,000 T~be 191P4D1281 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids.

and the end position for each peptide is the start position plus eight.

I1staiiq seq uence 1 s. Lo-HYiIJ I[NJFIQC LLGL DLl~z 181 ~FLfFFLP 12.194j LG7SNPPA] 8.4j6j K- 2I Eff 1[IFTL F7 ILLRFFI 2-7191 11281 MQMPWEG.85

L

F1 FIfYFYFF !11.576 IEE7F1TLY I 59..

FT [LLGLL [TK.57.

[F 21 FFLPFPLW 1.

00 00 Table X- V9-HLA-A201-9mers- 191 P40128 Each peptide is a portion of SEQ 10 NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. [Startil ubsequence I Sor 9.1.IQCLLLGLL 1 1.io0j F- 29QFM--EG-iL .09702 407 LE ME S HYVAf 1.02 L1 RGYFQGIFM 0-.5-71] L LVFFY-FY 0.337 GLL R I TF N- Fi 8 0 L [NPPASASLV 0.454j []jLL!SVRPL II 0403] [42J[MS AQ [t IDL 15*3 Fj- ILGTLSV IPAJ 0.255 158 II SNPPASASL. I F- 9-] ULjRITFNFFLF II 0.113 [L7LA-§LYAG. I 1l L!PiI. LLRITFNFF L0..

LYIYFFLEm-I 0.085 J NE68 ILLRVy J 0 .055 FRFIQCLLL1Lo~ L 91. ffIYFYFYJ~4 LzUL.Ly~IJI .039 i 31 ISASLVAGTL 10.0391 F126 iIGIMQAPWI 0.387 PLW[ F-F 0037- Ii[ HCACFESFT, .0395 [Ti-f LAGILLRIT ij. 0.033-1 f119 q E RGYF QG 17 0029 ITbe X- V9-HLA-A201-9mers- 9iP4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

F07] LLGLLK1VRP]F 0.025 Iii II V QGVD 0O.0167 Li F§ SFKRKKKL 0015_ M4 ITFNF _ff 0.010 QAgLELLG;I 0.008' qHCCFE1 0.007 [0.-006 L IELLG§SSNPPIf_0,004~ I P.IIFFLEMESHYI LOP0

I

l.A 7 I qAGELS RI 0.003 l.FI9J 5 IIL9 0.002j L5TI LELLGSS NPLO.02 4 [i lL-VAP.1 EEEM2RKLL-A!9qGI[ .0.0021 I P8jLQHQGVN Sj 0.002 11 .1 LFFLP ii1 0.002 RI ]GNCDCE RII. I FT6 F7fAC:2 EThI .001TO §I51 GSP PA§, oFPo I I x FflEMVE 0.001 K1LHA97 DL-9! 1 F[T~i GILLRITF7F 00 0 0 Table X- V9-HLA-A201-9mers- 191 P4D128 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

IStartI Subsequenc1.e.j 7 .LfZ I.S NPPAA F Oq-7 Fl1177 Ic .9 F~ P000 1 Lu. ItKSCDCE9L 9.~ ;fTable X- V1O-HLA-A201.

9mers-1 91 P401 2B Each peptide is a portion of SEQ ID NO: 21; each start Iposition is specified, the length 1 Iof peptide is 9 amino acids, and the end position for eachI peptide is the start position plseight.9 Seuence'1-r I .7 RLRMVPPL 11.03 'F7iF RVMVP-PlPS K 0.2:4] 11 .000 I F-47 L VMVPPLPII *K0:00 Table X-Vi1 -HLA-A201 Each peptide is a portion of SEQ ID NO: 23; 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:l I Susqec _§roJ LG TSDVT 11177K2 F5-- FAGELGqT S DV F0029.

00 00 Table X-V1 1-HLA-A201- -9mers-191'P412B Each peptide is a portion of1 SEQ ID NO: 23; each start position Is specified, the length of peptidle is 9 amino acids, and the end position for each peptidle is the start position *plus eight.

StarJj ubequence 11[Score SCPAGELGTS! 0.0 77P-AGELGTSD jj 000 V[7R-CPAGELG 1. F.-0 9rers-191PQ12B3 IEach peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptidle is 9 amino acids, and the end position for each peptide Is the start position pius eight.

jStart j Subsequence lScorei [TF7jc GCSTLTT ILRm9 PEG-CSYSTL 0.014 Jj IDfEEGCOSY [000I 11.1 IIMSEPGCS [Eo007o L Table X-V1 3-HLA-A201- 9mers-191P4D1 2B_ Each peptide Is a portion of SEQ ID NO: 27; 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 !9rtII. ubsequenceJScr 1~ SQVTVDVLA 1[.0.50 Fl7 WA PQDJj.25 3FflVDVLADP 0j.003 LADPQEDS 0 II OOOJ Table X-V13-HLA-A201- 9nmers-191P4D12B Each peptidle Is a portion of SEQ ID NO: 27; 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.

Star][SLs uence.1 _scoreI [E[I[ADPEI 11 FOO9J Table X-V14-HLA-A201- 9mers-1 91P4D1 28 Each peptidle Is a portion of SEQ ID NO: 29; each start position is specified, the length of peptide is 9 amino acids, and the end positon for each Lpeptide is the start position plus eight.

[Start ffu Subequence F--iril-~ F(7] NPP ASASLV jFO.

ffl S nPA-sALjJO.- 13flJ L17 GSSNPP-ASA1 T. 0q032] MI] ASLVAGTLS J[ .900 F1 L PA§V I 0.000 ~fTable XI-V1-HLA-A201l0mers-191p14Dl2B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidleis 10 amino acids, Iand the end position for each peptide Is the start position _plus nine.

I359.i LCLV\y 4256 358 j L-F-cLYY924. 6741 244 LES~Ii2 S230FL-LQDQRITHi 1167.2481 81 LLSYG9LHV IP 8.2381 215 1 SMNGQPLTC 115.534 IFTab-le XI-V1 -HLA-A201 I Omers-191 P4D12B 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 pRlus nine.

ISta tisbsq u e[ 1.

ILHVS FL AE A 73. 8 L -PAW 72.031 252_ GLEDQLHI~j4.2 [Y6j? civwvj[!:. FI 3LT9T EH SG lY2Y9j.0

RLDGPLPS

RL 27.821 3 5-4iIGv -IAA! L L124.93 L!441LYVPPLPSL 20 LL4LASFTGRCJ 15.4371

VTDTEVKG

181] 1377 11Q .i VGALcL[.8I IPE 8.453 VI QDAKLP1 LP85[Z QENGTLR3.18 [381LT 7.560j I 447 _ILTYIEIET. Ji 350 VVVVGVIAL Fo9J 5L IALLFCLLV1 .4 [2 7 GQPPPSYNW 6.3 WGPEAWLLL .4 158 IALEEGQGLTL1 5.6095 NEFSSRDSQ 164j [LLAS T 96:8:]~ 00 00 Table XI-V1-HLA-A201- 10mers-191M4128 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. LS it. a-rtSubsequence I rF 1.34 IjLVSASVVV1L_49j9 [1 VQADEGEYE; 3.511 WVGVLL1[YV 31 78_ jLsIWLLU.LLLAs 112.917 1118 I LLLLLASFG ILZ.Z.!9.

l[T2 IIYEqfRV§TtPAI 2.5771 t132] LFASQAL L[7I LETSO) VV 2:16

FSSRDSQVT

P21 r.RISQVT2.088 L1TTP2QRLLR.l 1.8- 79] iL. [M79w TWGQA L.891 FE ]Pj LALHSKYGfl .866 9KANHFVI 3J AvWG .775] 211 M TSEFHIiIF.721] Pii! 1:SV1 FN VJ=.608 [370 VGQA J 1.2220 ~OjFMSR F 11 208 32 LGEETSDVTJ Pf991i [T~f E LTLT TNSI I. 0.782 [39]1 VVTVVLGQD 0.739 Table XI-VI -HLA-A201 I Orers-1 91 P41 2B 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 LrtISubsquen~e] IS(creJ (353]VG-VAALLFC.69

YNWTRLD

280]GL 0.692 [3:iT_QRiTH7IL'10.60471 IPTLTc vvSHPGti q. 5o04 6, VAWARVD 050 [I 2 F GQGTLAAS 050 7TE I [1 APSVTWDT I 0.454] [T's EAWYLLLLLL I045 [176PAS

T

W 0.365~ E1MNG Q PLT C V 1 [f8]qKYEEE-LTLj_ .3I L~mi1CLSEGPPP~~0.306] L29JGL,_QDQR [.j0.276.

1501 PIWsLN PqtALj 0C.237 M GP 0.226 ;112 IK VLLRNA V9QAD .216j T6i 5GLTLAS~i .A§i 119 LL-LLASFTGR I. 9Y7.

.~1FTGRCPAGE [017 36J QE7DSGKQVDJ[0.6 9- E Q _P R N -1 2 'i 4_ YSTLTTVR~ 014 1 41SVRGLEDQ .4 249 .S99i L.140 F Table XI-V1-HLA-A201l0mers-191P4D12B.

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. ,[Sai Subs equnce SFcor e [K][NPLDSL 0.139 4]091 RSQ7PEESVG 1 0 FLk]GF9 LL 0.1391 156 GPALEEGQG 0.139 14511 IVLVPLp§SLN L:3 Table X14V2-1-1LA-A201 -1 l0mers-191P4D12B_ Each peptide is a portion of SEQ ID NO: 5; each start Iposition is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start positionI plus nine. I1 MIt Subsequence Score] MIJL qAKLPCL1 2.23] 10 LYR DSGEQ]I o FLQDKC R 10.0031 V]L__LP CLYD [p j [T7 DAKLPCLYR [0.000 Table XI-W-HLA-A201 F Imers-191P4D12B Each peplide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position .usine..

[t7 jj.Sy!bsequence. Score [F_7!sQSEEPEGRSII 0.004_' 151 00 00 [Table XI-VW-HLA-A201 1 Omers-1 91 P4D1 28 Each peptide Is a portion of SEQ ID NO: 15; each start position. Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

EStartjj Subsequence Score F iJ1lHHTDPRSQSJ, 0.0006 1 [TDP R-SQSE 0.0 F H HTql)s9 E LQO F T HSTPRSQ!:LPF0.PPJ STable XI-V9-HLA-A201 -I nrs-194P 11 Each peptide is a portion of SEQ ID) NO: 19; each start] position is specified, the length of peptide is 10 aino ais and the end position for each peptide Is the start position plus nine.- IS LStub;sueq u e nce Sore FI1YFYFFL1 7861.87 HFLFFFLPFPL 2108.811 FiEL LRITFNFFL[3.50 PLYFF 1 .429 2] jFMQMAPWEG[F23 [T3JIf FLEMESHYVI18.538 LGLVRPL1670 DC] IL ITFF J2- 4.898 157-1%12 K FPLV F Lt3 .j I-T± IAL -VAGT7LVJ 1.680, 105] IlfY!-LQH9 11161 ITable XI-V9-HLA-A201- 1 l0mers-191 P4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Subsequenlce IFssorei II F53 ]LEL--GSNPPA7 9 .j .1 3][LEMESffyV [:O600j IF! f -T 171. 30 1 D I T FF fYfYLP.4 0T] 5 I jPWF lF[ 09.32 9 I 102j[GLLKVRPL9 j F.276j F7I. VAGTLSVI-IH 0.270

GTLSVHHCA

08PLQHQGVNS [0.251.I E-l[ Ii GILRIFNF I 17 [Tf]lLSSNPA I 0.127] [5I FLDA. V F I 126j GIFM(QAAPW[ .7042.

[4j MSY 0. 040] F 80 -11 <RKKI I*09 LTable XI-V9-HLA-A201l0mners-1 91 P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

[S t artl FSu-e qu en~ 7ce .]e F 2] .SVHHCACFE 00O38 AGTLSVHHC][ .032] 62- 1IAS Y.iLp R-0 ILE

M

EHYVA 0 01711 43J ESHYVAQAG LM7.1 FF LF-FFLP]17 000 FI KALE-LLGSSN [o.007Jf FT 4]fY4jIFQGWAPfJI.O07 liii AGILLR-FFN 0.006_ EF] JPA ASLY AGT I121 [RGYFQGIFM 0L024

_FE

LF7J C EGYF 0004] 7 IHHCACFEFTJI. 97I QQGVNSCD [0.003 GVNSgDCER J[0 ]0 [I96][§L LLLGLLK .LP. 03I 19LQHQGVNSC .03 M4 J ESHYVAQ 002 G 0.002 00 00 Table XI-V9-HLA-A201- 110mers-191P4012B3 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length Iof peptide is 10 amino acids, ,and the end position for each peptide is the start position t[StaJl Subsequence lj SoRej I !-flLLKVRPQHQL.992 L-E-LLGSSNPPi0.00i I7 RPLQIIQGVN 0.002 [TFj LGILLRITF 0.002~ 47 i GiLLL 0.002 tF iNCDCERGY 0.0 16jj NFLFFFL 1 0-q L71 FLj K zmi -EF~RIJ0.0 0 1 I !P IIK2LKT< I 01 i 3 ]jASLVGTLSF 00 j 5~1. G1LELLGSSNP !LO.001 i Ff71iISVHHqcACFEL.001 1 F17Jlf-YFLEEHY LOO-jJ 121 FfLFPLF A 001]I I f9 LKKARFQC]qEL F11 FLL /PwQ 0001 [125j FQGIFMQMAP 0 000 GSSPPAS 0.000 STable Xl.V1O-HLA-A201mers-1 91 P4012B Each peptide is a portion of SEQ It) NO: 21; 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. WI9LGsDvy)vTYYJ AK? IJ9JlKIS0Y3 I 0.499 Table XI-VI 0-HLA-A201 L. lmers-191P4D12B J Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peplide Is 10 amino acids, and the end position for each peptide is the start position pRlus nine.

MFWL-GTSpV\'il 0.2201 MF]PAGELGTS-DV L92-L 0.006- LLTGRCPGELGLoJ o Table XI-V1 1-HLA-A201 I Omers-1 91 P4D12B Each peptide Is a portion of SEQ ID NO: 23; each start position Is specified, the lengthl Iof peptide is 10 amino acids, 1 ,Iand the end position for each p eptide Is the start position plus nine.

ttartjL:S9se 91Lnc 9 el ]VMV-PP LSLN I001 ALRLRVMIVI 0.073 f05 LRVMvPP LI1j.043 [-4IIR!LR,VMVPPII .00R3 110 yPLPSLNPLT..2I =6 LR YvPLP1I 0.1 [7 ]LRVMVP.PLP.

f7ULAR LLRVMVPI JB0000 I TbleXI-12HLA-A201 lms-19P4D1 26' Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length, of peptide is 10 amino acids, and the end position for each peptide is the start position p lus nine....- Start Subsequence iiL.cr EIGCSYSTLTIVJ 11.044.I i MSEEPEGC1 .788, 1 Table XI-VI 2-HLA-A201 L 0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 25; 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. L[Lta~j Su c e L~il!~Fs 049 FL- LJEEPEGOYSTj 04 ]10LEC§T. =O901 Table XI-V13:-HLA-A201- L i mers-1 91 P412__ 1 Each peptide is a portion of SEQ ID NO: 27; each start Iposition is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Dart bequence.I[cofeJ MLQFOVDVL- I~ [Wt~iSwoDVL 002J [I][vLADPQEDS]ffK]T E4~I m'M =0001 D 1 PPQEGQ[ 0.0001 Table XI-V1 4-HLA-A201

-I

1 Omers-1 91 P401 2B-- Each peptide is a portion of SEQ ID NO: 29; 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.

00 00 LTable XI-V14-HLA-A201-I 1 0mers-191P4D12B Each peptide Is a portion of SEQ ID NO: 29; each start position is specified, the lengthi Iof peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

1Startj ubsequencejj score.

L1ull AS-L VA-GT LS L F15.6 F41 NPPASASLV L 0.541 L-Y 000=8 (Table XIlI-VI -HLA-A3-9mers-1 F1-Z 191 P4D12B 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 [FLrtj[ seque ce I c r L[7 LA F LG]~ 000J fTJ51VMSEEPER .000j L~zo LM RYHR [aooo 1 IILLAF IL 4. 00 L3~zL~~wvv.LjI .050j L 92iLT LJI 4.OO I L qY 99A~ 3.000 1)821 SVVWDTVK O 0 IK L~YQVW I L1.800 368]jIVVLMSRYHRII 1.800 Table XII-V1-HLA-A3-9mners-1 191P4D12B 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 subseq-uenc~[ e 136-311 Y 5LLvL FE.350-.

t1-61SNE-SS-R_]jF 120k [252 GLE-DQNL Vl_1.200q IL 78 IL I1.200 I[366Pj j WMSYL.900j9 T1Jl KckY.I m 900 :115 IL LL~LLfl=.900 j89 I HVPAYER FL 6qT [485 II FV-qENGTLR[-0 F~TIREQPPP 1 0. 600 [392 q P.ENIR 6Lo 0 351 -j V V AL[ LLq I 0.3 040 31 =Y.vj039 1)12I yLRNAVA L .300 L2T PCl FPPLnj 0 LL6!I.I =LLSJ .3q0 P EGVIL~ j 0- .270 FIf =51 F9 2~:70J 1 5 51L qN--v H-1 0.21 6 fl~~ [yyV VIA 1- E2180 I 1861 EK-GTTSSR II.p89 1y9. SFHYPRJ L0.1801 I ii L -LL LLA 0.150219 I. 7 UQELALHSK i~:~i I Table XII-VI-HLA-A3-9mers-1 L: 19P4D12B Each peptide is a portion of1 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.

378 IFL QA7TKYII 0.12035 L~IL!YSFLE o FT.1U p LLLASTG Y-290 P35 ]IGS FALRL 0.090 LFTII DLVASMVW Tn.00 L 4FEPLPsGJ[y68 jL-- IXJLI][PHSKYGLH ][0O760 [3lyji I I -9 [IJL 7SRSFK] 060j 121g. IILRAVQAD TLo C I 345 LVSKsVVWJ[-=060_ LF7-iI 0\Y i 0= 1471 D KCFR [0054 PI 20 LTTESM 00460 D39 A1LTLT R 1 0.04 00 00 ITable XIll-VI -FL-3ges Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino adds, Iand the end position for each peptide is the start position L pluseight [StartI[ SLbsequence rej 1.-8fllALEEGQGLT jt05j L66 II.AMLKCLSEG =1..045 Fi--iRL ~n~ 2 J _0=640 I? ISFPLKD NS S 1 I 0 .0361 1276 EYI.?~jliI 0.036 F3~ I7fLFAAvR n 0.030 I ITable XII-V2-HLA-A3-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 5; each start position Is specified, the length of peptidle is 9 amino acids, and the end position for each peptide Is the start position plus eight.__ I Strtil Subsequence 11 Score j Di K[CYRjGDSGEI 0.100 Wi]E GQDAKL C L FI-m91i LPCELYRGD ][1E0.] g[III_LPCLYSI0.004 ]L LYRGJ =0.000 L 7-1 LYRGDS9GE] .000] ale XII-V7-HLA-A3-9mers-1 191P4D12B Each peptide is a portion of1 SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each1 peptide is the start position I p -__lus eight. _j [E i aL squene[ScoreJ 1181 SQ: EEPE GRI .180 9 j HDRQE[T.00 7I Table XII-W7-HL-3mes 191 P4D2 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino adids, and the end position for each peptide is the start position plus eight.

[07 9SEPEG LoOO Lg1l HHTDPRSQS EL.S7 I lli R 9EEJ 0.000 I]PRS QSEEPE 0~.001 M] SHHT D P RSQ 7000 TbeXII-V9-HLA-A3-9mers-] Tb 191P4D12B Each peptide is a portion of1 SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight._ 1P Strt ubseqyence II Score [ul_I1FFFF.000 PILL(Iu±LLKV~fl 6.750j Liii LLRTFNFjL6.-0001 F] LFHY .500 P- ]EFPfFW j 4.50 0 WL. LI 'FiflKRITFNF=FLF 1.800 fL13[GN SCDCERI 1.200] Fi-ffCLLLGLLKV0.0 ]L o.54o9 82 j F KRKKKLK j 0.500 FTable XII-V9-HLA-A3-9mers 191 P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino adids, and the end position for each peptide is the start position plus eight. Fs-taRt I[Subsequence IL cy .1.1FLFFFLPFPI 0.450J L ]KFRffQCLI 0.405 0 F-.Tn L ELGSNPP 0.2001 M12 I[ ql-LKyRPLj 10.1 '35 1 FI~ 4 -IYyV 9L9 [0121]j [80:I ESTKRKKJ0.7 g69jj GlSVHHCW j 0.0680 F! -5I FFLFFLLF0.054 17I[]LffFfFLPFI0.054I [F.1L 5 f.945 1 [2KjFIFYFi[ 027 I FYFE E10.027] =24I ff iL-24 FoFL 9 y 00ELGLV P 7201 Fj1 LSHCA NLO.0 FW~~1 L 1F oi [86l IFET-q KKLKF If_0 .012 11051 KVRPLQHQGI1 .009 1191 LFFFLPFPL 'I 0.009- 00 00 ITable Xl-V9-HLA-A3-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptidle Is 9 amino acids, and the end position for each peptidle is the start position plus eight.

[L-t~q S-ubsequence _I Score 11.DFLRITFNFFL- 008 96JLQCLLLGLL 8 1.741 HHCFF! F0- 0008] LI~.ILMEHYV IF 0.006 1 IiN3LSFDER GYQ F T 9 RELAGILLjjt005 [A21z N4 WEHY VAQI 0.005 I6.1GSSNPPAESA J6L-5- [lj.-IFFFLPFPL .0 I 40 EMESHYVA P0.041 F!7 L N AFRFQ II0004 LF i I HCACqFESFT jO.031 [T6i NFFFF!FLP 1.003] LI. I FFLFPW. =0.003 [63 ILSASLVAGTL I0.003 I T 67 VGTLSVHH 0.002 ffl]K RGYQGIFM JLO02j 0.002 [47 II YFF LEMESH JR 0.9927 1621 ASSVG 002 112 RLGFQGIFMQJ_0.001 .RKKKLKKAF Ii 0.001o Table XII-V9-HLA-A3-9mers- L....191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptidle is the start position plus eight.

F24jPFPLVVFHJ om i 9jE RGYFQGIF L9f. i 1 Table XII-V1O-HLA-A3-Smers- I1P4D)12B3 Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptidle is the statpsto plus eight I[Start ILSubsequence I Scorel [T7 [GLGTSDWT I LO.oo43 7YJ] [OPELT][ 0.0041] ~1L ~!§YYTV CI01] FT7171f-- I9~ D Ilq.99' EP GEL] 10.0001 able Xil-V1 I -HLA-A3-9mers- 191 P4D128 Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptidle is the start position ~us ight. F1. tart Subsequenc L crel F±@PPLPSL 3.38J Fl] K-RLVMVP 101 FT][ QARLRLRVM- 0-001 [Table I -I 1 -H LA -A 3 -9 me rs 191P4D12B Each peptide is a portion of SEQ ID NO: 23; each start position Is specified, the length of peptidle Is 9 amino acids, and the end position for each peptide is the start position plus eight.

FS-iart Subsequence][gTor L FA 1LRLRVM7VL~ ]F .LLRVMVPPP [00 Table XII-V1 3-H LA-A3-9mers- .191P4D12B___ Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is 9 amino acids,.

and the end position for each peptidle is the start position plus eight.

WELLP9 Ps-J 0.020 F -1H1Ipy 9_Q .002 _YALP Joo1j 4Y]LYPVLDPQE 0.0 Table XII-VI4-H L-A-9mers-1 191 P4012B Each peptide is a portion of1 SEQ ID NO: 29; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position eight.

I tartj Su§ bsequence ;Iscr 7i7[GSSNPPAS II 000 NPP SSLV I 0.00 [P LSNPPASAS[-

KLO.

[77 L SLAGT Fjqq000 2771 SSNPPASAS 11 OJ000 00 00 ILL LVAGTL S .09L~ lTalex XI-VI -HLA-A3-1 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 pius nine.

[Stalubsequence IIc e1 K2 3jAI)LDDGK -LO M9 [ILLLSFTGR 18.0003 LMSRYHRRK][9.go! [25211[9 GLD-QN ml I 181 100of I II11 TLRENSIRR 18.000* F1 Ti~iLLLLLLLASFE1.500 I IF 81 M WqP wLLFl4-050 I KOC LRLHSHHTOPR[ 4 000 FC6 J[IGREAMLKJI 4.000] [3s911 LLFCLLv~vyvVIL.0-001 U LE L7TL.80] [L2LG~LLQIDq i] =1.809] LYfl[WVLMS-RYR][ j- [2[1VLnQDA[ L1.500 [362] cL50 [L 754 GVIAALLFCL, FT .2151 12571 NLWHGREGA. II .0-j :yLhv ;sRJ.F 0.9001 [239 ILSLAEJ[0. LE:CoiLTHR!. ii 90~p F1674 STE I O.600L P68j1 LVVLMS Y::R0] t] RAEGtHPDSLK I 0.450] 8 L FCLVk VY Y 0450 Table XIlI-V1-HLA-A3-lomers- 191 P4D12B 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 Runine.

Ir W:Jj GVIMALLF1IOAOOI] F4--flEQVGQVAWARf =.36o.

J[ 4[ R GPLs~ v] .300 1 I FT4ILPW-A-Mqi. ILT29K Pq PI LDNSSCSVJI 0.270o I[Fl?0j LA IGEIT 0.27001 llIF4!1L~VI LPQ0.19.

I

[-3IL11__h~qyENS I ]wO] vSFLEASV il0.200 F51 LLL sFT10.81-9 T4]LMy 0.090]8 F A-L-LHSKY -T i~~ 1789 G S I 0..120 Table XII-VI-HLA-A3-loers-1 K191P4D12B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, ad the end position for each peptide is the start position .jusnine. Fi-irJtLy~eence [(Scre] [±6!LQNGTL RA2KLo.09-0 [Th 11 'YqK- I -0 [3D6[ IHIL U28JLRsTFPAsfl 6.961 127011. CLSEGqPPPS j[N060 I 47J[GIQAMNFV I0.060j I. =jF~NTR I0.060 g205) TSEFLVPSR] 0L.060 I-Ill GPEAWLLLLL 1[ITOR4] [218 LGQPLTcYHI 615a41 L99LTh9?tPTTE] 0.045 [272] QPPSY 1[0.045] 135 GFQARRLR 0.045I a~j]LLLWWVL ]ff041] [341 ]QYQLVSAS§ j 0.0413 N5][ EETLTR [36 Iq IT ILLEg. 3 61 I I cwOi 03 Trable XII-V2-A-A3 -1 mr t 191 P4D12B _i 00 0 *Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length I of pepfide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

LStartll subsequence Score [Z,.iU19CD ]L9L F366Q [6_7[!PLYRGDS 1v 8) I77I PKC pYR 0,.012 1-I IGQALC II QID NYRO:GV 15; eachstar Eapeptide Is ar poition o plus Ine. 1 [st-art L ubsequenc ej[Scor e [Z11]F SQSEE 00] LFJ[]PSQSEEPEI 0p.0900j ,jffHHDPRSQS§E 0.0001 K]LRSQE EPEG IR 0.00 Table XIII-V9-Hla---I-mers- S191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

I startI Su bsequence II.Sco~n I 8 I FY F 1L40001 18 II FLFFF P 9051 Table XIII-V9-HLA-A3 10mers- 191 P4DI2B" Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peplide Is 10 amino acids, and the end position for each peptide Is the start position plus nine, M [ldqueneILF cor 900 I 6. LYVF:F .100 'IF GR-ILLRIT FF I4.050 I 2! RFFLFF L3.600J FL E S FTRKI .250O 182 j FKRK KKLKJ[2.000_1 [T.iT9J:LT CACF [12:000 F! t. FLLKVRPLqH] 800I [911ICLLGLLKI i:20 I qL 1.20 L-3_il F LVAThSVEIO7K P~J LLGLVP 1 o] I 7[CCFSFTK I LjjI HVA L.ELL I9 130 II 7FFYF L001 FU I REGL] 0.54 69 ITLSVHC~c 0.45 Table XITI-V:9-HLA31Oer Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Lstartj I Subsequence] 1.103 !LL~v Lo745 I66jLVATLSVH± _L3 0 [Igj GIMQPAtWJl 0.030] (~221 GYQGIFQA j0.027.

7EL.'GL iI 0.022 I. 7]YFLEESH q[0.00 I181 CFYFELEJ[0.018 112l1y _±~~oo2 I 1!9]LfEPLFQGIF I 0.010 Ti]T2 R-KILJK.00 79!j FFF c LPFP i[.O6 7I r F TIbY G F I Fgi 119A qE IL0 I 9iLL SfmHY( .006 I 8 L~KLKAFRII .004 I..i PAALVAL 0.004 I L 0.003] 5?UT -j ASL LSV 1115.1 t NSDEGF o07 00 00 Table XII-V9-LAA3Iomers- 191P4Dl2B_._ Each peptidle is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position 1..art§Sbsequence JF7coe J1 RFICLILqlGL 0.003] 32LYFYFFLE (0.0031 FT 80 EFTRKKKL- II 999Z Fi! L 1ESF -RK FL.00 [Z1[YAQGLEL 10.00 f 61[ FFFEMESHtPO2 123 ['LQGlFMQAA.001 [6i[ASASLVAGT 10.001 [7 I2RELLAGLLJ[0.001] 8j _KKAFRFiQc 05.001 1l F2[AfRffqCLLL j 0.0011 1109 [LqtqGyNSqPIL01 FT ~jE§L Aq~q10.001 L87. !KKLKK FF 0.001j [11[ IIVN C DC EYO. 01 W2 IIHQGVNSCD I EIP00-1] [IO7JIP LqHQqGVNI 0.001] 27[I 4IL FM QP 0.001 LFEMESH YvJ=000 [iI FYF FFE]~ 0.000 ff.! 11 RGFQGI FMQ [L7O(? ITable AX1l411IO-HLA-A3- Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length Iof peptidle is 10 amino acids, and the end position for eachI peptide is the start position plus nine.

[S-tarti Sub-sequence IKc2Re I F LTDVVIq.T8R GSDVWTWL7 .13 5 F71. iE-LGTsDVVTT L.001 FT2 11F qiRcPAGEGT FO.0011 Table XIll-V1O-HL.A-A3- -1 0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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.

ELGTSDV-'0000I [T AGELGTSDVV 9J1.0001 [77RCPGLGTS ffYOjO ,[IfjGRCAGEj0. 02 fTable XIII-VI 1-HLA-A3l10mers-191P14ID12B Each peptide is a portion of SEQ ID NO: 23; 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.

10JI fLVIPPL P F [2Dl. QARLRLRVMV E0002 L itFQARLRLRVM 19.0011 Table XllI-V12-H L-A- 1 Omers-1 91 P4D1 28 Each peptide is a porton of 1 SEQ ID NO: 25; 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 S _rtJ Subsequence I.r7 0ii:Ei CSYSTLTrvR Kio-oo-1 IM SV.~MSEPEGq 10.030 V 1MSE EPS 10.030.1 5[ EEPEGCSYS]P7I990 LfLPEGCSYSThT.l.09 [T]1j E9STIIL9OOOI0001 STable XIII-V13.HLA-A3-1 10mers-191P4012B Each peptide is a portion of1 SEQ ID NO: 27; 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. Su_§bsequence7Lscrel 77 QV-TVRYLADP JO.0051 I[77_ DL EDS 1O [2 DYI Pi[.021 v RVLA D qo F f b v: L7J[ IPQEs ~ooo 900.0 I H ~llI .9 9~ I ~c~Xi IN01 0WMA~d 9LZ F i66 iF ozi I L~~Iiij ]F6v- I 1 N LP16 Z o6o II ij iL li- I6~ Ii/TVi I v~hiiii4Li 8 F Mildl Foqlso pel VdaH1A si op9 1 4088 Jol U11sod e eql 'S8 Ouwe6] a1iddK i jo to6i66a awoupsfls uoilso *JBI p Peg d 3 jo uoipiod e sl apdad qos flZ 10d1lU-5JGW6 I 9ifoOI 1dF1 I [8Cr I-2Z07 .1 r TA I-e6 i z 00JNAAA__ F6Fo F6i 1o dHA-1ie F oeooi~i9~i L\7[* IAS0lAi1 r6o WA 1-646i~ 1069, sniduofllsod piels 941 sl opIjded 14058 iol uoI~fsod pua 9qi pue 'Sp!Oe Ou!we 6 sl epijdad jo q)Bue I~ peij'p9gods Sf uoqjfSOd IJ'Is qL059! :O 01 03 DS jo uolljod e SI opqded 4083 S9- 1.OVd 1.6 1.-SJGw6 R601 9Wd6JA V6U I oo*Q IIS.L!A 8.

7091.0 MilmA 4H8SnA9 I 6E]I N V6 0 I jA]'6' WJddd6AA R] IO f ILOMIAS091 pea jol uoilsod pua 914) pue I'SP!02 Oulwe 6 Si opildod 'o 141Buaj 941 'peijpeds 5! uo, sod jo uofljod esSi opn~ded qoea L O0 _VVHU.-AX elqei 1b6oo Fi6VS 6_Ir I F 6: Fiido If V§~ri r ib& lv~s I~ 0-11 FJd6Y1 RXIVSdd] LI etju snld uoilisod liesS 814) Si ap!ldad 14059 iol uolSod pue 914 pUe ISJpi0 oufWe 01 sipided jo lb 14! aqjpaIj!0ds si uoilsod )J8)s 408!6Z :ON 01 03S jo uoilJod e si api~ded 14059 -CV-VIH-71A-IIIX Glqsj 00 00 00 00 Table XIV-V1-HLA-AI 101.

9rers-9P4D2Bj 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.

Startj Subsequence 11 Score 69]RVDAGEGAQ~j 0.006 362 FCLWVVVL I .0 -131-1TFP7AGSQ .0 357-1 -AA'LLFCLLV 0.00 j 1771(ULLLLLLASF i 0006 77"! FQENGTLRAKII .006 iOYiI Fq FP PL -E HJI-O6 rTable XIV-V2-HL-A-A1 101- Each peptide is a portion of1 SEQ ID NO: 5; each start position is specified, the length of peplide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

[sar I Sbsquenc 11 Score7 D-]j DAPC P LYRILffO 24 [9qDAKLPCL 0 .018 [T1LYRGDG T 180.00 LFLP G] E 000 D] PQoc9E[oo Table Xl V-V7-HLA-Al lol- Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.____ F{Start Susqyuencej[ Score 8,F§9I E P G R 0. 120Lo Table XIV-W-HLA-A1IO01- L. 9mers-1 911P4DI283 Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position I Plus eight.

Start I .Sbsence I L e I -TiJ TDRSQSEEE F TO.00 I- tHDP RFS-. 96 Table Xl V-V9-HLA-A1 1017 I 9mers-1 91P4D1 2B___ Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length 1 of peptide is 9 amino acids, and the end position for eachi peptide is the start position ____plus eight. I SusequnceSc-ore 1:3 Jj GVNSCP9E III.200 L 1 77 I,1.ACFESFTKR 0.08 7-A7 ELL-GUR] 0.07 8L769 iIGVHCAI.045 L2. .1 DTT fLf 000 I i YYA9 FL 241 66 _LVAGTLV 921 31 I FIYF YF YF] 19.0161 ITable XIV-V9-HLA-AlI 01- 9mers-1 911P4DI 28 Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight. LS!a rt.Subsequence j[Score F86 IFKKKLKKAFRJ[02, [i71 LFFFLPFPLO2 [98~~7 [iCLGLK 0. 121 F91 KAFRFIQCL JIO.01'21 FT SLA G TLSV 19:91? 110.ii LLKVRP LQH [9CO8I I[ IL. LLGILLRI 10.-08-1 195_1 FIQC LLLGL E=0081 H371 -iFYY 9k9 LF-1 F FLPEFPLW E0.06 [14T 1I.TFNFLFFF- 0.006 FA- KI FESFTKRKKK Fq9.P97 17JffLFFFLPfJL006 1I.FQ9GIM-QM- ][.079J 105 I1jSRf !9 II9PLQ G K Ji31iFQ~l F. OI9 41 jq:1 LRITFNFF 0.004 M2 LF-x 87 LKK KAFR FO.T93, 47] VAQGLELL 0=.002 _76-1 SCDCERGYF 19921 00 00 1[ Table XIV-V9-HLA-Al 1OI-_ L.9mers-191P4D12BI Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position ____plus eight. j~arti Subsequence Iscorel Lf1 67 vAGTLSVHHJ[000FC L 727SVHHCACEJ[002j L 63 ]SASL AGTLJp0 F7i]RFIQCLLLG 1102] ___]__1LLRITFN- .002.9~ I PLFF ic..

L 33 YFYFYFFLE 0.001 1 F48]j AQaGLELLGJl[5.001 88j __]KLKKAFRF FO9691 116 j NFFLF ffLP joOj II_5JLELLG-SSN.0011 I8 ]SFTKRK L 0.011 I] FL LR1TFNFFL 110.0011 L~p~ i RPLHQ~v .0aoo 1 128 J{ QAA q E.~0o I I iii fLFT PFJO.R011 RRELAGIL[07.0012 T0jijqPHQGVNSq 10.001 'L QM PWEGT 0.9211 =1i EIQGVNStCECIKp901] LF i ALLRT jol1 421_] MESHYVAQA 110.9011 7 SHVQG.0001 F1il LLEGL LKVRP iFO.oool L F TSHHA 10.90 49 QGLE LLGs 0.000! F127 Jj LFM~qMWEjg DOO~ L SS E.IsA~ I.709 Table XIV-V9-HLa-A1 10-1 *9mers-191P4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight. FStar 7d S ubse qu e nce] soe F[7h1LsN'HLKA F 60.666 FT]IRKK LKKA7IOO Table XlV-V1 0.Al101-9mers- 191.P4Dl26 Each peptide Is a portion of SEQ ID NO: 21; each startI position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position pius eight. Startj Subsequence I Score' II9_J GTSDVVTVV 11 00 0 I 6LGELGTSDvvjIF.0031 2FIT ROAEGJN.001.

JF Ei GTjDVVTVj[000 =5L EGSDV L9 T ELGTSDV [ooo =71711 GRCPAGELG3[a=0O [7111 AGLGTSD 000 Table XIV-V1-AI1O-9mers.] 191 P40128 Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 amino acids, and te end position for each peptide is the start position plus eight. I_Subsequencel[Taores F7 ]f RV VP.PLPSIO.2 j7~ _RVMPSL0.006 JD7 71 MVIPLPSN I 0002 Table XlV.V1l.A1101-9mers-] Each peptide is a portion of SEQ ID NO: 23; 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 _FARLRLRv m vjo. 6o6;7 ILLR~VM4~ 0.000 I Table XlVMVM2Al101-9mers- 191NPD1213 Each peptide is a portion of SEQ ID NO: 25; 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 PI jjoC Y_ 0 01] ~F I FL~~C SY S ogoo [77VMSEEPEGC T00 7~ZLPECYT .0001 [711 EEG 0.000j1 f Table XlV-V1 3-Al 101 -9mers- 191P4D128 r Each peptide is a portion of SEQ ID NO: 27; 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. J [rt Subsequence Score I..1_j[EQ T yLAI T LJ9YTVDVLAD j[ 004] [Mi[ PYVL: 9D. I E.02!.

00 00 Table XIV-Vi4-AI 101-9mers-1 !95-41Bt Each pepide Is a portion of SEQ ID NO: 29; each start position is specified, the length of peptide is 9 amino acids, and the end position for eachI peptide is the start position plus eight.

Start JISubsequence (Score!I F- SALA GTL Lo:0021 [fl~sNPPSAj 0..01 AS VA IF.00 A- ~PrAS SL 1 0.00 17i7] A 1 SLL 199 [T][ASLV GT~0.0001 SSNP ASI 1.000!, ~7~JjPASASVAqIO00 LTable XV-VI..HLA.A1IO1 1.

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~ F usqec Ico rel 401 F y-ooj~.5 0-1 364 LVVVLMSRR1l1.2001 f #L HIREGAMVLK1080 §YVMSEEPEGRJ[O.BO0 I T IAQ LALLK Joo0 I 4 1LRAEGHP!DSLK 10.76001 L .I5WLsRyiHRRI Fq6016 PT] 'VLDPQEDSGK Fq10.400; I ~0JL~~r~N O. 0300 ~354i GVIA LLFLJI0.2701 I LOj.( RLHSHHTDR P0.240 I LLLL A§FTGR IF..120 ITable XV-V1-HLA-A1 101-1 j Omers-191P4D12Bj 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.

Rl-12 ILKrqsY!wT.] 10.00 IR~j 48 [YEEGT JF270.0 KGTTSRSFK I0.090I P1112811 L- RVTPG FOl.0.9P ![lj Q19 QDGYCJ0.060 [352j WVqALF 1 0.040j F 301_VVPVWLMS R 000 lF?5P]LLyylQyIT 10.0361 T q~ iL~~Asv I0.030i F1 1-111 GrmnGRGHI .0 Ef2J[ gLQnLHI (1.024.1 QVfl GQVA A R 100207 lF2-i GRVEQPPPPRH 10.0241 [Table XV-V1-HLA-A1 101- 1 Omers-1 91 P4DI 28 Each peptide is a portion of SEQ ID NO: 3; each star position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

LStartl__Subsequence jscore 1479.11 tED Q DE GEIT.0 _tL TSHR 10.0181 [F58JITELLSPGSGR 0.-018 14 [QDAKLPCFYR 11.018 LT9Jl qIqAMN-FY] 0. 012] [Eljz. 1. QMNFJlO.0121 G1_LTLAACT _91 21 18 -kI YLHV~spA 0.012i L284 Ij RLDGPI-P§GY]051 'I 1 [RYHRIRKAQq 0I.012J 25 DLFIRCPAGL]Fj!OJ lj TCWSHPGL =0.10I [2-'f[_nuLHYLF 0.010! F L5EL41cLL"17P.i0.98.

FI !l..ALEEGqGLTL ff-08D 257l [NLWHIGREGA =0.008 (315 jC YSNE FSSRjq06 NI88][LHvSPAYE GR I F-6-66j- I 156f GPALE =GQGIJ(0.006 358 IO. 0LF 0l96i I. P1.l1. IYINGRGftlV j.006 I 79JI._ LHSKY9 i1295 I 493LWAKTGNGlFj[.006] FkAziI L CLLW 0.006 d II RVEPPPPRj006 3 62] 0.LW V 0M0L06 12941 =VDO.006bos 161FLLLL L16.6AS56.1~ 00 I 00 ITable XV-V1-HLA-A1 101- 10mers-191P4D12B

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.

Kt Subs-ybequence I Scorel 1312- 1LYCHVS N!EF EWE-06 FI 69 I RYAGEGAQ 9 006 L29. _LRVDGDT 0.JIO006: 1223 1 CSPLqpf-- 10. 006 [I7MGPAWLL10,05.1 GO.RTLRA KPTGN 0.0051 IL!!HSFLAEA I 10004] F426] §LKDNSSCS 10.0041 IILVPPPSLN IL..004!, ~_Table XV-V2-HLA-Aii11.

l0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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.

~t t L sbs eq e ne I scReI E2 G1QD-AKLPCLTY E:6:81 [3 QDALPCLYR] 21.08 1i :61LLY-RG5 SGEQ P6149~ DI1I.nKLYRGDs 99-11 [i]LI CLYRGDSGE j0.001 DAKLPCLYRG L0.00~ AKLPCLYRGD 000 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine, SStart S1 ubsequepce-IS-core] [711 SQSEEPEGRI 0012 ;7F71 DPRSQSEEPE IaOOOJ j:]7F7 HHTRSQSE P000 TI~able XV-V9-HLA-A11O1- I lmers-191P4D2Bj *Each peptIde is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide Is the start position plus nine.

i 82 Ij TK RKK KJ 2.000] ELE1L E LLLGLLK .209P I. .Th.IF CACFE FTK jja60]0 L lRELLAGBR[l 18 I-El SFT_ KRIKKL 100] [27 JjyVFIYFYj 0.090 DI8EE 9!JLf [LLK 00 ff~F Q0.q90 L7UIiJ1L LLFNF 110.054] IF7 ES qFFT7R Fo0.040] 11102 _GLLKVRPLqH 0.036] 1121RITNFFLF 10.024 3111 F[YfYfYLP 1L04J 787, C CFSTKRK]Oq Table XV-V9-HLA-Al 101.

lomers-191P4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

f Stat Subsequencej JScore F66.11,LVAGTLSVHH F10.02 ii -4.1LR ILqUL.FI2: [91JJ~FRFRILL[.-O1 29IVFFIYFYFYF 1F0012 11 JL~LRTFNffL ]E.12 [45 I FYVAQAGLE I .012J F23] TFF LPF-P LVV]lE. 0128 16 I NFLFFFLPFIK 19081 F33 lfYfYfFLE!J0 2 12QGVNSCDCE06 -1 FY.Yil0 251 FPiL VVTFF g.711006] I 79CLLLGLKV lt.096 L J L~FL 19.006 F L ILRT 10.004 19 l FFINF LFPFLI 0.004]5 II 7 j TSVHHAC 0.0045 F92 ]AFRFIQCLL .00 38 Fii EM. !l'I[9.031 00 00 LTable XV-Vg-HLA-AI 101- I Omers-1 91 P4D1 2B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids.

Iand the end position for each Ipeptide Is the start position plus nine. j Fstart L~usqjecescoref 13 ii YFYFYFFLEA 10.0021 (81 KKLKKAFRF [.oo21 D71 RELLAILL] 01001, [AQAGLELLSIOO1 1_26 jPLVVFFIYFY .0011 I. 7JjMQMPWE .00101j F-7( ASLVAGTLSV 0t.i90i F1. ~ni~ KKARF90JI.1 (11911 ERGYFqGIF 1!~I F1, FMQMAPWEG I O~OOO [LSSNPPAS. M00 F10011 LLGLLKVRPLI1K000 581 SIFNPPA!SASLVJI0.000I (103 1LLV-RPL-QHQ (.000 !F2 121FGYF "P K670O1 r F7 KRKKLKK b7O00 F17 J. FFLFFFL5PF.00 Table XV-V9-HLA-AI 101- 0mers-19P4DW2B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino As, and the end position for each peptide is the start position plus nine.

Startl Sfubsequen~e coe 9 us f[N CtcERYF 0.000( 63y SASLVAGTLS (0.0001 7-T] MRRL-LAGII.I0.000J 7~T] VA GLCII.009J 7~]LAs~~GI._)[0.0001 Table XV-VIO0-HLA-A1 101 Each peptide is a portion of SEQ ID NO: 21; 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.,_, E[IE:TpyYTLyY' L 0:P.

[~7[ELTSDVTV F01] I7L9 LG7TL9LYVP7.p00 LVGRCPAGELGT ILO.oOO 11 Table XV-V11-HLA-A11O1- 10mers±91P412B Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is '10 amino acids, and the end position for each [peptide is the start position j_ plus nine...

art71 Subsequence F-77 RRVVPPPL 110:901 AO: 7i1 KPLPLy i 110.0041 Ff7* LRLRVVP .I02oo 3 7 ALVMVPL 11.001 F]LVMVPPLPS 110.0001 ITable XV-V12-HLA-A1 101- IL__0mers-191P4D128__ Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, Iand the end position for each peptide is the start position .plus nine. fLs 7aq Susquence JScore 10 CSj YSEtLTTVR.0081 IDTM] GCSYSTLT 10.0001 3 MEEEGCSY ~ooo =5i1 LEEPEGCSYST 0.9OI ITable XV-V13-HLA-,AlIO1- 1[ 0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 27; 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.

IF7LTIDVLA DP 7CE 0.002 WLQpy.l-AfO2 F]DgWJl9- 00 00 Table XV-V1 3-HLA-A1101- I Omers-191 P4D12B Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position P -plus nine.

[S~taRtJE Sbs quence I s- rel -TVDVLADPQI ,021 7T] DVLA PO GI0.000! [7 DSQVTVDVLA!j0.000j ~J 6 VDL ADPQ S K l 0 00 r FTabe XV-V14-HLA-A11OI L_1 Omers-1 91 P40128 IEach peptide is a portion of SEQ ID NO: 29; each start position Is specified, the length of peptide Is 10 amino acids,1 and the end position for each peptide is the start position plus nine. __j ItrjSubsequence- !LSoe E.57 NPPASASLVA.04 Li. il UAVGT SV 1P011 fT ]SNPPASASV o_ o 7W] A SLVAGTL 119.0001 LFi7IiI.SASLVAGTL 0.000I L771LGSSNPPASA 1.00 L7.ss~~Jj~pp I S7]GSNPPASAS .7 7W] PPASASA 10.000] FT7 PAALAGT .R990] le~ XVI-VI.HLA-p,4..gmers- 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 Star.t Subsequence I Score 1501 IYINGRGHL30.0 111241. EYECR VS fP0990 ~Table XVI-V1-HLA-A24-9mers- 'I 191P4D12B j Each peptide is a portion of1 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.

rt ,,tSubsequence Score, 7I 485 Q KEELTT Li .0 Iio5RNLDGSL 12.07001 15 8 JI_tKYGLHYSPA _I 00~ [i 1 21 _RLRVLVPPLjI .E6 9J 10 _q P I F.6470I i 53621 CLVVVV'/L 8 '.00 I~5i~Y~Y~ 1L.4200 1141 AWLLL [T7200] :F5'2 WLGQDAKL 6.600 382j MITQKY E E L L6. '600 _R7J AGE GAqEl .I[6336 11200lR AAISEFIL.16L1 :12221TCWHGL.o I'53 F 6.0-00 'l j IETQITELLII6.00.

I i11ALHSKYjLII 60001 I2 I VTSEFHIiOOO00 [1111 GpEAWLLLL 1L6.o.

1251LAEASVLL60 LO ff7 AALLFC LL i l_5.76I 12811LNTLGk 4.800.-l 1111 EAWLLLLL. .800-o 135iVIALFCL 49- FYI A-4.800 I261 TGRCPAGEL f7 4.400 EMWGPEAWLI4.000 I '12941 L Table XVI-V1 -HLA-A24-9mers- 191 P40128 Each peptide is a portion of SEQ 10 NO: 3; each start position is specified, the length of peptide is 9 amino acids, and Ile end position for each peptide is the start position pj lus eight.

Star Subsequence Scre 11 35!'[.SFQRLRLI400 [211 ARlLDRD LL4.000 1? 9! HGREAML.! I4._00 17411 EGA QELL 114.000 S 4~l1L~A 000 '1 7jL9M H F 0~ IA LL2 .00 L5 45) D KPCF I2.000 b 2] YSTF ALS 1 0 14951 KP TGNGYIL?:T090j L QlI LTLRN~ IPIL TVRE 11.650-1 l~iILVVVVLMI1.001 WLLGEi O 01 11571 EGGL ~0.8641 Y12I AYGV~t EI F.750J 111 IFPAGSFqAJ 07501 [I7LEFH LVIDI 361LFCLLVVVV 110.600] 3931 T RE NSR 0.6001 00 00 Table XVI-V1 .HLA-A24-9mers-I 191 P4D1 2B 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. Star Subsequence Score 2371[ HILHSFL] i0:6001 53 LFYRGDS GEQ P I-50 L301EFSRSQV 1 050 F195][SFKH§RSAA If 0.:500 L2.!~I SRMNGq Lj[ O. Oj [297 I[.qDTG FP P l 48 250]1YRGLEDQIN1L 0.480 [384 1-KYEEELTL 7 0.480' F?2i 1. R GLEqDQNvLW [o0.-457? 341 [KQVDLVS AS .42 L3?8 LKA QqMTCqK 1ii .39 6- E I L9CPAGELETIA3 q_ 014[Y-LPLPLIEtO.300 E3].RfITHLHVS !Fi 0.280I 58 SGEqqVGqyA! I. .252_j F16 [LP LPLN 02.216.] 110~ 1 "'--LNAJ0~~ IFI-01 FOPLC j 216- I3T1LqpPPSYNVT I I[VTWALqQPAJF 6-Ti LableXVINM}LAA 24 9 mers- Each peptide Is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each ipeptide is the start position 'I plus eight.

[starjasubsequenceI Icre F][iGQDAKLP-CL IF00 Table XVl-V2-HLA-A24-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position .pus eight.

lFatI Subseq uence I.P9 .F yRI)SqQ ~0.550 ;FT LPCLYRGDS r0.100I 77 QDAKLPCLY 02 D711 K L m lTable XVI-W7-HLA-A24-9mers- 191 P4D12B_ Each peptide is a portion of SEQ ID NO: 15; each start iposition Is specified, the length Iof peptide is 9 amino acids, and the end position for each Ipeptide is the start position _plus eight FStartj .Subsequence J co q D ][TD §R II 0.002 17_TtDPSQ002 LI] TDRSSE j0- [Table XVI-V9-HLA-A24-9nmers-I K 191P4D12B.. J- Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight..,ar; ubsequence IiScore I 32 IYFYFYFFLU203 i, TFYFYFFLEM j 3a0001 Table XVI-V9-HLA-A24-9mers- 19P4Di2B- Each peptide is a portion of SEQ I D NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position .pus eight.

[Start Isubsequence 11 coe I LF FLPP 24.000I Fi FLFFFLPF [T7 J F FlY 8I1.002i 'I'd FNFFLFFI1 5.000 [*5J P AFRFClqq1! 9.609] F5 I SNPPASASL 1 7.200j IL 6,1 FYFFLEM[I6.00 (1q~i[GL~L 6.000] LS![ FNFLPFF 57601 63 7 SAF SLVAGTL II 5.600 I 9:61F IQLLGL 480 Fk] R2 RIFNFLF IA:0 F 711 F S t qjI 3.000 OI LRITFNffJ 2.880 23- 3 LPFPLW-FF280 I. il LKKFF 1=.400 176~~i SODOE YFI 2.000 Ii23iLYFQ(LFMQ? ij-t260

I

WT FFELEM--L0-0 Pl. 1 L F VVJI 0.90 II75I1HYVAFlE _1_0.750_ 00 00 ~Table XVI-V9-HLA-A24-9mers-I L -191P4D12B Each peptide is a portion of 1 SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight._ Fi11LRITFNFFL (0.600]

FFFLPFPLV]O

VFFIFYF 1(f0.600 IFz. II w KKK( F O.60 1122 G QGFQ1051 4 P LWFFIYFQ q 0.360 F11 RFPQQGF 10T F7 FPLVFFI II 0.252 Ij 7 iLHAE F ILPSfl ot7 ,L769LGTILSYHHCA 0.210O I [T201 ERGYFQG(IFF .o Y. FIQLLLG 0.10 LF..ILFLE:MESHYV 1[.10i L6.iU AsLVAGTL F(01561 I CERGqi YFI 1[ 0.144K F I i FQGIM2 0120i 9 M09 FTLQHQGVNSC L9 1!_0.1 111'5 1NSCDCERGY '_0.12 I 56 SSNPAS F .100 P- gSSNPS Fq[67ThoI Fj?1LMQAAPWEGT [I 9:Iq7 EfYJT[[ HGVN SC DC -0 0 [Table XVI-V9-HLA-A24-9mers- Each pepffde Is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each Lpeptide is the start position plus eight.. -1 [i26 f[~FMMPWF .1700 [7ATLSVHHC j[05.iqp LLGSSNP l[.100t 9I 1 FMQAWE [I06:6'I

FT[F

8 ST jL~ g. [~Y1 FFEMEH [0.507, [Tqfl]!KVRPL-qHQG ]L .7i K "L [0F.0722 I 106 _QHqGy [L 018 FL4o]J~tMESHYVA jj0=.18 I LLSSNPPLO08 1 43JI SH AQAq jf.01-7 ffTljfGVNSCCR_ .07 Table XVI-VI 0-H LA-A24- S 9mers-1 91P4D1 2B Each peptide is a portion of SEQ ID NO: 21; each start position Is specified, the lengthI of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

IStart[ Subsequence 'IScorel l77[Grso w.W 0.1681 AGELGS~y 0.1501 !FT-F7] oLTDV :1.10 FTable XV1-V1O(-HLA-A24- 1 9mrs11PD2B J Each peptide is a portion of SEQ ID NO: 21; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each Ppeptide is the start position plus eight.

LS!9rt]I Subsequence ji®cre F iL EG YI JK.ooI PA-GELG IjL0100 F 7_ ZL~I~ pI Iqk:: GL OL03 Table XVI-V 1-H LA-A24- 9mers-1 91 P4D12B Each peptide is a portion of SEQ ID NO: 23; 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. St art( Subsequence I r Y MVP1L 8[.000 V_]MVfPPSL 17.2001 F ][RLRLRRVM .OI E23 ][.LRLRVMP Fi r_1[LRVMVPPLP ~0502] E7 LRRMVPP I[.02j Table XVI-VI 2-HLA-A24- 1 9mers-1 91P401 2B Each peptide is a portion of SEQ 10 NO: 25; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position p lu s e i g h t t.=tartI Subsequenc Scre F7IP=EGCSYST [..o501 111111~

VMEPG

7§7LCSYSTL t~O.12O [77 0.1 00 00 Table XVI-V1 2-HLA-A24- 9mers-191 P4DI2B Each peptide is a portion of SEQ ID NO: 25; 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. Start Jlbsquen.~c rel EL L2~TLT II0.1001 K-7 SPEG SY S 0 J.018 [77IEEPGSY P.018 Table XVI-V1 3-HLA-A24- 9mers-191P4D12B i Each peptide Is a portion of SEQ ID NO: 27; each start position is specified, the length ofpep ie is 9 amino acids, and the end position for each peptide is the start position plus eight.

IStr jI Sursquce I §;oRe F iL D i0...20; Zli LADPQEDS IO,0i12i 71111 PQy~j[p 01 9mers-19 1 .01 2 1 Each peptide is a portion of1 SEQ 10 NO: 29; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position L- plus eight W[Subse quence [Scjr& F 3J[NPPSS [.0 7Ti FK PASAS7LV [q[0.!5]0 F 7711 FTPAS-LVkG-T] Fq.120 FT] GSPPS 0.1001 FPASASLVA I0.010 PASASLVAG0.001~ FTable XVll-V1-HLA-A24- 10mers-191P4D12B 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 plsnine.

1Ir:I Subsequencej corej 1[ 3 3]RYHRRKAQJ 60.000 1 [TL42jRSQPEESV9Lj--.i0 KI'GVLSP J000 ifl705jR iPLDG F[6][EQPPPP P L FK4 0j I31FjC-FPU'LLVL I 7.200k !I1q8.jAL GQGYLL 17.'200 Iji j~GPwu~~IK7.00 GVIAALLFGCL .9200.

k~IEEGRYST I=.000] [-2F =ITWsHPGLLI 2311 LQQRITILJ5. !F3] FYRGDSGEQ 1 so] I ~I[F~SRGL1 .001 1249EAWYLLLLL1[4.8001 23 41F LERENSIRRL1L4.800 1YNWTRLDGP J4 I LO 4.800 12351 RITHILVSF 4.800 Table XVII-V1-HLA-A24- 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 Su. bsequence 1 Scorej 9MWGPEAWLL 14.8001 '12961 DTLG FPPE 1 II4.800_I Li56I GPALEEGQGLU[4.800 TqKEEEL 14.4001 1132 ?I TPAG SFQARLIDL4O1 261. .ITHILHVS7F.I401 [2211 y§ LTVV -GLI 14.000 128 [IIRVSTFPAGSFI1 4.000 L134 [GSFQTR~ 4.000 [501MYNRGH 4. 000 F EM GP AL 4 118L..L .000 3~3Fl!;[qEEELTh L9. 0 [E5 1g PGPA1L FA9.- 11611 F LLL LLAIF L60 I~ 47 61EIQMH .0 [IE2 I WGVMLLF J 2.000 [E2 .9h!DQNLWHI I 1.800j F?239J[QRTtH 1.800 I4JI.LEQTELLJ1

'A

F3LG- Q 1.26.2

I

3 9P ILTTLTRENS!J1 2=01 I?!71IHGLLQDR 1. 200 1124 ]YERV STFP 1105 473l- =QDGIQA LQ8I Wj01[ Q ??!THs 10.900 113 6 RL R Vl F- o 76 00 00 [Table XVII-V1 -HLA-A24l0rners-191PD2 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.

Ltrt Subsquence Sore P24 FIR -S Q-VTV Dv Q FT .800 27 SYNWTRLDG (11 RRVLPPYI1O .720! I[6 0] FO LL YV-VVV'J.7001 F( 51-j FVRIE-TqTE jI 62j GREGAIVLKC 0.0 SWHIGREGAM 00 _201 EFSDQ I0.600 §.27 PPSY vTrR 0.600 F !AEMWGPEAWI '1 L 0.600

VOAGEGAQEI

I 34KQDLVSAV! 0.

5 0 4

J

SfLWHIGREGA I L7 LRAGS [LJ0480 P2]SRSMNGQOP 0.480 [3 QEDSGKQVD1 L43 NH EL 1.0 '4001 L29 YRVDGTLFIO.360 LI 9j[ sL YS vsF [0.3081 [;97]RVEQPPPR 0.300 [11RSMNGQPLT~ 0.3001 I~ D 28 1R ELT(.300-1 (41 PESGLRA [L0..

(43j RA PGGy 0.240 11123]K GEYECR V7STF *0.240 1 Table XVII-VI -HLA-A24i0mers-191PD12B 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 __pus ne.

I LVPPSLN F-.2-6j [274 T 0.P N O216 [Table XVII-V2-HLA-A24- 1 Omers-1 911P4D1 28 IEach peptide is a portion of SEQ ID NO: 5; 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.- [tatSubsequene] cr [T1 LGQDAKLPCL] 7.200 [i~I KPCLRGDS 0.3001 [f DKLPCLYJ 0.120 FLYRGDSGtq1 0.011 [7]LPLYRGDSGI .1 [T-]FPCLRGDSE([ 0.002 IF QDAKL-PCLY F0507, Table XVII-V7-HLA-A24-1 IEach peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine._ ;F~7_QEEPEGR A120 R-8 SE1 PEGR IFo.0307 7T T R9~E!41 F Table XVII-W7-HLA-A24-I I l0mers-1191P412B j Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

l.1i~siHD~i~Q1[O0 I I 1 HHTDPRSQsJ0.010 1( T T DPR S Q S j 0r o 1_ 3 [HHTDTPRSQSE IToqool I17IL! -Q EPEG l[0.ooppj

F

T

abeXVV 9

-HLA-A

2 4 j 1 Omers-1 91 P40128 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide Is the start position plsnine.

(Start I Subsequence- Pjcj( IL 451 HYVAQAGLEL 330.0 1 F Q LL 1720 -'j ID T7LNF!FFFL j320 I .Fl~T~YL118.000 16]NFFFLFI1.2.o FLqR!FQLLI 11.200 LT.-YIyfY-FYF 1110o.0001 [j].-GYFQGIFMQ 8.400j 5 IUL- ],F.200 M12]RITFNFFLFFIK[T800 11811 FLFFFLPFPLJ(M.01 ESFTKRKKKL 9 WGL RN L4.400I 119 1 ILLRITFNFF ]4.000 00 00 Table XVII-V9-HLA-A24- 1 Omers-1 91 P4DI 2B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position I- plus nine.

[startlE Su bsequ e nce J LScre I L4-61.nA~qA LL ]J[,4.9*99 Uoj L_LLKVPL I .0001 I E-V IL T!Yy IF 3.600 I.2FLPP LW FF][ =.600 [3 fj YFYffyFLkEM Gfl 2.400 1118 fDCRGqFqGI F2.160! W[ LL~ILLI ~2.10 01 [i3i jf rNfFLFFF1200 3 i_LP~fLWFTFI l~.680 1 I KAFRFI .601j LF1iI2 YFQG Fq 09001 Las. YFYFFLEMES][ 0.660j FLi][MLLAGILI 0.76-.o r [Y[LFFLMESHY [.76 [36 f FFFLMESHILO=.5001 86 jj KKLKFRF 1 P.

[711 ][LRITFNFFfj~.36O1 [~~Z~iIF KKKA I I ff60

I

j1105 IKVRPLQHQGVIIO28 L77lI vHHCACE~sff I02406 1 -19J CERGFQG-Lf F[ 20 SNPS-V[A0 [j-TT][QCLLLGLLKV ifoIl5 .j53j1 ELLGSS',NP-PA1FO.1j50 7Table XVII-V9-HLA-A24-I 1 Omers-1 91 P401 2B__ Each peptide is a portion of SEQ ID NO: 19; Eac tr position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position F647LSLV GTLSYI0.-15O I FLLFE:-EtYA 1.0..150] I 2TLFQApW. G. 114591 ri:2 LPV] L1. 6 y R~9 1FO150 681 J]ALEHaA 0-14 I~qA 24O- I.f)Y94 7iC .YflYY1I0.120_1 WI] LLGLRT -0.120:' 55 1LGSSNPPASAj 510gp 0 [114 IIVNSCDCERGYI 0.-100 I [9 ii GNP=PASj 0.100] [E3A.YGTS~ 0.100] LJIL GTLSVHHC F0- o1-0 0 F127 IRIMQMw?)G]LO.0831 .i~i LE0Yqql_.050 1 1oi][ILLKVRPL4o992t] ][RGYFQGIFMQ 1L2.q20 I ii9jCLLLGLLKVR FO 018 F7 I VAQAGLELLG .0-O187 I. 12 IqqYSCDCERLQ2 51 .L~ELI-GSSN.?j[ 0.015 I 26IPLVVFFIYFY 10.015' F able XVI I-V9-HLA-A24- L....I9mrs-191 P4D1 28 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Dad[subsequencej1 Score I F211 II KVIRLgHJ[0.1 [l JkV L~v~ AcF E I 1 15 j Table XVII-V1O-HLA-A24- 110mers-191ND P4B -j Each peptide is a portion of SEQ ID NO: 21; 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.. FSt][f Subeq uen ce cr [10] GTSK3V11-00 [7IFG LGTSDWV 0.1591 L=91[K9TS D V VTV V0.1091q [LALF LGTSDVVT fl 7 2 PAGELGTSDJ 0.01 F7]lE PAELGTqpVj =0.012 LIT]19..CPAGELGT 0.012 17 Ifp- T CRCPA GELG 0.0 101 Table XVII-V1 1-HLA-A24lomers-191P4D12B Each peptide is a portion of SEQ ID NO: 23; 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.

1 Startj Suseunc__ o 8 VMVPPLPSL F7-7LT-LRMVPPLJ 777.FW IFQALLRVMII-o..0 00 00 Table XVIl-V1 1-HLA-A24" l0mers-191P4D12B__ Each peptide is a portion of SEQ ID NO: 23; 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.

L__iiSubsequence. te [7i7 LVM VPP LN .0.2161 7Y] QRLRLRMVII.1 20 [FT7 RLRVPPLLP]0.0181 FF77 ILRVM VPPLPS] P0.951 7__7 ,ARLRL RVMVPI{0:0.021 Table XVII-V12-HLA-A24l0mers-191P4D1.2B.

Each peptide Is a portion of SEQ ID NO: 25; 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.. ILta.1 l.Subsequence core 1111 sYTLTfvR~j[0.50O 111]EEPECSYSTT 1 SEEEGSY L.o181 =0 oLqSTLTTVR !R.121 [7F L EGCSYSTLTjI.

I

L Table XVII-VI 3-HLA-A24-1 10mers-1 91MP41283 Each peptide is a portion of SEQ ID NO: 27; 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 rne [I strtj[ Su bseuenc e I Table XVII-V1 3-HLA-A24- Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is 10 amino adds, and the end position for each peptide Is the start position pius nine. 7T W9LMLVP7P 0015 7~7[TDVLPD0QE 1 q t1 L Table XVI I-V1I4-HLA-A24lomers-191P4D12B Each peptide is a portion of SEQ ID NO: 29; each start Iposition Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

[-strtJLSubequence lScore EN SSNP ASAS L 0. IT ~FZ3- PASN ASAST F0_1 I7CfLTA_As G y CIL I Table XVII I-V -HLA-B7-9mers- 191P4D12B 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.

Subsequencoe Ij 292 GVRVDGDTL]20.0 HQPPPPRNPL 00 IL -PDSL IFO.p2 26 LT9q.PAGE i 60.000 LISIL RRV=LVPPL Pl 9.929.

[L17 [GPEWLLLL 124.0001 LD.lI VVGVIMLL 12.0001 LV\KILL 12.0001 TfL7 2 9AQL 112.000, F7i1]1 DALLHSKYGL [11.L001 1E6LMLLqA -112.000J 13 LGSFY RLlI.0 11] _EMWGPEAWL IL6 992i Ii45jLP PLPsL]I 6000 45_0]LTvREIETq:TJ] .000i 222 1 TCWVSPGL 5-67660 [25]1 D SoVrVD vL .00 1287]GPLPSVRVJ4.000 I F~ICLVL I4.000 E[1 IEWL .0i L260]1I.HIGREGAML II kLP0i 41 0] L:ESVGLLPc00 I 741 EFQ4AL.±0 0 07 FJI T~qKYEE L4.000 203 F3_00_051i [F2.7]5 F2.SNW J000]1 322 1_SRDSqVTVII 2.000 AALCL I1.800 I 00 00 Table XVIII-V1-HLA-B7-9mers- 191P4D12B 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. [Strt ubsequence 1 Score, [3ft MSRYHRRKA 11 ':oo 1331[ PAGSFQARL F 1.200] 493 ,.AKPTGNGI 1[1.200 L1I AWILLLLL .0 36 ]LPVVTL IIoi 4-3L EIETQTELL I 1.200 L'iI PALEEGQL 1 .200 F 7 j A\SVVWGVI 121.001 F?249II SVRGLEDQN 1.1.999.! T74lIHRRKAQQM F .OO200j ii EGRSYSTLT IFT0001 345 LVSASVVVV 111.0001 S 1.000 j[YQVAWARVDA if 6o Ii1[_PPRNPLDGS 1 0.6001 I8ALLFCLL-W ;0.6001 .17. APSVTWDTE [0F.6C0 501 IYINGRGHL 10.600 151 PSLNPGPAL IL.6001 ]CFYRGDSJ1E5 I439i EPEGRSYST 10.6001 LsA~sVVVVGV I[ 609J W9 SWVGVIA J~ 050O L GV LFC 40.500 I ASFTGRCPA j[ L450 i L CPAGELET.S i0.400j 7i1 STLTTVREI 11 P0.0 GDTLGFPPL 110.400, jl.3 QaoRIHIL jf0.400 263 REGAMLKCL /10.400 1 [28j WRLDGPL I0.400 1 39J LTLTRENSI .1L9A00; t~J HFVENGTL 0.400~ REIETOTIEL j 0.400 [T3 j( QKYEEELTL 1 0.400! 1Table XVIII-VI -HLA-B7-9mers- 191P4D12 B 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 puseiht.

Pt7q 1§y sejuqnce )I Score] 302 IFPPLTTEHS i0..00 23j THILHVSFL IL0400 250 I VRGLEDQNL .400 GEGAELA .001 a ~WGPEWLL- II OA9O 3371 EDSGKQVDL 0.4001 Iflj.ILPSRVGj[ 0.300 LT:iI GSVLLRNAV o 1ij AVQADEGEY 0.300 MNGPLTCV 0~~F B137II~FQAR~LRLRVI.3001 F7 ]WARVDAGEGIIO.300 342 [QVDLVSASV 10.300 4q2 IISPGSGRAEE F300 IiY1[ IRSMNGQPLTI 0.3001 211 VPSRSMNGQ [0.200 17][ NGQPLTCW FO.2opi FO. VIW 11020-0T .1]1 ETSDVVTW 1.v00 iil PPLNPGPALEEG LO.3PQ Table XVIII-V2-HLA-B7-9me 191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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.

LtartI ubsequence liScorel ?1 GQDAKLPCL [11ooi0 IFDL yLPLYi j0.600j AKLPCLYR /I:9iS [F]Jf CLYRGDSGE ]701 17jiL LRGDSGEQ 0.010 i L.D KLPCLYRGD 9j WF7411 KLPPCIYRG 1.0031 Table XVI I-V2-HLAB79mers- 191 P4D1 2B Each peptide is a portion of SEQ ID NO: 5; 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 =2L QDAKLPCLY IL.021 [abe XVIII-V7HLA-B7-9mers- 191 P4D1 2B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Startl ISubsequence Scre [7111 PRSQSEEP j.000 I71SSEEPEGR /101O II LHHjDPRSQS J0005 K HTDRSQE e.0031 FLIL SHHTDPRSQJ 0.001 F PRSQSEEPE g0.000 Table XVlIkIV9iLA-B7-9mersI 19D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

[tailSubsequncetll re I6 ILA~LLi .0.000 I2 1i FRFI9Q4L 112.000 L4iI VAQAGLELJI 12.000 I. 2J NPPASASLVI1 4.000 [qJ FICLLLGL 4.000 [iL IQCLLLGL!][4.000 15s FNFFLFFFL] 4.q 00 00 [TablIe XV I I I-9-LA-B77-9mers- K. 191P4D12B j Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each pepide is the start position plus eight. KSar Su-§bsequence score 'i01 L-GLLKVRPL4.0 F5'81 SNPPASASL F400] 11IfRGYF-QGIM1.00 FK-VRPLQ HqG I[O. 500: F 9Z!IF qPLQHqGVN [0.400 L[i 77LFTPFF Fj 00j KLKKAFI I0.400 j L 1 SHVOAGL FT_00 TT171sF TKRKKL ff4o0 1 L-.J.FVFF Y 0P.40071 FT IL IYTF7YFFFL Lo.4j0] RELGL 7( .40 Wi ICRGYF QGI ii .400 F71 FFE CL 040 I111LF NFLEo40 [M71 LAGILRI 0.3001 f1 SLTSVL .301

FT

1 I LRELAL 0.120 E8[2L LQNS L 0S.100] 659 [G TL_ [E2.OA0O EOI lV H4j 1.9lMQqo.

FI =F HCC E 10071 Table XVIII-V9-HLA-B7-9m~ers-I ___P4W2B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position plus eight.

ItE~rjI Subseqence score [7 t-.AG ILLR I TF I 009 0 F flQPAGLELL W, 66] E F1 SLVAGTL 0.06 TPFEjALELLGSS FO0060 WLLYT ATLVH IA( 50 F7fl I-11AF JR C I L L THR [9030 [2]FYLPFPLWF 1 0.030] [7T]IIEY!I CF J S F030 I 55 LSNPPASJ0.2 21 LffLPF PL V!0.2 E2 iL RITFNFfFLIO01 I7WiL 1T F=NF i020 Li SCDCERGY

J

fl ITFNF] a020 F 13i M j q_9 F ITYF-F-YFF-7L0 [iP GLLRPLQ 1. 0.015.] Ai1 GFQP IF-l .1 L281FMQMPWEG 0.1 I[ __LFFF F 0.01 F!iq.oi LLLVRP 0.1 Iip!.I LLKVRPLQHj 0.0 TbeXIIi-V9-HLA-B7-9mers-1 191P4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 9 amino acids, and the end position for each peptide Is the start position plus eight.

Stat Subsqec I 83JLTKRKKKLKK 010 1 90O LKKAFRFIQC 0L01 i [L2]Ki MESH LAQAL 0.01 1 L±]LS nAGILLR- 10.010 K-L o-io 1 n [*IIL1I LGPP 11 0.010] E1J±LnS-cDCERG .o LFIl]. SODGERGYF 1 =0.009] F?17]IP I-FMAPWE][0.004] LTable XVII-11-A-7.

__9mers-191P4D12B__ Each peptide Is a portion of SEQ ID NO: 23; 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 rJt [ubsequence SIe F RLRVMVPL 0.000 M]?AIRLRIRYI3000 W-80EPPLP.?L 6.000 ELK =VVPP .450 DII RLLRRVVPYD T !L W ARLRLRVMV', FT90 1 LD] LRYVVPLPJ i0001_ 00 00 Table XVIiI-VI 2-HLA-B7- 9rners-191P02 Each peptide is a portion of SEQ ID NO: 25; 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 Su bs e-qu en ce IS EPEGCSY~Ti 0. 600 9Li CSYSTLTTV IL0.200 [Fl-7-T TJ 0,100 17L_ _UE 0.1001 tE99Y§11 0.040 Table XVI 114V13-HILA-B37- I9mers-191P4D12B J Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length 1 of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight fstart] S u bsequence] -score.) [qVTVDVLA II 0100 7~7IVLA PQED I .5 EyI qV TLDVLA7CI 0050] 17710 LVAPESL030_I 111yVT V 0.010 EWDL Fq igL-G q TalXII I ypyk HLA-B7I I [Ta _9mers-191P4D128 _1 Each peptide is a portion of7 SQ ID NO: 29; each start position is specified, the length of peptide is 9 amino acids, and the end position for eachI peptide is the start position plus eight.

I ar~j ubsequ nce]Screj Fk J L VL 7. F 2000I FT able XVIII-14-HLA-B37- 9mers-1 91P401 2B Each pep tide is a portion of SEQ ID NO: 29; 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.

SatSubsequence IIScore rT][NPPASASL II4.000j ]ASASLVAGT]I030 §2NAS FF/A 0: 1 I*ALVGTLS 6~0.6 ASASLVAG E.03_j ITable XIX-VI-HLA-B7-1 Omers-I Each peptide is a portion of1 SEQ ID NO: 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.

K1t![ usauencj Sc ore 1249] SVRGEDQI 0 o I DPRSQPE2SV060.00 IEPERST 24.O000 35itVVVV GFMA.i 20.000 3501 WVGV I 0WJ FL[q1. GVIANjLFCL =4666600 1 .'I1 EAW YLLLLL L I1@:.9 I IF79]LA~LLHSKYG9L]I. o000 Table _XIX-VI -HLA-B7-1 Omers-I 191P4D12B EIach 'peptid 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 99] EQPPPPRNPL_ L-00 F1 3781- QARLRLRVLV 1 9.000 19 LY~NniL8.000 PT?7]HPLQQ 1 i[00i 1_I~qG 1YL][.000 25 FTRPn I 09 LTfIAMWGPEAWL 5. 4001 14021 ]RSQPEESVGL 'F4 4000] F IL1. EP9Y 14.000 F 8 1 WGPEAWL LJL.095I I 83lQiELTLi 4.000] L7JL9~~Y~lI4.7000 1 E ITILV FL ±69i 91iSRVGT 4.0001 10 WGPEAWLLLL I 4.000 1 2jqLIWsHPGL(L 4.000~ SPSRSMNGQPLI.00 I280. Y T RLDP l40001 M21 I LTCWVSHPGL 1 4.0001 IMALLFCLL l~ IFQTQKYEE! _LJ 4.000 [T6]5 IT D 4TV [i~fl ALEGQGLL60 CO I~ F 1? AEAE _L360_0 [E7 )L\A9IIq 30001 176I~PAPvTDT I2.oool LQQHILv 1.200 SAVVGj[.200 11291]F IREGA MLKC F1.70001 00 00 Table _XIX-V1-H1L'-B7-1 Omers-] 19PD1 2B IEach 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.

Stat Suseue c o jSoe] 397] SIRHHT [i00oo 61_I[QVGQVAWYI1.00 441EGRSYSTLTT 100 189 jHVSPAYEGRV lL.. 2J L~2j[TOLLVVVWLM_11SY1.000] :133 PLTTEHSG 9~.1 P] 1~d QPY9Et:GLRF LO60 0 I IMLLCLLV ]0.600] 358___ ALLFCLLVVV. 0.600 I4 75FVQNTRA 0~.500 TVREIETQTE 10.500! 292 GVRDGDTG [0.500.

FT2[LASFTGRCPA I 9A 5 9 REIETQTELL]L70400] 3241 RDSQVTVDVL 0o.40 L7o: VOYAqGGAEL IL[O0 IjJI APLSLG!_EMW 0.4 0 0 r? ILT GEGM T i 0.400.

F. LGEGAQELALL 0.400 [Tj~jKPTGNGN 0.400J F i1 LRAEGHPIDSL I040 7 MWGPEAWLLL 10400 483 NH FVQQENGTL 1 0.40 l l* rLLQDQRIWTH I LRLRVLVPPL I .401 ]4~jYSTLTTVREI .0.4001 JF342, QVDLVSASVV LO.300 2SMNGQPLTCVj]q0.307P, T, D1 DGEGAQELA!Io030 2141 RSMNGQPLTCl 0.300 IASVWVVGVIA 030 01 I lDGSVLLRNAV ,1 0.300 Table XIX-VI-HLA-87-l0mers- 191 P4DI 28 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.

[Sart] 1. uj un e][Score L1679 [ASqAEGSP ]Fo0.300] [~fl[~PAEGRVEQ L:300 UL ff AE SPA P [0 2 0 FSRSQT LO.200i Table XIX-2-H11A-B37-1 Omers- 191 P4D128 Eh peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acds, and the end position for each peptide is the start position .plus nine.

[[cixi~q~qG][.200j ET IL PLYRG L2 0.030 D 10.039] AKLLCLYRG I=.003] I[T]QDALPLR [2a002J PAC LRSG 0.001 ITable -V47-H11A-B37-10mers! 191 P4D1 28 I Each peptide is a portion of] SEQ ID NO: 15; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plusrfnin.' [start[SubsE FnjScorej 171[PRSQSE EEI200 ~Tble XIX-W7-HLA-B7-10mers.

191P4128J Each peptide is a portion of SEQ 1D NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plusnie Fs-tartl fi ubsequence I[re]I FA- SQSEPEGRS 0.030] [fRQSEEPEGR fW1o HSHTDRSQS.005] 5 TDI!PRSQSEPI 0.001 [[]PSSEEPEG. L7 P~9 Tble XIX-V9-HLA-B7-10 Orers-] 191P4D128 Each peptide Is a portion of1 SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

IEtrtL sq uen ceJ =co] 46 IWA QAGlELQ 20.000 I ii. AFRF i..9DLF MlkJ i.000 F6 jAS±SLAGTL j[ .O OO [105ERIKPLQHQGY 1000 L1 qPJWL~LLK~fI 4.000] 80K ESFTKRKKKL [00j lAF5'_S[fQGLL 4.000 I. 9 ,NPPASASLVA [L0 L64T I[AkA~SJ 0. IT FFJ[ LAGVNSI 0. 400] 114~ L iiFF LFFF I] .400 00 00 Table XIX-V9-HLA-B7-1 Omers- -19IP4D12B_ _J Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 10 amino acids, and the end position for each peptide Is the start position plus nine.

[SrtjI Ln? Suseue c L~oreI [_FPWFFIYFJIF040 1_RHQCLLLGL ][000 1.TU[ [YVAqAGLEq LO40 I[ KKFFIqQCl 0.4001I I[~2VAGTSVHHIFO 300 IFI-.994 6LLLGLKV-J[ 0.200] 8 1l SNPPASASLV F020 11281fMQAPWEGTIF .1501 FL iLGSSNPPASA1 0.150j 'p 118]LqCERGYFQGI11F-.i20JI IWFFIYYFYIL.100 j IELLGSSPPA 1.100 11_12 ]V CACFES j 0100 I -i1]T!R KKA Iol 2 jjLyLfF!YFI0.1:9P LU290jI EGYFQGFMJ 0 100 L11._AGILLRIT1F ]i 0.0901 [ilL AGB..LITFN 0.060] AGE-LLGSN 0.060 [T j1L%ELL§§[ FO.6- AQAGLLLGJ0.040 56 GSNPSCDEGY 0P030 Li 77IACFESFTKRK 110.030 Table XIX-V-HLA-B7-lomersj 191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Sube unceScore T9 ]I FLEM ESHY VA I 0.030 I] F- I -LP0.LF 020 [iij 9ERGYfqqIW L2O I F.jT5ILQqlIFM9AEW 0.0201 rK7]Es-A4HI A0-Ln GUIITFNF.] .1 2 [Ifl21LTFNFLFF T .02.01 [11]LYTNCFLFFFJ( 0.020] LIFFLP FPL .07201 F~~~11_1 KLKFI 0.1 jiOB VSOIL.0 0] IW IQLLLLKVI 0.0157 L8U I LKK jlql90'5 jF11[LLVPLQHQVS I 0.010 ~iF-8-9 -1 l0.010 L065 L VATSH 0.010 L.i LLLLLVi0.010 FIo-91 I LQHQqVN.ScDI *o5oqi 1-0Q1 GVN ScDC!L0.010oJ IFLF .1L H YCE1If 0.010! F. D26[9!!1QA V EI aoio I [T E LFGF0~ 0010 112 IFq!EMQ P~ 0.10 T~able XIX-V9-HLA-B7-1 Omers- 191 F4D12B- j Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

fft7ad I Subsequ-ec Scr~e .7 jfCCFEST 010 I F 112JLQGVNSCDCE FOO-070 L _hI FLPFPLV 0,003] F12LRWIIq~ IFM MPW 0I-03 I 40O I MEH.YYAQ ED 3 F i7 iCLSDERYF1FIaOo3 IFTable XIX-V1 0-HLA-7-] I1 0mers-191P4D128 Each peptide is a portion of SEQ ID NO: 21; each start Iposition is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[Start &!se quencel Scre FLLT -SD 0.2001 E7]KTPAGLELG .100 IGEL9TSPYIA 1 M RCLAGELGT [o Table XIX-V1 1 -HLA-B7- L 0mes11~P1B Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 10 amino acids, and the end position for each pepti'de is the start position plus nine.

F~aJ Subsequenc Score LRVMVPLPSL90.00 LiF-9]-2___ 0.99 00 00 Table XIX-V1 1 -HLA-B37- 1 0mers-191P4D12B3 Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each Ipeptide is the start position pls_--e I start Subsequence IScorel 1_T1 FQ RVM 1.00 IELLPR vMPPL -1 K M-_-LRmvmvPPLP LO.io D 71[MVPPL PSLN P 10.075 FflLLRVMVPPL]F [0.03 Tabl XI-VI2HLA.B7- I1 10mers-191P4D12B Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, Iand the end position for each peptide is the start position plus nine. [start]1 Subsequence [Icorej 6_ EPGCSYSTLJ200 ][CSVMS EPEG 1101 1..i ifGCSYSTLTT 0.200 [IJIVM§EEPEGCS F[0.030] [fEEPGCSYST 10.010] I.i7K. ILSySTLTTI J90.1.

7LMSEPEGCSY Fo.oo6 SEEPEGCSYSII j [Table XIX-V13-HLA-B7- I] 0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 27; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plu nine.

IIS.~.tII Suseque c lcorel jDVLADPQEDS 0.o150] [T]I TVDVLADPQE 110.015j Y VLAD DS .01 1 ijAjyDPQEDSG LO03 WI. VDVLADPQED R oJj-- 01 [Table XIX-VI 4-HLA-B7l0mers-1 91 P4D12BJ Each peptide is a portion of SEQ ID NO: 29; each start position is specified, the length of peptide is 10 amino acids, and the end position for each p eptide is the start position plus nine.

[LStart Subsquen [j cre 1[7 ASASLVAGTL 11 2 d00 SSN3 PPASASL '4.0 00 Ki-i [ESL VATLSV I0.600 IF71]1 NPPA-SASLV 1LO-01 !IT]IL9.SNPPASA FL1 501 F~jI] -YAGTLS I0.060 Table XX-VI -HLA-B3501 -9mers- 191P4D1 2B 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.

iftftFu en -F r I 78 AQQMVTqKYI1.00 MM I RSAAVTSEF ][1o9-ooo TbeXX-V1-HLA-B3501 -9mers-I 191P4D12BJ 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 su bsequei~ne] L ?7 K FT 81i7 P R Lg-.00-0 L5iLRKPTGNGl oo LA22IL~.~9ulYI 6000 L1j,]RLRVLVPPL L6.000I 71 J[DD EGA-EL I 6.I [135 GSFQARLR L 5.0F0 4.00; L287] GPPSV J 4.00 L~il AQADGEY LO 3.0 26 IITGIRCPAGELI 3.000J L2 I.A VTSEFHJ .0 1 I =QNLW I3.000 29 I AE LES [L--00 0 13 EAL LLLLL 3.00pj 356 D77 ILIMNHFJ 3.000]1 =7 F~Jgs§PWsvrw LD=-566 FA-II VVV-VLM SRYIIZOOO I L75IILqPs-YNW]Lo00 li7 J1. 2.000. 1 Liiilj WGPEAWL 2 000 L1.?F-PL-=H 91-~F 00K1 Ezz T' Fj Y qZR 0 zo 00 00 T able XX.Vl-HLA-B3501.9mers-1 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, tihe length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight._ StartI Subsequence Sco re LF44.]ItRSYSTLTTVI WOO0 [-419-1EGHPDSL 15800 1 t6 il TD WL R1500 L3:1 J[qSR YHRRKAI 1.5001 F.L4iw~~ II E19 I fo LIYIIL-LLLlLA F I1.0 F P M F l 7o6 6 EjlF2. TQKYEEELJ]1.900] =L6 2L Ep OK~ Li1~ LS j1.90 F-L4-1LAwi:VID _[T1.o ~~LiF 1.ooo 'U38J D[SG Q VLj[F. 000C L 2 iL IjHV 1. 0 531 EFLISSC~J FO.75 3 57 AALLF CLLV .11 0.6*00 LTable XX-V1 -HLA-B3501-9mers-1 191P4D12B 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 FL-tiI FSubsequence1 Scre 4 F J RIFEEG IO60 39 IL SYKQ. IL0.600 I Fi450]F PPVRNPLDGSQT- 0.600 F 72 T F0:600 1 GQPSN I0.500_ 19! [I ILQC 9.

[I7 Kf.-r§i 1q07 =83 jj TQKEELT =L.520 I =.400~iF1~' 1 35 I E TVVL 0.400 Trable XXV2H A-3309mers-] Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the s__tartpositonplus eight.' LPCLYRGS 2 .00 7F2GDA LPCL 0.3007O jFF-KPCL 71 09 W I4 RC-LYRGG 0.01 Z-ZLJtCLRGj Loi L able XX-W-HLA-B3501 -9mes I 91P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

F t rPi S ubs eq u-en cejScr Ii 7oDSQSEEPL=.6001 FL771 _q9EEEI j .1 50 1 Fi77KCLS~tGRI 0.030 F7 71FrIffDPRSQS 0.0201 177 IH-I T PRSq IL7T 2 [Table XX-V9-HLA-83501 -9mers-1 191 P4D12B IEach peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the I start position plus eight. '7§tartliubseuen~i cr I 0 Lj [iVP=CER=Gy1 20.000] 6- [TJITKAR=FIQCL 1I: .ooo] [if jr[LYVHHCACF 5 .OO 1OD7 1LII NPASASLV 4.00 UL11[ RGYFqa~fy ][4.00] F-F I[ _LTf NFFJ L.-=.000 IfrLl LVAGL 3.000 1FiI KLKKAFRFI 2400O JIFFIYFYI 2.000 F [hITNF FL 1FlxY-KQ. GL 1O00O F J FNFFFFL] FI.200--] 7j[ AGU=RITF [1L1.oOd PE~ILPFPL-NF ]I.00 L~i!.lLm~.IL Two0 L 11 GLLqRPLjII.' -0 00j .~LI f1YXY± f 1I791 q 00 00 Table XX-V9-HL-A-B3501.gmers.

IP4DI2B Each peptide IS a portion of SEQ 1I) NO: 19; each start. position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

I~J Suseqencej Score I..-jI SNPPA SASL 1.000 12!VVFFIYFY .000 ILLRITFNF 1 .000J 13iJ ITFNFFLFF .19 I 96 ICLLLGLL F-1 6-6 .85 1 RK tKKKAF f .600 E57]jj GFMQPW I=SA 0.500 I62t ASASLVAGT050 L6 4]...A.S-AGTLS! (561 GSSNPPASE 0. 0- SQAGLELLGS 0450 Il F LLAGLLRI 01]P 4 00 L' MES:Hy.JL0.400J 2 Z AfRFIQCLL L 300 L A1.j LLRIT 07.=300 DO MRRLAIJ 0.240 L P[ ASASLVAJ 0 .200 98 CLLLK 0.200 34I FYFYFFLE E.O~Ooi LVAGTL SijL 20=0 AGLELLGSSLl0 [297 VFFIYFYFY L o oo- 1191LCERGYFJI 0o Lvi] TLSVHHCAc 0100 ll HQGVNSCDC .0 FF IYFY F 11i. LRITFNFFL 010 LGSSNPpA P A 0 32 IYFYFYFFL 00 I26! PLVVFFIYF 0.100 ;FTT.b 1 ~eXX-V9-HLAB3501-9rers- 191 P4DI2B Each -peptide Tis a portion of FSEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end posiion for each peptide Is the ,sar poito plus eight.

[Sta Subse Serce F1- TFNFFFLFF F 0 LIo I 1447PSHAYVY H jG 0.00 109l TLSH CA~?9! [..100.

TT] FFLFFFLPF 0~j.io00 1[11I4] QGIFM~ 0100 ?74 Z41.t ACFESF1 ~o 7 HCACFEFff]F -090.

[120J FEGYFQGF 0.100-- 68! AGTLSVHHC o,0 129 1MWEGTI .100j 100__ fL] gL SHYV .090! j[.84_IL KK-Li-KK 00- 1105.! [TYRP9HC2 0.060 WIRRELLAGIL .060 SjESFTKRKKK 0.050 43ESHYVAQAG L. 050 If6 7lCACFESFTKI 0.045 [-C2 FTKRK'K-KLK 0.0p30 51 GLELLGSSN. 30~ I 90j[ KKAFRFIQCJ[02 L?.9.JLFLPFPLVJ0.2 [Tl LEMESHYVA ~2_ I16 VRPLQHQGv L 0 2 1I114JVN-Lc cER~L01 I x 1 02[ EA JT72 =~HFj 01 18 FLFLFFFLPFP 1[0.010 I 2 5_QGIFMQAAp II0.-010-1 TbeXX-V9-HLA-B3501.9mers.I I Tbl 19IP4Di2B Each peptide is a portion of SEQ I SD NO: 1; each start position is specified, the lngt of peptide is 9 amino acids, and the end Position for each peptide is the start position plus eight.

Ia r SubsequenceLco 1 97 ii CLLLGLLK 0.010J 'I1 2HKLU yRPLE1S 0010 ITable XXVOHL-30 19P4DI 2B IDNO: 21; each start position is speifid, helength of peptide Is 9 amino acids, and the end position for each peptide is the start Positionjplus eight.

OStarti Subsequen~ce Score7 FiCPAGELGTS GTSDVVV L .400 IsjfLGTSDVVA y]0.300j RCPAELGT 0.200 57~ AGELGTSDV 6 tLTSDW7I 0.020 WL~~~i~p j .ooJ IGRCPAGELG0. 1 Table XX-V11-HIA-B33501.

IF 9mers-191P4D128 FEach peptide is a portion of SEQ ID NO: 23; each start position Is *specified, the length of peptide is amino acids, and the end Position for each peptIde Is the Start Position DIus eloht- 00 00 Table XX-V1 1-HLA-B3501- 9mers-191P4D12B j.

Each peptide is a portion of SEQ ID NO: 23; each start position is Lspecified, the length of peptide is 9 amino acids, and the end position for each peptide Is the sart psition plus eight., FI. jj[Sibsequeflcell ScreI 651 FLLRYVYVPPUF 0.001 Table XX-V12-HLA-B3501- 9mers-191 P4D1 28 LEach peptide is a portion of SEQ ID NO: 25; 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- L7CLTOmmd 11-0-i- F~fl~.SEEPE CS 0.0 0.100 7T][EPEGCSYSI 0.020 LTable XX-V1 3-HLA-B3501- 9mers-1 91 P4D12B Each peptide is a portion of SEQ ID NO: 27; 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.

Liii SQVTVDVL 0100 'F Y8-L pPFR pSG [9.q jf7YIILIYDLADPQI 0.03- 91- qK F-.

I Table XX-V14-HLA-B3501- 9rers-191P41281 Each peptide is a portion of SEQ ID NO: 29; 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.

IF Susequence i~~ 4]~SV~T. F.000 I 7T] SNASL-ASL I TP q 2 0.500 ElF SSPPASA Tgso_ 1 5 ]AASLVA L.201

PA-LA

1 03 Table XXI-V1-HLA-83501- I Omers-1 91 P401 2B 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 Score F!9I[ R AKPTGNGIY 3.6.000 Table XXI-Vl-HLA-B3501- 1omers-191P4D12BI 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.

!a~iL eLun-ce 11 [Fj3 L~I 3.000 201 [5L vvv SYJ 2.000 F276 IPPPSYNWTIRL 2.000 I?]IRVSTFPAGS _F-ZOO1 FT,1_ ET-sDRvT-WL I 100 E62] _LLVVVVLM][2.000 LIN ]THLHVSF QWO [144 RVLVPPLPSL 12.0.00 [9J STLTFVREI.j[ 10-q~ [WGPEAWLLL j 2.T009j L§ iljAST.IL9PP U 9.7. RNPLDGSVLL Di2.000 24]j FLAEASVRGL .000 IIVRVDGDTL Lf.500.

F192 jSSRSFKHSRSj 1.500 [1 21_SRMNG PJ1.600 I7 kI EMWGPEAWLL TT99 [j426 ILKDNSSCSV 1 1.200 141Ij QPEESVGLRA 120 1.1.03I[ PPRNPLDGSVJ 1.200 LjI..§Y VVVGVI L9.

[Wfl[DQDEGIKQAMJ[1.200 I IT1___LT !rHiHVSFL1 [1.000 1 flj UTCSHPGL_1LI9q2i [L??zFI jPGLLJul -OK0 Fjz]II CPGELi 1F.000 VSASWW-VGV IL1999- LPY. 1[ DSGEQVGQVA 111.000 FTI 94Jj RSFHSRS AAI jI7000 K M34! I..§SMNGQPLTC ILT.00 1581 qmi QMTQY- EE J[ i o 132 GPALGQ Lj 2i 0.0001 [4091 RSQPEESVGL j 15.000] L4O7LDltRSqPEESyjI12. 000 I FT] L i 10.00o0 I F1 6]LjI HPGLLR 9!.000.J 'j 32 TRENSIRRL 1L.000 383 Q-K(EEaLTli 4500 F[2 PSVTWDTV. 4.000 KFT5EGNGIYIN ',L4.00J [T7Lf] LSEGQPPPSY~j L.0P..- :1_9 L LtKY9 7113.000 00 00 Table XXI-V1-HLA-B3501- LPmrs-l91P4D12B 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.

[ar]l _Subsequence. Sore, F375[ VIAALLFCLL F157000 I 35-JL WGVqIAL 1_1.0 ,I 352 1_ WGVIAALLF D~:00.I -L .VVIAL_ 1.T7p 1 L~zi SNEfSSRD S- .1.000 .1 [501GIYlNGRGHL]I 1.000 E,,L LLLLLASF iOq9J 579 i qffPPRNPL 1 .1.000 L 41ylWG1DAKL .'=00o [Fi 1Ij AGSFQARLRL i'1.00 GIKQAMNHFjEOO '12i[FSSRDS Q\1V 1A.000l L1_ AVSEFHL-V- LO.900i r 7]LWRYDAGEGA .00j K-~QLVA..1 0.800 1 L~9ILSCTAES~j[ .750j 1. iiDAGGQ I 0609j5 [2331 V 0QIHLY[.60 _1 F1LP GQ91-Tl 0.600 L!561[ jAALLFCLLV j.09] I 2 [HDsLKDNSS .60 L-O 30911 HSGIYVCHV I[0.50 FO 28J[ AVRGLEDQN IF 0500] 1 74 !EGSAVTW I 0.500 425 jjDSLKDNSSOS I .020 13811 DSGKQV0DLVS d-O:0:1j lj 71 JI EGQPPPSYNW0.500 [T7 GAEMWGPEA i0.0 Table XXI..VI -HLA-B3501 l0mers-191 402 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 Start, Subequnce i 339i52!9Y ]'FA0.45 0 j INPLDGSYL~LR P.

RKAQQM 91TQKY0.400.] 452][_REIETQT1ELL ]f 0.400 389 D 1LTLTR ENSI Table XXI-V2-HLA-B3501-1 j1iners-i 91P401 2B Each peptide Is a portion of SEQ ID NO: 5; 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.

Strt Sbsequence T] LGDAKLPC L F52000-] 711 LPLYGDSG1 020] ]I DAKL-PGLYREj[ 0.090 7]CLYRGDSGEQ I 0. 0151 711111 CLYRS jI01 10.00-11 Table XXI-W7-HLA-B3501 -I lomers:91P4D12Bj Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position plus nine.

[arj ~~sqecjscore FEPRQSEPEL q-i.

WLJ. EEPEGRS 1I0.200] [11-]_RSQSEEPE9R]ff. 0J HSHTDRSQ[ 75] [Table XXI--HLA-3501- K I mers-191P4012B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[stiart ISubsequence jco9e] 4i HTDPRSQsE il 0.003 [7ITDPRSQSEEP I0.001

L

1 71 71 1 PRQSEEPE9_JI aooOJ Table XXI-V9-HLA-83501- L 0mers-191P34D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

I~ .r lrE eI!~p [.7i SNPPASASL J' 5.000 I L~9 .T-'L I .6?L NI!AQT..L 5.000 I IPLIRPLQHQGVNS I 4.000 [UIaLGILLITF- 000 Li LITFNFFLJ .oooJ [2Ki I[2_ I [LU_[Y§CD .000 105LK LQQ 1.2000 L~iL.AYA5-0 -0 1.

M Ij. fLffFLPFPL j[.000 [1p 1 _L !KVPL LL99P J [1171LRITFN..l 1. T000] I ~~JI.2 TT9b- -1 0 00 00 Table XXI-V9-HLA-B3501- 10mers-191P4D1Bj Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the .start position pus. nine. _j IS~ttI .Subequece ScreJ L~i.FIYFYFYF 0~ 27 3 LVVLFFIYFYF 100 LPFLVVFF [1.000 I 86 KKLKKFRF .600-, L125L~GFMQMWJI0.5001 [T LLGLR 0.400.J II1[:19]. CERGYFOGIF 0.300' S.ASLVAGTLK][ 0.300 F~i _AFRFlqClL IL I49 1qAGLELLGSS I -Too0 l I ERGYFqGIF MJ .200 F58 S- Np-PPAsAsLVJ L[F2i [TO ,I KKAFFQCL]E.200 j [33] IYTIXLfM1I .200 F506 AGLELLGs SNJI 0200f F26 [P.LVVfflYFYJ_0.200 [T7 FjFLMEtl-IY F___2.9 [~ji.._RfIqCLLLGL COO.20 lAB jAQGLELLGS 1 0.150 [21J[FLPFPVVF 1.1001 Lf019 _Iff!YFYFYFF F- 910-0- F §Y JI f SVHCE .100 ,I LGSSNPPS _q.10f1 69 TLSVHHCAC F0.1001 451 HVAQALELJ 0.1001 16 jFNFFLFFFLPF o io I 2 NFFPEG 0j 0 AGTLSqj VHHCA I 9t92J I Tabe XX-V9-LA-B33501 loes191P4D128J Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

W AGI qlLLRITFN ~I .1 LLGSSNPPAS] _0.100J VHH cCFE 6O.166J UL2] FFIYFYFYF JI .PJOO I WLA LLRIT1L010 E87] .iI 0.080 LJ:81f IY !SYiL 0.0601 WI KKKRFIQIL.6 =2 I_ RELLAGILL 0.060 E L1 SVHHqCF_ [q05osj F~TF\~QAGELL]I .030 Ifl ASASLVAJI 0.030] IFT6Lq~FESFTK J aO-30 I II~fl~rKR~KLK I0. -03-0 _LKKFRFIQC ILQ~ ILEFEMESHYAQA IL09Pi 139D _yLEA SHYVAiLj 0.30 [111RYFqGIFMVQJ 020 E4 21FPLVVFHI 020 D_9

IJLLFFFLPFPLV

I .?9JFFLPFPLW][9020J? O 113 LqvnYsPqEGL:9[.1 I 11ii H9vysUll.916 .1 _jl]ffFFLFFFLP 10 oo T9 LLLq9LKVRPJi[.9q19.

LVAGTLS§VH- [0.0 1 [FLL4KvrPQ o Efi LL L g[ GI[ QAPEII I l979jL tK I(.00.

Table XXI-9-H11A-B33501- 10mers-191P4D12Bj Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is- 10 amino acids, and the end position for each peptide is the -start position plus nine. .102 [_GLLKVRPQH1 0.0101 Table XXIMV 0-HLA-B3501 -1 lomers-191P4D12B Each peptide Is a portion of SEQ1 ID NO: 21; each start position Ist specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

1[itg_ Susequen c c~.

$17 1OJ[GSDVMTVL IL 2.000 RCAGE L L Sry 0L.300 1 i _il o .SD[ 0.200 fl]IGETSIDV V IL.20 1 IK-EGTP 0.10-60 IT1L 2I.19oe [7]TGRPGEG 0.030 IFLTRPAGE_ LGT .9 1 1 LEE U Y Li 0.010

I

FTable XXI-Vi1-HLA-B3501- 1 Omers-1 91 P4D1 2B Each peptide Is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the sta rt p o siio n plus nine [Start L-Sbsequ ence IS.ore .1 L ]9RLRLF Y~YV_1.800_J =9 FlLVIV PPLPSLNFT0.10I 75 ]LRRV VPL 0.06 LtIIVPPLPSLNP.IL=.010.

M LRY- IL2 I WAR LLRM PI 901 00 00 Table XXI-V1 2-HLA-B3501 1~ 0mers-191P4D12B SEach peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide isl amino acids, and the end position for each peptide is the start position plus nine.

Start Subsequen c L e' L]L ECYSLI_6000 2 [tVMSEEPEGC§SILO.200 [TiSVMEEPGCI 50 J E f[ ICZ[GSYST LnT I~ 0.100 K771PE-GS L 0.001 [T TTTR E 0.001 [Table XXIMV3-LA-B33501lmers-191F4D12B Each peptide is agprtion of SEQ ID NO; 27; each str osition is specified, the length of peptide is 10 amino acids, and the end1 position for each peptide is the L-start position plus. nine.

[Sart LSubsequence j Sco re FEI DSQ VT V RVL: II 0.500 [TQEy ?Fi9EDS 0.100 f YI~~Di~oi[ .020:* '[211 QTrDVLAD]32 F0.015 [T7VDVLAPQEJKII0 2 ~IT able XXI-V4-HLA-B3501- L. 10mers-191IP4D12B Each peptide is a portion of SEQ1 ID NO: 29; each start position is spcfethe length of peptide is 10 amino acids, and the end position for each peptlde Is the start position plus nine, j [St a- Subsequ ence 1. Sce I~ASALVAGL 5.000.

F .0 1G- AS IIGTS 0- .00 MW AGSII. 0J Li7ISNPA-yI 0.200.

LIIIIJP PSASL VAGI 0_.020 j Tables XXII XLIX: 00 00 TableXXII-V1-HLA-Al-1 9mers-191P4D12B 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 437SEEPEGRSY 1107 PDGSVLR 21 305 LTTEHSGIY 21 3 TTEHSGIYV 21 159 LEEGQGLTL 20 52 GLEDQNLWH 2

HIDPRSPE

SYGLHVSPAY 1 262 GREGAMLKC 19 SPEESVGLRA 19 6 VqENGThRA 494 AKPTGNGIY 191 I GPEAWLLLL ELALLHSKY 1 2 SEGQPPPSY 1 VLDPQEDSG 1 YEEELTLTR 1 TSDVVTWL 7 AQELALLHS 17 1 DTEVKGTTS 1 SVSHPGLLQD 1

LSEGQPPPS

F RVDGDTLGF 17 38KQQMTQKY 1 f SGEQVGQVA 1 SALEEGQGLT 1 7 SDSQVVD 1 SWVVLMSRY 1 SQIELLSPGS 1 1QDAKLPCFY 1 46 MSEEPEGRS Li~ TableXXII-V2-HLA-Al- 1 I 9mers-191P4D12 1 Each peptide is a portion of SEQ ID NO: 5; 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 QDAKLPCLY I17 GQDAKLPCL E 0 TableXXII-V7-HLA-Al- 9mers-191P4D12 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

IPosi 123456789 score 3~ HIDPRSqSE 20 TableXXII-V9-HLA-A1 9mers-191P4D12 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

[os 123456789 scorel 25 FPLWFFIY 21 2 VFFIYFYFY 2 11 NSCDCE 191 FFLEMEHY 16 1 ITFNFFLFF it1 1 LWFFIYFY 1 11 SCDCERGYF 1 211FFLPFPLW 1 FLEMESHYV i 1Z 1 GLELLGSS N 1 11 CERGYQG 1 4 ELLAGILLR 1 SSNPPASAS 11 SSLVAGTSV 11 9 FRFIQCLLL 1 9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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 123456789 score SAGELGTSDV 13 SGTSDVVIhV 1 RCPAGELGT B

GRCPAGELGZ

TableXX I-V1 1 -H LA-Al 9mers-1 91P4D1 2B 00 Each peptide Is a portion of SEQ ID NO: 23; each start position is specified, CK1 the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. FP;~ 1234j 5 6789 Fscorel TabeXXIIMM2-LA-A1 cK1 Each peptide is a portion of SEQ ID NO: 25; each ri start position is specified, 00 the length of peptide is 9 amino acids, and the end c~-K1position for each peptide is the start position plus eight, Pos112456789 TableXXII,-V1-3-HL-A7I- 9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptidle is 9 amino acids, and the end position for each peptide is the start position plus eight.

[Posj 12346789 re~~ [7I]LADPQEDSG I DIJTVDVLADPQ] EJVTVDVLADP 9J IEAQVTVDVLAD 1 7 511PPASASLVA 7 D~S~PSS [FGSNP] S 1 00 00 TableXXIl-V1-HLA- A0201-9mers-191 P4D1 28 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 123456789score 53 EIETQIELL 16 IYINGGHL 16 [I GPEAWLLLL 2 PEAWLLLLL 15 LLASGR[ 15 E GELETDW I DSGEQMGQV 1 SEGAQEAl.L 15 j FQARLRLRV 151 0 RLRLRVLVP j MNGQPLTCV 15 NGQPLTCW 1 0 LLQDQRITH 1h LHVSFLAEA 15 270 CLSEGPPP 15

PLTTEHSGI

HSGIYVCHV 1 VLDPQgDSG 15 3 RAKPTGNGI 1 TableXXIll-V2-HLA- A0201-9mers- 191P4D128 Each peptide is a portion of SEQ ID NO: 5; 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.

[os 123456789 scorel 1 GQDAKLPCL 17 -CLYRGDSGEL3 KLPCLYRGD 13 4 AKLPCLYRG 1 TableXXIII-V7-HLA- A0201-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 3 HTDPRSQSE 8 F8 SQSEEePEGR lI1 SHHTDPRSQ 4 FJRSQSEEPEG 3J TableXXIII-V9-HLA- A0201-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 98 CLLLGLLKV 31 5 LLAGILLRI 29 6SLVAGILSV 29 95 FIQCLLLGL 26 39 FLEMESHYV] 21 46 AQAGLEL 21 47 VAQAGLELL] F21 91 KAFRF!QCL 21 99 LLLGLLKVR 20 101 LGLLKVRPL 19 1jjMRRELGI 18 58 SNPPASASL 18 63 SASLVAGTL 18 88 KLKKAFRFI 18 18 FLFFFLPFP 17 21 FFLPFLW 17 22 FLPFPWF 17 4 LLGSSNPPA 1 96IQCLLLGLL 17 SELLAGILLR 16 9 ILLRIIFNF 6 F-44 SHYVAQAGL 1 62 ASASLAGT 16 6 LAGILRIT 15 8 GILLRITFN TableXXIl-V9-HLA- A0201-9mers- 191P4012B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 11 LRITFNFFL j 100 LLGLLKVRP A0201-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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 re SGTSDVVTW 201 F--3 [8 LGTSDVVTV 19 [5 AGELGTSDV [6 GELGTSDW ELGTSDVVT 13 SCPAGELGTSI TableXXIII-V11-HLA- A0201-9mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 8 VMVPPL.PSL 29 5~ RLRVMVPPL 2 ARLRLRVMV 17 3 RLRLRVMVP 14 TableXXIII-V1 2-HLA- A0201-9mers- 191P4D12B 00 00 TabeXXI Il-Vi 3-H LA- A0201 -9mers- 191 P401 28 Each peptide is a portion of SEQ ID NO: 27; 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 Fscore 7 QTeXIIVV-LA A0201 -9mers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 29; 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 124679score F- ASASLVAGT 16 FL1] GSSNPPASAF 10 F-4 NPPASASLV PASAS LVAG TableXXIV-V1 -H LA- A0203-9mers- 191 P401283 N-lolResuitsFound.

[TabieXXIV-V2-HLA- A0203-9mers- 191 P4D12B -NoResultsFound._ [TabieXXIV-W-HLA- A0203-9mers- [191lP401 28 NoResultsEound. [TableXXV-V9-HLA- A0203-9mers- 1911P401283 [P os 12345789 sore] NoResultsFound.

[TableXXIV-V1 0-HLA- [A0203-9mers- S 191P4D12B

]I

TabIeXXIV-V1 1-H A- F A0203-9mers- 191P4D12B E os 124679score] IFNoResultsFound.

TableXXIV-VI 2-H LA- A0203-9mers- 191 P4D128 99124567core No~suis~ond. TabeXXIV-VI 3-H LA- A0203-9mers- 191 P40128 Po134679score NResultsFound.

TableXXIV-V14-HLA-1 A0203-9mers- 191 P40128 TableXXIV-V4-HLA- FA02 03-9mers- NoResults ound TableXXV-V1 -HLA-A03-1 9mers-191 P401 28 Each peptide is a portion of SEQ ID NO: 3; each start position Is specified, the length of peptidle is 9 amino acids, and the end position for each peptide is the start position plus eight 14 0RRRLP 2 180 VTWOEVK 451 TWG DAK 2 24] DDLF 2 F2-1 LLLAF 2 459 DLSP9G 13621 CLFV\V 19 M Fj415 EQN1 [1] 00 00 TabIeXXV-V1 -HLA-A03- 9mers-191P40128 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 123456789 I9GLEDQNLW-II 19 g QVDLVSASV 1 Isw l SWGA[191 366LMSRY 137RKAQQMTQ]19 FVENTLR][ 19 ELETSDVT 1 8 QVAWARVD 18 I1QELALLHSK 18 RVSTFPAGS 18 29HLVPSRSMN 18 SHIGREGAML LJ1 lI1RLDGPLPSG 13 TLGFPPLTT 7i SGIYVOHVSN J[ DLVSASW II GVIMALLFC I i1 j~JLLFOLLVV Lj1 365 J 8 417 GLRAEGHPD 118 4TVREIETQT 1J 491TLRAKPTGN 1 F-2] [ZI PLLGMW 17! [6LLLLLLLAS [7 F-16 191 LLLASFTG 17 158 ALEEGCLTI 17 17641 GLLMSCT 77 3511 WMGIAA 171 368 W LMSRYI 97! WLLLLLLLA 16 81 ASKYGLH 16 1971 KHS AAVT 16 224 WHLLQ 16 TableXXV-V1 -HLA-A03- 9mers-191P4D12B 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 o D ISLGAEMWGP 13 431VLGQDAKLP L E 491KLPCFYRGD 13 F SKYGLHVSP 13 F 124 EYECRVST F 13LVL 139 ALLVV 1 23AVTSEFHLV 13 210 LVPSRSMNG 1 236 ITHILHVSF 13 27NLWHIGREG [j1 120CLSEGPP( 13 3041PLTTEHSGI 3ssDSQMTV 13 391DVLDPQE 3 D DPQEDS 13 LDPQEDSGK 13 151WWVG!!M 13 371LMSRYHRRK 13 SRSYSTLTTV 113 I4GIKQAMNHF IIi1 TableXXV-V2-HLA-A03- 9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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.

[s123456789 SKLPCLYRGD

II

[11QDAKLPCLY j 1~ TableXXV-V-HLA-A3- 19mers-1 91P4D12B 1 00 00 TableXXV-V9-HLA.A03.

gmers-191P4D312B3 Each pepUde Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 sor FIGIL-LRITIN [14 26 -PLvvFFIYF IF14 F28WFYFF 1 F53 ELLGSSNPP f 14 F72SHCCE 1 F76CCEFK 1 88 LKAFI 14 [11 3VSDCR 1 [126GEQAP 4 F21] FP F1 131 [861KKKKFf

J

38 FFEEH 12 EFKKK 1 23 LFWF 11 571SPASS 1 F63SASL 7GTL 1 TLSHHAC 11 FQLLL 1 107 PLQHGVN 11, [TableXXV-VI 3-HLA-A03 [9mers-1 91 P4DI 2B Each peptide Is a portion of SEQ ID NO: 27; each start position is specified, the length of pepuide is 9 amino acids, and the end posl~on for each peptide is the start position plus I eight.

[pos 2468 76 DVADE I~ VLADPQ 1 VLDQEDS 121 00 00 TableXXV-V1 4-HLA- A03-mers-191 P4D1 28 Each peptide is a portion of SEQ ID NO: 29; 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.

os 123456789 score F -2SSNPPASAS 11 8 SASLVAGTL 11 3 SNPPASASLZ 9 9 ASLVAGTLSZ 9Z F NPPASASLVZ 8 PPASASLVAZ 8 I GSSNPPASALZ 7 6 PASASLVAGZ 7 I ASASLVAGTZ 7 TableXXVI-V1-HLA-A26- 9mers-191P4DI12B 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 42 WLGQDAKL 19 L84 184 DTEVKGTTS 19 E3 294 RVDGDTLGF 19 331 DVLDPQEDS 19 337 EDSGKQVDL 19 54 GVIAALLFC 19 365VVVLMSR 19 1 EMWGPEAWL 18 60EQVGQVAWA 18 71 DAGEGAQEL 18 145 VLVPPLPSL 18 2361 ITHILHVSF 18 237 THILHVSFL 18 313 YVCHVSNEF 18 44918 191 TTVRElETQ 1(18 VTVLGQD 17 13281VTVDVLDPQ 17 3551 VIAALLFCL 17 411 TWLGQDAK 1 57 DSGEQVGQV 16 11301 STFPAGSFQ. 16 =1 298 DTLGFPPLT 16 13271 QVTVDVLDP 16 3491 SVVVVGVIA 16 16 MTQKYEEEL 16 [450 TVREIETQT 16 1413 EESVGLRAEI 1414 ESVGLRAEG 15 14731 DQDEGIKQA 12 PEAWLLLLL 14 [14 AWLLLLLLL 14 [17 LLLLLLASF 14 VVLGQDA 4 F16ol EEGQGLTLA 14 2601 HIGREGAML 14 345J LVSASVVVV 14 367 WVLMSRYH 14 TableXXVI-V1-HLA-A26- 1 9mers-191P4D12B 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 sore 387 EEELTLTRE 14 (437 SEEPEGRSY j 141 452 REIETQTEL 14 1472 EDQDEGIKQ 14 476 EGIKQAMNH 14 484 HFVQENGTL 14 485 FVQENGTLR 17 F1 GPEAWLLLL 13 45 GQDAKLPCF 13 109 DGSVLLRNA 13 135 GSFQARLRL 13 142 RLRVLVPPL 13 146 LVPPLPSLN 13 161 EGQGLTLAA 13 222 TCWSHPGL 13 249 SVRGLEDQN 13 320 EFSSRDSQV 13 329 TVDVLDPQE 13 344 DLVSASVW 13 353 VGVIAALLF 13 393 TRENSIRRL 13 421 EGHPDSLKD 13 438 EEPEGRSYS 13 446 STLTTVREI 13 459 ELLSPGSGR 13 o501 IYINGRGHL 1 13 TableXXVI-V2-HLA-A26- 9mers191P4D128 Each peptide is a portion of SEQ ID NO: 5; 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 I GQDAKLPCL 1 2 QDAKLPCLY 11 00 00 TableXXVI-V2-HLA-A26.

9mers-1 911P40I13 Each peptide is a portion of SEQ ID NO: 5; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptidle Is the start position plus eight.

[PoI 123456789lIcr J 0A-KLPCLYR F TableXXVI-W-HLA-A26- 9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos] 1l24567891 [sr TableXXVI-V9-HLA-A26- 9mers-191P4012B3 Each peptide Is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is59 amino acids, and the end position for each peptide Is the start position plus eight.

IP-o s 12 34 5-6 78 9 96iQCLLLGLL j 14j 4 TFNFFLFFF lFi1- 151 FNFFLFFFL 1 F26 PLVVFFIYF][I) F381 FFLEMESHY 13j 931 FRFIQCLLL 1 101LGLLKVRPL]1 105IKVRPI QHQGI 13 TableXXVI-V9-HLA-A26- 9mers-1 91P4D1 2B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight.

[Pos 123456789 F27 LVFIFY 2 28 WFFiYFYF F24 13 ITFNFFLF 2 46 YVAQAGLEL 2 120 ERGYFQGIFF 19 23 LPFPLWVFF F18 FIQCGL1 ESFTKRKKKF16 F91 KAFRFIQCL ]F16 [ELLAG LLR f15 7 AGILLRITF 115] 66LAGTLSVH 12 RITFNFFLF 14 29 VFFIYFYFY 14 TableXXVI-V1 3-H L-- A26-9mers-191 P4D1 28 Each peptidle is a portion of SEQ 10 NO: 27; 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.

DI~ DVLAQD 1 DVTVD-VLAD7 I~ TVDVLADPQ 12~ [TabIeXXVI-V1 4-HLA-] A26-9mers-1 91P40128] Each peptide is a portion of SEQ ID NO: 29: 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 fsc-~ D SASLVAGTh 1 L:1 ASASLVAG D PAASVA F-

I

00 00 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 F0 Q PPPPRNPL 13 GPEAWLLL 23 1 PPSYNWTRL I 1106 NPLDGSVLL122 287 GPLPSGVRV 2

KPTGNGIYI

4I1 LPSLNPGPA 1 EPEGRSYST 1 1 MPLSLGAEM 181 8~ EMWGPEAWL [T QPPPSYNWT 1 LPSGVRVDG 1 33 EDSGKQVDL 1 RLRVLVPPL j 151 PSLNPGPAL 16 [2 TGRCPAGEL 1 TSDVVTWL 1 GEGAQELAL 115 3 PPRNPLDGS l 132 FPAGSFQAR 1 145 VLVPPLPSL 1471 VPPLPSLNP f 159 LEEGQGLTL 15 SAWLLLLLL 1

SPAPSVTWD

APSVTWDTE

21311SRSMNGQPL 351 WVGVIAAL 14 362 CLLWVWL 12 PEAWLLLL 1 1 EAWLLLLLL 1 CPAGELETS 1 42 WLGQDAKL 1 74 EGAQELALL 13 ISPAYEGRVE][ 131 1 RNPLDGSVL] 131 GSFQARLRL 131 1 QARLRLRVL 1 161 EGQGLTLAA[ 11 TableXXVII-V2-HLA- B0702-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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 L1GQDAKLPCL 13 6 LPCLYRGDSII 11 TableXXVII-V7-HLA- B0702-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 ore D PRSQSEEP 1j TableXXVII-V9-HLA- B0702-9mers-191P4DI2B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 LPFPLVVFF 21 60PPASASLVA 9 NPPASASLV 17 SYVAQAGLEL 14 AFRFIQCLL 14 RELLAGILL 12 [is FNFFLFFFL 12 22 FLPFPLWF 12 [3 YFYFYFFL 6 GSSNPPASA 7i/ 5 SNPPASASL 2 SASLVAGTL 1

FRFIQCLL

00 00 TableXXVII-V9-HLA- B0702-9mers-1 91 P4D1 2B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus I eight.

7Pos 1l23456789 -icor 1FI QCL LLG7L T 12 1 017 FGRP 12 72 RRLAI 11 13FITFNFFLFF 1 FFPPV 11 11 47VQGEL 1 81 SFKKKL 1 911 KAR DQL 1 96 ICLGL 1 1179 EEEFQI1 [129 QAP EGT 1 1 FLP0L11 [42MSYAA 1 TableXXVll-V1 0-H LA- B0702-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 21; 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 2468 [1F CAE3]S 1 [ELGSVTL2 D j 'TA'TV 1 TableXXVII-V1 1-HLA-1 130702-9mers- 191 P4D12B Each peptide Is a portion of SEQ ID NO: 23; 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., IEos 123456789] score] 1 [TabIeXXVII-V1 2-H LA- B0702-9mers- 191 P4012B Each peptide is a portion of SEQ ID NO: 25; 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.

H I 123456789soe DI EPEGCSYST 19~ STableXXVII-V13-H LA-1 B0702-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 27; 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 123-4567891 scr D] SQ V V DVLA EF EA1QVTVDD F-4 ,D1VLADPQEDs 12] TabIeXXVII-V14-HLA-1 80702-9mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 29; each start position is specified, the length of peptide Is 9 amino acd s, and the end position for each peptide is the start position plus eight.

[P PA [TableXXVIII-Vl-HLA-B08- 9mers-1 91P40128 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 337 EDSGKQVDLF 23 4791TLAPG 4777GKANF 2 493 RAGG 362 CLVVV 19 00 00 Table)(XVIII-VI-HLA-B08- 9mers-1 91RPDI 2B 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 Pos F 1234-56789 scor F1T1 G PE AWLLLL0 17 EALLLL 17 2 T GR CP AGELQ 17 F7 1] ALPF 1 LE AGEAQE 17 124 EERSF 1 145VVPPL 1 [2 PPS7-7] 17 78~1 QD9RPL7h F1 PALE G G 16 247 FAV LE 16 265 AUCS 16 267 MLCLEG 356 EALCL 374 YHRKQM 6i 439EEG YS[ I [453 ITTEL] I 7DALCYR[ 1AA5OGf [Fol PPR6L 15 231 LQQTI 15 245LESVG 15 2 60 HIREM 15 355 E9ALFL 1 1369 VLSYHRt 1410O 15ESGLE~ 113 LRAQ D 14 202 AAVT E 14 1411 AEHPDL 14h TabeXXViIl-V2-HLA- 808-9mers-1 91 P4DI 2B lEach peptide is a portion]I of SEQ ID NO: 5; 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 Epos 123456789 Iscorel F- G QDAKLPCHO F211 STableXXVIlI-W*-HLA-1 B308-9mers-1 911P4DI28 J Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

rTableXXV I I -V9-LA- 1 BOB-9mers-191P4D12Bj Each peptide Is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start posito pu eight.Id 11031LLKVR-PLQH J F*821FTKKL 88 KLKFRILi LLLVRL 9 F8 11SKRKL j KRKKKK4]I~ [86 DKAKAR12i 110 LLITNF 63 AVAGTL [1 J 91_ KAFRFIQCL ~5 TableXXVIII-V9-HLA- B08-9mers-1 91 P4D12B] Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight._ 123456789 Isor F95 FIQCLLLGLl F2MRELAG 14 26 PVFIF 1 80E SF KR K 13 E A ILI 12 F58 ISNPPASASL Zi 1 TabIeXXVlII-V1 0-H LA- B08-9mers-1 91 P4DI 28 Each peptide is a portion of SEQ ID NO: 21; 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 eigh B08-9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 aino acids, and the end position for each peptide is the start position plus eight.

[Pos 12345678 RLVVP [24 [D RLLRVMV 00 00 O0 TableXXVIII-V1 1-HLA- B08-9mers-191 P4012B Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 amino adcids, and the end position for each peptide is the start position plus eight.

[PosI 123456789 score] 1 []QARLRLRVM 191 D VMVPPLPSLI 11 TableXXVIII-V12-HLA- 808-9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 6 PEGCSYSTL 10 EPEGSYSTI 81 4 EEPEGCSYS D TableXXVIII-V1 3-HLA- 0B8-9mers-191P412B8 Each peptide is a portion of SEQ ID NO: 27; 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 Iscore 7 VLADPQEDSi 7 8 LADPQEDSG 4 SSQVTVDVLA 3 2 QVTVDVLAD 3 TableXXVIII-V14-HLA- B08-9mers-1 91P4012B Each peptide is a portion of SEQ ID NO: 29; 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 e SASLVAGTLI 17 [SNPPASASL 12 TableXXIX-V1-HLA- B1510-9mers-191P4D12B 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 237 THILHVSFL 22 208 FHLVPSRSM 20 259 WHIGREGAM 18 374 YHRRKAQQM 17 393 TRENSIRRL 17 I 36 TSDVVTVVL 161 1362 CLLWVWL II 1 135 GSFQARLRL] 151 1308 EHSGIYVCH 15 1337 EDSGKQVDL 15 100 QPPPPRNPL] 141 106 NPLDGSVLL 14] 1138 QARLRLRVLI 14 145 VLVPPLPSL 14 245 LAEASVRGL 141 277 PPSYNWTRL 14 325 DSQVTVDVL[ 14 1501 IYINGRGHL 14 I 8 EMWGPEAWL][ 13 26 TGRCPAGEL] 13 71 DAGEGAQEL[ 131 S74 EGAQELALL[ 131 1142 RLRVLVPPL ][T13 11511 PSLNPGPALI 13 1159 LEEGQGLTL 13 197 KHSRSAAVT][ 131 222TCWSHPGL][ 131 292 GVRVDGDTL 13 297 GDTLGFPPL [13 351 VWVGVIAAL 13i 356 IAALLFLL 13 1403 SHHTDPRSQ 131 404 HHTDPRSQPI 131 14101 SQPEESVGL 1 TableXXIX-V1-HLA- B1510-9mers-191P4D12B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adcids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score [419 RAEGHPDSL] 13 [9 MWGPEAWLL 1 1 GPEAWLLLL [73 GEGAQELALII1 [821 LHSKYGLHV [88 LHVSPAYEG J[ 121 los RNPLDGSVL [121 [133 PAGSFQARL 121 [2131 SRSMNGQPLJ[ 121 1382 MTQKYEEEL 12 384 QKYEEELTL F 2] 14221 GHPDSLKDN 121 452 REIETQTEL 12 453 EIETQTELL 12 484 HFVQENGTL 12 10 WGPEAWLLL 11 12 PEAWLLLLL 11 13 EAWLLLL 11 42 WLGQDAKL 11 0 ALLHSKYGL 11 1157 PALEEGQGL j 1I 1223 CWSHPGLL ii 1226 SHPGLLQDQII ii [24J LHVSFLAEA (I 1315 CHVSNEFSS Ii 1352 WGVIAALL j[ III 355 VIMLLFCL Fi0 1401 LHSHHTDPR ii 1440 PEGRSYSTLj[11 [4831 NHFVQENGTII I 1232 QDQRITHIL 101 1236 ITHILHVSF 1250 VRGLEDQNLI 101 1260 HIGREGAMLj 00 00 TableXXIX-V1-HLA-

I

B31510-9mers-1 91P41 281 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.

263 REGAMLK-CL 10 281 NTL L 1 3631]LVVVM 1 4] QDG1QA 0 ITableXXIX-V2-HLA- B1510-9mers- 191 P4012B 1 Each peptide is a portion' of SEQ I0 NO: 5; 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.

[TableXXIX-W-HLA- 83151 0-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide Is the start position plus eight Po-sl F123456789- (cr TableXXI-9HA 8151 0-9mers- 191 P4D1 28 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of Peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Posi 123456789 scr F74 H A F 16 F46 YVAQ GLEL 14 F11 LGLLKVRPL 13j 32l IYFYFYFFLI[ 12 58 SNPPASASL 12 F631 FSASLVAGT-L] 12 96 Q-CLLLG-LL 12 F- RRLLGI 11 19 EEFPFL 1 F9j FL F 7LF 11 F 21 L-I LVF LiiEq F-1 KARFQC 1[ 0 U1 TNFFL1 TableXXiX-V1 0-1-LA- B1510-9mers- 191 P4D128 Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 9 amnino acids, and the end position for each peptide is the start position plus eight Pos 123456789scr F-9 GTSDV-VTVV 6 EL SVV E GSW11 F-8 L4SVTVf~ ElRCAEL EN PGEGS i~ AGLGSDV 1 TabeXXIX-V1 1-H LA- 8151 0-9mers- 191P4D1I28 Each peptide is a portion of SEQ ID NO: 23; 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 1234 5 6789 Fso-~ D MPLS 14] jj1 LVMPL 1 TableXXIX-V1 2-HLA- 81510-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 25; 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.

TableXXIX-VI 3-H LA- 6151 0-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 27; 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 124567-897 D IQVTVDVLA0 3 D VLADPQED)S LA~ SQTVVL j j VDVLADPQ E TableXXIX-V1 4-HLA- 6151 0-9mers- 191P4D128 Each peptide is a portion of SEQ ID NO: 29; each 00 00 start position is specified, the length of peptide is 9 amino acids, and the end position for each pep de is the start position plus eight.

[Pos 123456789 Iscore [3 SNPPASASL -71]

[SASLVAGTLI]

TabieXXX-V1 -HLA-B2705- Smers-19114D12B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 393 TRENSIRRL 26 250VRGLEDNL 1251 452REIETQTEL 22 135 GSFQARLRL 21 213 SRSMNGQPL 20 [77j RKAQQMTQK j19 42WLGQDAKL 18 9RVEQPPPPR 13 2621 GREGAMLKC Th 1351] VVGVIAAL 111 I376 1RRAQQMTQ1I 181 j3991 RRLHSHHTD 11 11 [g1 E2AWLLLLLLL IIi 17 LLLLASF 17] 11051 RNPLDGSVL 111 11421 RLRVLVPPL ]I17 201RSMAVTSEF 2061 SEFHLVPSR] 171~ 1294 RVDGDTLGF 11 17 12971 GDTLGFPPL 11 17 141911 RAEGHPDSL 1 17 14981 GNGIYINGR I~i F41 TWLGQDAK 16 S451 GQDAKLPCF 16I F8 ALLHSKYGL 1 96GRVEQPPPP 106] NPLDGSVLL 1 11451 VLVPPLPSL 16I RITHILHV16 TabieXXX-V1 -HLA-B2705- 9mers-191P4D12B 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 [243 SFLAEASVRII 1[ 6 261 IGREGAMLK 196 [293 VRVDGDTLG L16 301GFPPLTTEH [jjN 337 EDSGKQVDL LJ 384 QKYEEELTLD 4761 EGIKAMNH [3 1477 GIKQAMNHF 71 E HFVQENGTL ]I f lii GPEAWLLLL 13 0LLLASFTGR 1 [61 QVQAWA 31 I7111 DAGEGAQEL IIis 7 EGAQELALL 75 GAQELALLH 15 7 QELALLHSK 15 1 PLDGSVLLR 1-5 1 8PAGSFQARL 15 19 5ARLRLRVLV F 7 141 LRLRVLVPP 15 1 KGTRSFI 15 189 GTSSRSFK 1 151 227HPGLLQDQRi 15) 271THILHVSFL I 123REGAMLKCL][ 151 281TRLDGPLPSI f 71 1(31 LDPQEDSGK][ 7j1 [365 VVVVLMSR][ 15j 3921 LTRENSIRR 4661 GRAEEEEDQ[ 15 [4921 LRAKPTGNG~J 15 [j A IYINGRGHL ]ER IJ EMhrWGPEAWLL FI 11 EAWLLLLLL LZ1 TabIeXXX-V1 -HLA-B2705- 9mers-191 P4D1 2B 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 [sce 27 GRCPAGELE[ 14j 71GEGAQELAL 1041 PRNPLDGSV t 14 [11411 LRNAVQADE [14 1201 ADEGEYECR 1 4 [13LRVLVPPLP[ 14 [151 PSLNPGPAL[ 14 157PALEEGQGL 14 [15 LEEGQGLTL[ 141 861 EVKGTTSSR 11 SRSFKHSRS LiI 199 SRSAAVTSE 23 ITHILHVSF 127PPSYNWTRL .I 1286 DGPLPSGVR 1 1292 IGVRVDGDTL J[ 141 13131 YVCHVSNEF[14 132311 SRDSQVTVD 14 3681 WLMSRYHR[14 F451 HRRKAQQET1 378 KAQQMTQKY j[14

YEEELTLTR

[408 PRSQPEESV 1 [410SQPEESVGL 711! 48 LRAEGHPDS 14 40AEGHPDSLK 1 [4441 SYSTLTTVR 1114~ 1459) ELLSPGSGRI 141 lE1 MPLSLGAEM I13 S121 PEAWLLLLL 11131 S261 TGRCPAGEL 13~ 36 s(TsDVVL [1 131 S781 ELALLH SKY 1113] S861 YGLHVSPAY 131 10 QPPPPRNPL Li1 12 EYECRVSTF L1 12 VSTFPAGSF 13 00 00 TabieXXX-V-HLA-B2705 9mers-191P4D12B 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- 123456789 1 13 FPAGSFQAR(( 13 1138 QARLRLRVL 13 202 AAVTSEFHL 208 FHLVPSRSM 13 129QPLTCWSH 113 T WSHPGL 13 23 LQDQRII 13 SGLEDQNLWH 1 SEGQPPPSY] 13 PPPSYNWTR 1 P1T1 HVSNEFSSR 13 WGVIAALL 13 I VGVIAALLF 13 3 IMLLFCLL (13 F WWLMSRY 1 38MTQKYEEEL (391 TLTREN SIR i 3 [~JRENSIRRLH ]I9 F44I0 sHHIT 13 1111 QPEESVGLR 13 481KDNSSOSVM [Th PEGRSYSTL][1 FVQENGTLR[ 13 [JQENGTLRAK [71 [101 WGPEAWLLL 12 47DAKLPCFYR LYRGDGQ 12 6ARVOAGEGA 12 127 CRVSTFPAG 1 4 AGSFQARLR 1 192 SSRSFKHSR 1 22 PGQDQRI 245LAEASVRGL 121 255DQNLWHiGR [259 WHIGREGAM 1Ej HIGREGAML i TabieXXX-V1-HLA-B2705- 9mers-1191P4D12B 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 [os 123456789 score] 281 NWTRLDGPL 1 308 EHSGIYVCH Ii 3-25 DSQVVDVL fj1J 55 VIMLLFCL 1 F33LLVWVLM 7i SVLMSRYHRR Li1 370LMSRYHRRK [32SRYHRRKAQ[ i (396 NSIRRLHSH

I

435VMSEEPEGR 14511 VREIETQTE EI1 471 EEDQDEGIK 2 474 QDEGIKQAM 7 RAKPTGNGI 1 491AKPTGNGIY TabieXXX-V2-HLA- B2705-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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. Ps123456789 soe 1 GQDAKLPCLtII SDAKLPCLYR Li1 F-11 16 [~JAKPCYRG E8 TableXXX-V7-HLA- B2705-9mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position pius eight.

[PoF 123456789 s D PRSQSEEPE 1 L SQSEEPEGR 12

RSSEEPEG

TabieXXX-V9-HLA- B2705-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position pius eight.

Pos 123456789 sco 2 RRELLAGIL 27 S931 FRFICLLLI 241 S1 1I LRITFNFFL 2 120ERFGF2 [I MRRELLAGI 2 77ACFESFTKR 2 87KKLKKAFRF 2 RELLAGILL 18 SELLAGILLR 18 SKRKKKLKKA 18 8RKKKLKKAF 18 91 KAFRFIQCL 18 LIi AGILLRITF 17Z 23 LPFPLWVFF II17 83TKRKKKLKK] 171 99LLLGLLKVR 1 171 [I ILLRITFNF 1 161 [0ESFTKRKKK] ]j6 66KKKLKKAFR][ j.6l [11ITFNFFLFF jI SHYVAQAGL 81 SFTKRKKKL 151 97 QCLLLGLLK 101 LGLLVRPLI IGVNSCDCERI 151 121 RGYFQGFM 00 00 TableXXX-V9-HLA- 82705-9mers- 191 P4D1 28 Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

1Pos1 123456789 Iscore F12 RlTFN FFL-F 14 1 5 F NF FL FF F-L 1 F 1 91 L FF FLP FPL 14 F22 FLFPLF 14 28 F FI-lY FY 14 32 IYFYFY-FFLI 14 F37 YFFLEMES 14 46 YVQGLL1 F58 S NPPA-SASL 14E F63 SALAT 14 92AFR FI QCHLL 1 4 9-61 IQC LL LGL DF1 4 I71 LLGILR 13 [1-71 FFF F FL P F 13 27 LWFIF 17 [31LFIYFYfYF 13 j FFYFFLEM 13 F47VAQAGLELL 1 66LVGTLVH 13 76E9 EST 13, F79 FESTKKK 13 F9 5 FiQCLF 13] 122GYQGFM 1 [TableXXX-V1 1-HLA- 82705-9mers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 23; 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. [os 1 23-456789 IsEl 5 RLRVMVP-PLI 16 EFVM-VPPLP-SL 1 DLIRRVMP 14 E9 LMPL7P 13 LE QALLV 11

DLRLRVMV

TabIeXXX-V1 2-HLA-1 B2705-9mers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptidle is the start position plus eight.

os 124578 score :D EG6 YTL 1 ::SEEPEGCSY1 281 GCSYSTLTT] ZI6 :91 =S6T~ TableXXX-VI 3-HIA- B2705-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 27; 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.

[gs 1l23456 78 9-1 jjEAPFQEDSGK 16 TableXXX-V14-HLA- B32705-9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 29; each start position is specified, the length of peptidle is 9 amino acids, and the end position for each peptide is the start position plus eight.

[Posij 123456789 Esog D~ SALVGT 14 D GSSN PPASAE: (TaIeXXXI-V1-HLA- B32709-9mers-1 911P40128I 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 peptidle is the start position plus eight.

Po0s 1234567891scr 139 ARRLRVL 22 250 1VRGLEDQNL I1 211 F39TRENSIRRL[21 2SRSMNGQPL 2341 QRITHILHV 104 PRNPLDGSV 19 408] PRSQPEESV 18 135 GSFQARLRLF 17 142 RLRVLVPPL 16 287 =GPLPSGVRV F16 399 RRLHSHHFD 1 00 00 TableXXXI-VI-HLA- B2709-9mers-191P4DI2B1 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 pepbde is the start position plus eight s 123456789se 9 GRVEQPPPP 1 F RNPLDGSVL i1 f~jGDTLGFPPL 1 5 RSYSTLTV I 4 REIETQTEL 1 11 GPEAWLLL 1 [jjAWLLLLLLL 1ZIGRCPAGELE[ 14 f GEGAQELAL [4 j ALLHSKYGL FT1' 22GREGAMLKC 14' 23REGAMLKCL [14 1GVRVGDTL 14 292 RVDGDTLGF 14 lICLLVVVVL It4 lZ1RRKAQQMTQI i 1 RAEGH1POSL11J SGRSYSTLTT I 4 GELETSDW lii [LETSDVTV ]l13 [i NPLDGSVLL1 1 CRVSTFPAG .i 941 LRLRVLVPP h (1 VLVPPLPSL 1il 1f53 PSLNPGPAL [13 1 TRLDGPLPS If13 324 1RDSQVTVDV [Th I3QKYEEELTL 1T 46GRAEEEEDQ 13 SRAKPTGNGI [J*1 fJMWGPEAWLL( i21 w VVLGQAKLII21 [lGQDAKLPCF 112 6ARVDAGEGAI[12 Ii1i1 GSVLLRNAV 112 1 PAGSFQARL EZ2 14 LRVLVPPLP [12 TableXXXI-HLA- B2709-9mers-1 91 P40128 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 i 17 PALEEGQGLI[ 121 1 AEGSPAPSV 12 200 RSAAVTSEF 20 AVTSEFHL F1 2 TCWSHPGL] 22CSHPGLL 2 237THILHVSFL fJSRDSQVTVD [WGVIAALL 71 2 ALLFCLLV 1 358ALLFCLLVV 121 361 FCLLFVVV 1 372SRYHRRKAQ 1 D501 IYINGRGHL 11II IN MPLSLGAEM E l 1WGPEAWLLL 11 12 PEAWLLLLL 1 13 EAWLLLLLL 11 2TGRCPAGEL 11T 36TSDVVTVVL 1 711 DAGEGAQEL 1]] 10 QPPPPRNPL [j1 115 LEEGQGLTL If1i 18 KGTTSSRSF] iii 191SRSFKHSRS [jj 19SRSAAVTSE jj1 23AVTSEFHLV 11 28PGLLQDQRI 11 231QDQRITHIL 1jj 25LAEASVRGL II1M 27PPSYNWTRL j-1 21NWTRLDGPLI 111 123VRVDGDTLG II1i SDSQVTVDVL l1l 37EDSGKQ VOL 11 l VDLVSASW] ill SDLVSASWV IIIvi TableXXXI-V1-HLA- 82709-9mers-1 91 P40128 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.

Pos2456789 o 348 ASWVVGVI 11 351 WVGVIAAI. 11 SVGVIMALLF ill 35 IAALLFCLL 1 j 359LLFCLLV 11 1LLW VLM 38IRRLHSHHT ILAI 4101 SQPEESVGL li1i 1181 LRAEGHPDS 11 I 148KDNSSCSVM II l STLTTVREI lil 47GIKQAMNHF 11 4 HFVQENGTL IJIP 42LRAKPTGNG 11 45KPTGNGIYI 11 17 LLLLASF I57 1DSGEQVGQVI 7 EGAQLALLI 101 14 LRNAVQADE 1129VSTFPAGSF L41I 131FQARLRLRV] 101 38QARLRLRVL] 11 [81FHLVPSRSM F ITHILHVSF 1 42 VSFLAEASV 1 26 -IGREGAML [45EFSSRDSQV 1l SLVSASVVW 1 U11 SAS D§GV j 1371 VIAALLFCL Ii Th6 30LFLLW V J QYHRRKAQQM 135 RRKAQQMT ~i MTQKYEEEL 30LTLTRENSI 40PEGRSYSTL 00 TableXXXI-V1 -HLA- B2709-9mers-1 91 P4D1 28 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 7Pos F 1234-56-789- [4511 VREI1ETQTE l 453EIETQTELLI 10 TableXXXl-V2-HLA- B2709-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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 13579 (TableXXXI-V7-HLA- B2709-9mers- 191P4012B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

FE 6PRSQsE E 10~ TableXXXI-V9-HLA- 82709-9mers- 1911P4D1283 Each peptide is a portion of SEQ 10 NO: 19; each start position is specified, the length of peptide is 9 aino acids, and the end position for each peptide is the start position plus eight.

IPos 124578 ore TabIeXXXI-V9-H-LA- B2709-9mers- 19 lP4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

FPo 1-23-456789 sor DIRRELLAGIL ,r F93 FRFIQCLLLI23 01MRRELLAGII1 106 VPQQV1 [871KKLKKAFRF L 91 KAFRFIQCLI 14 [1211RGYFQGIFMj 1-4 E1 RITFNFFLF 13 232PPVF [jjFNFFLFFFL1] h LFF1FP [2 84 RKKLKA 12 TableXXXI-V1O-HLA- B2709-9mers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eighL Posl 1123456 789 (cr 1 GRPAGLG14 6 GELGTsDW~ 1-i3 8 LGSDT 1 3 TableXXXI-VIO-HLA- B2709-9mers- 19IP4019R Each peptide Is a portion of SEQ ID NO: 21; 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 DGT-SDVVTVVr2 DAGELGTsDV E:j TableXXXI-VII1-HLA- 832709-9mers- 191 P401 28 Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 9 Lamino acids, and the end is the start position plus eight.

F12] RRRM 22 114] RL V V P 163 F-8] VVP 13 !I1 MVP E9L1 ,DRVVPPD 1 ITableXXXI-V12-HLA- B32709-9mers- 191P4D128 Each peptide is a portioni of SEQ ID NO: 25; 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.

[Pos 123456789 EqCSYSTLTTV F11 TableXXXI-V1 3-lILA-1 B2709-9mers- 191P4012B J 00 00 Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 2 QVTVDVLAD 4

VDVLADPQE

6 DVLADPQED 3 1l] SQVTVDVLA 2 F-3J VTVDVLADP 1 4 TVDVLADPQj 1 8 LADPQEDSGI 1 9 SIjADPQEDSGKJ 1 TableXXXI-V1 4-HLA- B2709-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 29; 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 3I SNPPASASL 1 8 SASLVAGTL ii 4 NPPASASL D9 TableXXXIl-V1-HLA- B4402-9mers-191P4Dl2B 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 7 AEMWGPEAW 27 4371 SEEPEGRSYI 25 12 PEAWLLLLL Il23 59 GEQVGQVAW[ 23 73 GEGAQELAL 23 159 LEEGQGLTL 23 263 REGAMLKCL 23 452 REIETQTEL 23 ITableXXXII-V1-HLA- 4402-9mers-191P4D12B 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 272 SEGQPPPSY 22 440 PEGRSYSTL 22 g LEDQNLWHI 21 470 EEEDQDEGI 21 14 AWLLLLLLL 18 413 EESVGLRAE 17 13 EAWLLLLLL 16 100 QPPPPRNPL 16 351 VVVGVIAAL 16 388 EELTLTREN 16 9 MWGPEAWLL 15 106 NPLDGSVLL 15 124 EYECRVSTF 15 138 QARLRLRVL 15 237 THILHVSFL 5i 246 AEASVRGLE 15 337 EDSGKQVDL 15 393 TRENSIRRL 15 453 EIETQTELL 5 487 QENGTLRAK 15 494 AKPTGNGIY 15 1501 IYINGRGHL 15 36 TSDVVTVVL 14 74 EGAQELALL 14] 78 ELALLHSKY 4 80 ALLHSKYGL 14 98 VEQPPPPRN 14 135 GSFQARLRL 14 145 VLVPPLPSL 14 151 PSLNPGPAL 141 160 EEGQGLTLA 14 173 AEGSPAPSV 14 202 AAVTSEFHL 14 232 QDQRITHIL 14 274 GQPPPSYNW 14 294 RVDGDTLGF 14 TableXXXII-V1-HLA- B4402-9mers-1 91 P4D1 2B 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 307 TEHSGIYVC [14 319 NEFSSRDSQ 14 362 CLLVWVVVL 1 387 EEELTLTRE 1 394 RENSIRRLH 14 420 AEGHPDSLK 1 438 EEPEGRSYS 14 PLSLGAEMW 13 8EMWGPEAWL 13 10 WGPEAWLLL 13 11 GPEAWLLLL 13 17 LLLLLLASF 13 LETSDVVTV 13 42 WLGQDAKL 13 77 QELALLHSK 13 86 YGLHVSPAY 13 105 RNPLDGSVL 13 117 AVQADEGEY 13 75 GSPAPSVTW 13 188 KGTTSSRSF 13 213 SRSMNGQPL 13 231 LQDQRITHI 13 251 RGLEDQNLW 13 348 ASWVVGVI 13 35 WGVIAALL 13 353 VGVIAALLF 13 356 IAALLFCLL 13 378KAQQMTQKY 13 38 YEEELTLTR 13 410 SQPEESVGL 13 446 STLTVREl 13 458 TELLSPGSG 13 468AEEEEDQDE 13 471 EEDQDEGIK 13 TableXXXII-V2-HLA- B4402-9mers- 191P4D12B 00 Each peptide is a portion of SEQ ID NO: 5; 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] F 2-345678-9 1 GQ DA KLP CL1 Li? 2 Q DA K-LPC LY 12 ]4F PCLYRG L FTTab -eXXXII-V9-

HLA-

FB4402-9mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peplide is 9 amino acids, and the end position for each peptidle is the start position plus eight.

Pos 1 123456789 scoe F J I LL R ITFNF 14j4 MI FLRIT FN FFLD 1 221 FPFLW 14J RKKKL-KK-AF 1 f93 FIQL 1 14 1 01 L G L LK-VRPL 14~ F 15 FNFFLFFFL 13 F17 FFLFFFLPF 1 19 LFFLFP 1 271 LWVFFIYFY 13 28 FFIFYF 13 291 VFFIYFYFY 13 FFIYFYFYF F1-3 F79FEFTRK 13 87 KLKAFR 13 961 IQCLLLGLL 13 11 NSCDCERG-YF13 11-6 SCCEGY 13 126 GIMQAAP 132 2FPLWFFIY 1 F47VAQAGLELL 1 5FICLLLG 12 [14 TFNFFLFFF r711 24F PFPLVVFFII 1iiL 31 FIYFYFYFF F11 38 LMESHY 11 E44 ISHYVAGL Dlj TableXXXI I-V9-H LA- B34402-9mers- 191P4D12B Each peptidle is a portion Iof SEQ ID NO: 19; each start position is specified, the length of pepticde Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 scr 461 Y VAQAGLEL D =1l 774 HHACEF 11 88 KLKAR I TableXXXII-V9-HLA- B34402-9mers- 191 P4D12B Each peptidle is a portion of SEQ ID NO: 19; each start position is specified, the length of peptidle is 9 amino acids, and the end position for each peptidle Is the start position plus eight.

FPo sI 2468 12 ELA IL 2 F GILRTF2 11-9 CEG F 20 F23 LFLF F91 KAR F 17] F13IFFLF 1 F5-8]PASS 15 F63 FLV1T 15 I 811 FKKK s 92j AFRFqc-L-L L-:15j TabIeXXXII-V1 1.HLA- B34402-Smers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide Is 9 aino acIds, and the end position for each peptide is the start position plus eight.

Pos 123456789 sore LD] MvPPLP~sLNF- TableXXXII-V1 2-H LA- B4402-9mers- 191P4Dl2B- 00 00 Each peptide is a portion of SEQ ID NO: 25; 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 Iscore 73 SEEPEGCSY 24 76 PEGCSYSTL :[EEPEGCSYS 13 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 71] DAGEGAQEL 2 245 LAEASVRGL][-23 1287 GPLPSGVRV [23 i34ZI SASVVVGV Lz 2l [493 RKPTGNGI

KPTGNGIYI

106 NPLDGSVLL QA2LRLRVL L 357 MLLFCLLV 19 11571 PALEEGQGLI jj1 S11I GPEAWLLLL I i9 F13 EAWLLLLLL F 191 2021VTSEFH

L

228 PGLLQDQRI (3561 IAALLFCLL Lf1 110 QPPPPRNPL 181 (27NGQPLTCW[ 18 (27PPSYNWTRLI Lip' II1DPQEDSGKQ I 8 345 LVSASV 18 419 RAEGHPDSL 1 ETSDVVTW F17 9PAYEGRVEQ IZ Z7 13 PAGSFQARL fjf fASVVWGVI 4RSYSTLTrV 46STLTTVREI 10 WGPEAWLLL 3GELETSDWI 6 [121 DEEYCV i [21911 QPLTCWS1-I]16 129LPSGVRVDG][i6( 135DSQVTVDVL][16 [1VDLVSASW][16 1 DLVSASVW] 16 39ILLCLWV J76T TableXXXIIII-V1-LA- B5101-9mers-191P4D1 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 scre] 36 0 LFCLLWW 13621 CLLVWL 61 (3901 LTLTRENSI D 16 LETSDVTV (65 VAWARVDAG[ 151 F79 LALHSKYG 1 151 [148 PPLPSLNPG 12311 LQDQRITHI] 151 1276 1PPPSYNWTR][ i5 338 DSGKQVDLV[ 13581 ALLFCLLW 151 lI1QKYEEELTh f's (407 DPRSQPEES][ 1411 IQPEESVGLR1[ 22LASFTGRCP 26TGRCPAGEL j 29 PAGELETS 31 AGEETSDV4 47 DAKLPCFYR 14 75~ GAQELALLH 1 141 [2LHSKYGLHV j( 141 1721 TAEGSPAPS 1 11761 SPAPSVTWDI 14 253LEDQNLWHI 286 DGPLPSGVR 1 ((FPPLTTEHS FT4 jPPLTTEHSG [7i MPLSLGAEM 13 3PAGELETSD 1113/ 3TSDVVTL 13 5LPCFYRGDSi 131 f31 EGAQELALLI 131 9VSPAYEGRV1 131 302 PPPRNPLDG 13 [1471_VPPLPSLNP L1 TableXXXII-V14-HLA- 84402-9mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 29; 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 Isi123456789scr SSNPPASASL 15 LI 81SASLVAGTL 15 ,E21 SNPPASASI Li7 -TableXXXIIII-V-HLA- LB5101-9mers-1 91P4D1 2B 00 00 TableXXXIlII-V1 -H LA- 85101 -9mers-1 91 P4D1 28 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.

[Pos[123456789 sor 1501 LPSLNPGPA -1-3' 177 PAPSVTWD-T]13 178APVTT 13 211 VPSRSMNGQ 1 275 QPPPSYNWT13j 300 LgFPPLTTE [13 322 SRDSQvTV 1 37 KQM TQKY 1 4 78 1KQ A MN HF V 13 r42 WLGQDAKL 12 ~YRGDSGEQvI 12 F86 YGHVP 12 101] PPPPRNPLD 12 109 DGSVLRNA] 12 119 QADEG EYEC 12 154 NPGPALEEG 12 1i59 LEEGQGLTL F12 167 LAASCTAEG 12 [168 AASCTAEGS 12 234 QRITHILHV F12 2-65 ALK C LS E 12 309 HSGIYVCHV 12I 339 SGKQVDLVS 12 46-7AEEEQD12 480 _QAMNH FVQE 121 ,F 1LGAEMWGP 11 58 SGEQVGQVA 1 F67 WARVD AGEG 11 103 PP RNPLLDGS 1 116 NAVQADEGE 11 137 FQ ARLRLR 1 139 ARLRLRVLVF11 201 SAAVTSEFH 11 216 MNGQPLTCV F11 247l FEASVRGLED 1 1111 1 TableXXXIlIII-V1 -HLA- B5101-9mers-191 P4DI2Bj Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide Is 9 amino adds, and the end position for each peptide is the start position pius eight 9os 123456789 score 285 LDGPLPSGV 11 296 DGDGpp 1 304 PLTTE HSGI 1 3-06 TTEHSGIYV- 11 310 SGIYVC HVS 1 324 RS VV 335PQDSKQ 1 35 1 W GIAL 3931 TRENSIRRL I1 427 LDSCV1 470 EEEQDEGi11 502 YINGRHV 1 TableXXXIIII-W7-HLA- 85101 -9mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position Is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position pius eight.

Pos3456789 f ableXXXIII-V9-HLA 85101-9ers- 191P4D128 Each peptide Is. a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos1E 123456789 score 59 NPPASASLV 6SASLVAGTL E2 101l LGLLKVRPL 47 VA(aGlL 9KAFRFIQCLI

J

LLAGILLRI E 21 FFPFPW iA 23A LPFPLWVflEF 25 FPLWFFIY 16 24PFPLF VFIF 10-O7 RPLQHQGVN jj~ fIVRRELLAGI j :LA-GNLLRIT j 60 PPSALVA fiji4 61 PASASLVAG 14j 67 VAGTLSVHHEE 98 CLLLGLLK 7 LI1CERGYFQGIfl 13 9QAGLELLGS F12 6CACFESFTK 12~ 20O FFFLPFPLVE:f 50O AGLELLGSS fnj] 121 RGYFQGIFM Tab~eXXXIIII-W7-HIA- B5101-9mers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

P34679 ce 00 00 TableXXXIIII-V1O-HLA- 851 O1-9mers- 191P4D12B Each peptide is a portion of SEQ ID NO; 21; 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. [PLGTsDVVT 21J-678f GTDVV 217 79LGSD 1 EFPAELTS 1 1IAGL3]D 1 F1JPAE TSL9 TableXXXIIII-V1 3-H LA- 85101 -9mers- 191 P40128 Each peptide is a portion of SEQ ID NO: 27; 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 scr

::]IADDS-G

ADQE ~j 213 VTVDL=5 TableXXXIV-V1-HLA-Al l0mers-191P4D128 Each peptlde 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 ~ER 36 KS D-VVT VL G][7 71 QELALLHSKY[ I7 3061 TTEHSGlYVC[ 17! 377 RKEQ2KY 411]QESGR 7 4 f DAPGSY 7 304 PTESI 6 32 VDQDGK[1 365VVLSR] 385KYELLT L__ .4571 ILSGS 6 85 LH PAI S [116NVAEE T [251 TEFLVSR LTableXXXI V.V2-H LA-Al-] l0mers-191P4D12Bj Each peptide is a portion of SEQ ID NO: 5; 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.

[P0s 123456789 l[IJ GQDAKLPCRYL [TableXXXI V-W-H LA-Al l0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

[Posl 1234567890 I~eR

[~DPRQE

TableXXXIIII-VI 2-HLA- 851 01-9mers- 191P4D128 Each peptide Isea portion of SEQ ID NO: 25; 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 [123456789scr F~ CSSLT 17 F EPEGSYST=1 PE GCSST .8 TableXXIV-V1 -HLA-A1 -1 l0mers-191P4D12B 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.

F1RS] 124680 sc-ore 271 LZEGQPPPSY -3 45GDAKLPCFYLZ 405 HDPRS QPEE F-2 493 RKPTGNGIY [-20 158 kLEEGQGLTL i 11GPEAW LLLLL F-1 72 1AGE GAQELAL [j8 E10 PLDGSVLLRN F-1 I3iE!ETQTELLS L1Z8 00 00 TableXXXIV-V9-H 'AAl l0mers-191P4D18 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 scor r281 W FFIYFYFY [F179 24 1PFPLW\FFIY1. 8 RE2 A ILL] 1 [37_1YFFLEMESHY][ 77 [_26 PLV F -YF 1 114 1\VNSCDCERGY 16~ 6-82 FTKRKKKLKKj 15 F391 FLEMESHjYVA IF13 116]jSCDCERGYFQ 1 118DCRYGI1 331 YFYFYFFLEM I 11 [41 EMEHYAQ 1] 551F 1z SNP1SS I9F RFIQLLL 102 96 FIQC LLLGK [j TabIeXX(XIV-V1 0-H LA-A1- 10mers-191p04D12B Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 10 ami no acids, and the end position for each peptde I the start position plus nine.

P-osli 12345 67890 lscorel Each peptide is a portion of SEQ ID NO: 23; each start position Is specified, the length of peptide is aino acids, and the end position for each peptide Is the start position pius nine.

fEos 1234567890 scre F-1 61 MPEP I=i TabIeX)(XIV-V1 2-H LA-Al l0mers-191 P401 28 Each peptide is a portion of SEQ ID NO: 25; each start position Is specified, the length of peptide is 10 amino acids, and the end position for each peptide Is the start position pus nine.

E9os 124680score [D MSEEIPEGS 19f TableXXXIV-V1 3-H LA-Al-I t 0mers-191P34D128 Each peptide is a portion of SEQ ID NO: 27; 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, IEos 1123567890 scoref D LADPQEDSGK [74] D1 TXVDVLADPQE F10 DNSV=TVDV=LAD 21VVDVLADPIQ 7 rTabIeXIV-V1 4-H LA-Al-j 10mers-191P4D12B Each peptide is a portion of SEQ ID NO: 29; 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.

Po1346790sc r SLVGTLV 1 [DNASAS-LVA1121 [TableXX(XV-V1

-HLA-

A0201-10mers-191 P4D1 2B 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.

E~ol 1234567890 244 IFLAEASVRGL 3-58 IALLFCLLVVV [291 3591 _LLFCLLVVVV][ 29 215 SMNGQPLTCV 27 158 IALEEGQGLTLI 26 F30] LLQDQRIT-HI F3-44 DLVSASVWVV 11251 33]1 ELETSDVVTV 239 ILHVSFLAEA 24 426 SLK N T 1C 24 1B LLHSKYGLHv][ 23 NJ~ RVLVPPLPSL Li f 252 GLEDQNLWHI][ z 12841 RLDGP LPSGV if23! 357 AALLFCLLW 1123 16g LLLLLLLASF 1122 350 WVGVM I_ 362CLVVVVLM Dj22! 392 LTRENSIRL f]22 l3Ij GVMLLFCL7 i j3551 VIAA4LFCLL j[ 211 79J LALLHSjKYGL F1 27 1236 THL'IF F3476 VSASWWGV F50oof GIYINGRGHL 2! 1411 LRLRVLVPPL 1912 351 WVGAALL -jj9~ 1356 ILFCLLV JI 1ii!9 f36 FCLLYVL7h 3811 QMTQKYEEE-L 19Th 147 GIKQAMNH7FV F1Th! [1 EMWGP AWLL 118 15 WLLLL A 1 10s-19=T1428f 00 00 STableXXXV-V1-HLA- A0201 -1 Omers-1 91 P4131 2B 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 ts.cor [1-71 LLLLL.LASFT [iI-1-8 [i121 VLLRNA6VQ FT18 1152 SLNPGPALEE [181 [121 TAEGSPIAPSV [[181 201 [SAAVTSEFLJ[I- 221 LTCWVSHPGL]I 181 [2-4J9SVRGLEDQNL F -78j F ]SASVVVW:GVl F 8 [36 0_ LF C LL WV F181 4181 LAGP J18 11 WGPEA FLL-171 11 LEAWLLLLLLL F 17 1 25 FTGRCPAGEL][-1-71 GDSG EVGQV[ F1 7] VDAGE GAQEL] 17 S73 GEGAQELALL Jr 177 Fj-3 FPAGSfQARL J[ 71 137 FQARLRLRVL]-7 1202 MTEFHLVJ1 177~ 2411 HVSFLAEASV]IIIP 305 LTTEHSGYV ILPI F3631 LLWVVV'LMS ILII7 13891I. ELTLTflENSI ]lZ 118 LLLLLASFTG 16 A~ F61 QVGQVAWARVI 161l [89 IHVSPAYEGRV II [1381 QARLRLRVLVj Il- 1401 RLRLRVLVPPI 16 11641 GLTLMSCTA J1r.iil- 11-66 TLAASCTAEG ]161 [257 NLWHIGREG j[ 161 1259 IWHIGR EGAML l[ 16 [341 KQVDLVSASV jF- i 1370 1LMSRYHRRKA IiI1 441 GRSYSLTTV IF 176 7 J AEMWGPEAWL[ ]5i Zii GPEAWLLLL ]Ej TabieXXXV-V1 -HLA- A001 -lomers-1911P4DI2B 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 1s-r [191 LLLLASFTGR 1 [34] LETSDYWVTVV LA 1721 AGEGA ELAL7 1181 vTwDTEVKGT E 2291 GLLQDqRITH j:j 262 GREGAML-KCLF17 299 TLGFPELTTE JD 321 FSSRDSQVTV] 75 343 VDVAV I 15 1349SVWVGVIAA] 15~-~ 397 SIRRL-HSHHT] 175 [409 RSQPEESVGL I(151 1445 YSTLTTVREIr71 447 TLTTV(REIET 1 7h 460LLSPGSGRAE flF1-5 5011 IYINGRGH7LVF 7 F-12] PEAWL LLLL.r 74 LLLASFTGRCELJ5 f[J LLASFTGRCP] 74 jETSDVVWL] 14 F80 ALLHSKYGLH F14 [871 GLHVSBAYEG 7i i [i-07] PLDGSVLLRN I[14 [iii1]1 SVLLR NAVQA][ 14 1113 LLRNAVQADE]L4 FI1501 LPSLNPGPAL] 14~ 1156 IGPALEEGQG-L][ 141 118APSVTWOTEV][14 195 SFKHSRSAAV][ 14f 233 QRITHLHV ][j r1j~ SGVRVDGDTL [T14 12981 DTLGFEPLTTJ 17 3-11 GIYVCHVSE]D 323 SRDSQVTVDV 14 324RDSQVIVDVL 71 13321VLDPQESGK] F141 1342QVLVAS JD 142 EIE TTELL II 141 TableXXXV-V1-HLA- [A0201-l0mers-1 91P4D)12B3 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, [P0s 1234567890 e [47j LRAKPTGNG11I 4I TableXXXV-V2-HLA-1 A0201-l0mers- 191P4D12Bj Each peptide is a portion of SEQ 10 NO: 5; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus ninel [10 LYRGDSGEQ4II14 CLYRG6DS GEQ D ,D1~ KLPCLYRG-DS-F 711 TabieXXXV-W-HLA- A0201-1 Omers- 191P4D128 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position pus nine.

[PosI_1245780 R~ TableXXXV-V9-HLA- [A0201-l Omers-1 91 P4D1 281 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.j [PosI 124567890 e [1001 LLLVRLF -2] E J LLAGILLRIT

JA~

00

E

0 0

O

(O

O

0 TableXXXV-V9-HLA- A0201-10mers-191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Posl 1234567890 Iscor A0201-10mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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 so 8]ELGTSDVVTV GTSDWTVWL 18 -ILGTSDVVTrVV IPAGELGTSDV 13 TableXXXV-V12-HLA- A0201-10mers- 191P4D12B Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the STableXXXVI-V1-HLA- A0203-10mers-191P4D12B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino adds, and the end position for each peptide is the start position plus nine.

00 00 IPOSI1 1234579 score 1160 IEEGQGLTLA[ 19 [-R4 RSHSRS F1 9 349 SVWVGVIAA 19 F59 1GEQvGQW F18 12391 IjLHVS 7pEA 18..J.

1671 EGQGLTLAAS [ThIj 19511 SKHSRSMAV F 177 350 IWVWGVIMAL 11171 LGAEMWGPEA1j 1-01 11 AW1L.LLI-LA j]iof -221 LASFIGROPA I 921 391 'VTVVLGQDAJF 10 I 571 DSGEQV GQVA F .1 I 63 GVAWARV0Al 10! F67 WAV6RVDA GEGAf10 711 DAGEGAELA]j. 10 F-84] SKYGLHVSPA] I 10 1-0811 LDGSVRN Fi 10 F1111 SVLLRN8VQA] F10 F125 YECRVS TFPA]. 10 FL301 STFPAGSFQAJ 110 JF49]1 PLPSLNPGPA]. 10 RLs9] LEEGQGLTLI 10 FW641 GLTLAA SCTAJ 7f161 F1691 ASCTAEgSPA.1 ][i0 F11931 SRSFKHSRSA riol 2-371 TH-IiH-vSFA 170 F257 NLWHIGREGA ][iIq 339 SGKQ VODLVSA 170 [3-J8]ASVWVGVIA][ 10 3701 LMSRYHRRKA][ ]iO F4-] QfPEESVGLRA[ -101 ELLSPG SGRA] ][10 4721 EDQOEGIKQAj I1- 01 [4851 FYIQENGIRA ]j70 G AEMWGJPEA F91 F-2]ASFTGRCPAG JF2l VTVVLGDAK I[ 21 F58 SGEQvG.QvA~ 219

QVAWVAGE

681 ARvDAGEGAi 9 F7-2] AGEGAQELAL 91J KYGLHVSPAY 119 TableXXXVI-VI-HLA-1 A0203-1 Omers.1 91 P4DI 2B 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,, [PosI1 1234567890 j[score] 1109 DGSVLLRNAV 112 V LLRNLV6QAD ][79 176 E~jRVSTFPAGLi 131 jTFPAGSFQAR 9I~ [1501 LPSLNPgPAL LI^ 1 1651 LTLAASCTAE]FEI1 170 SCTAEGSPAP [I-1 2381 H!LH-VSFLAE [21 240 LHVSFL.EAS FE~q 125 LWHIGREGA-M [2 7340 GpKQvDLSA~s][91-g 371MSRYHRRKA L79 412 PEESVGLRAEF 7 14-60 LLSPGSGR-AEIF79 473 DQDEGIQM 1486 VQENGTRA ]7 9 TableXXXVI-V2-HLA- A0203-1 Omers- 191 P4012B Each pepbde is a portion of SEQ ID NO: 5; 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.

PoIs 13579 cr 1101E DiTL FEach peptide is a portion of SEQ ID NO: 19; each start position Is specified, the le ngth of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Po 1234567890 123 YFGFA 19 41l EMS-VQ 18 124 F I1~M 17 59 NPAASV 681ATSHC 831 TKKKKK 0 12 YQIMAL~ 40LEEHVQ19 MEHYAG[9! 5=4 LLSNPA [9 =5 GSNPAA 60l PPSSLA Z 69GTSHCC[ 84 KRKKLKA TableXXXVI-V1il-HLA- A0203-1 Omers- 191 P40128 Pos 1235679 NoResultsFound. -11 TableXXXVI-VI 2-H LA- A0203-1 Omers- 191P4D12B [PosI 124679 Fscoi TableXXXVI-V1 3-HLA-1 191P4D12B J Each peptde is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is TbeXX-V9-HLA- 1A0203-lOmers-I 19=,=j 4D~12B=~J 00 00 TabIeXXXVI.V1 4-H LA- A0203-1 Omers- 191 P41I2B Each pepfide is a portion of SEQ ID NO: 29; 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.

rPo0s 12356790 e G S SNPPASA

PPASASLV

E]SNPAAS

TableXXXVII-VI HLA-A03lomers-191 P4D 12B 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 13457890 ]]score 152 LSL NPGPALEE] 203 AVTSEFHLVP 284 RLDGPLPSGV]1_20 345 LVSASVVVG 3521 wGVMLLFf21 369 VLMSRYHRR1~ 201 17 LLLLLLASFT 1191 135 VYVIWRY 191 4 1-9 R HP LK T19 F19 ILLLASFTGR 118 117AVADEEY][181 1442] PLSL 1 [3591 LLFCLLVV F 18] 400 RLHSHHTDPR [18 450 TVREIETQTE 1181 WLLLLLLLAS 17 18 [LLLLLAS FTG F17 42 VVLGQDAKLP 1 Bj~ LLNAVADE iZ 145 VLVPLPSLN J-7 jjgKGTSSRSFK F 7j 17KHSRSMAVTS 171 F24 RVDGDTLGFPF 17 3PLTTEHSGIY F 77 376 LVVVVVLMSRI 17 391 TLTRENSIRR 17 443 RSYSLTTVIR F17 460 LLSPGSGRAE Il1 f[ QEALHS 16 g LLHSKY GLHV -16 11 VLLRNAVQAD 16 F1-23 GEYERVSTFF 17 [C6jLV PPPSLN P 1 61 E SUAEg 00 00 TabIeXXXVII-V2-HLA-A03l0mers-1 91P4D12 Each peptide is a portion of SEQ ID NO: 5; 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.

P0s 12345678907 [scoree CLYRGGEQI 181 SKLECL~GDS 11 LYRGEQVII1 CZ QDAKLP~LYR( 1 j GQDAKLPLY 91 PPCRGD 81 TableXXXVII-WI-HLAA03-.

i0mers-191P4D12B I Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

1234567890score [HTDPRSQSEEL16 TabIeXXXVII-V9-HLA-A03- 10mers-191P4D12 Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Ps 123567890 [score SSLVAGTLSVH 24 102 GLLKVRPLQH 23 E2ILLRIN FF 21 6LVAGTVHH [21 98 CLGLKR 21 121 RITFNFFLFF 96 I61L]GLLK LII 105 KVRPLHQGV Li FL2FP21 FF 1 !I LL!GLLKVRP 18 TabieXXXVII-V9-HLA.A03 10mers-191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Po 1234567890 score L ELLAGIRI [i 21 FFLPFPLW F 17 [8 FTIRKKKKII 7 26 PL FFIY 2 WFFIYFYFY 2816 I GIIRJIFNF I 7 HCACFFTK[151 88 KLKA IQ IE9 [I RELLAGIR ]7I~ -16 TFFL I 14 F-27! LWFFIYFYF 14 39! LEMESVA 1 50AGLELLGSSN 14 51 GLELLGSSNP14 53ELLGSSNPPA 77 ACFESKRK 14 LLAGILLRIT 1 17RPLQH0VNS 13 31 FIYFYFFL 1 LLGSSNPPAS 12 [2ASASLAGTL] 12 8RKKKLKKAFR) 71 86 KKKLKKAFRF 12 F18 12 126 GIFMQMPWE] 12 1 FLFFFLPFP L 11 14!YVAQAGLELL ii 7SVHHCACFES[Jj 7FESFTKRKKK F71 811 SFTKRKKKLK Li1 100 LLGLLVRPL [i1l 103 LLKVRPLQHQ 11 125I 11 0 r9 1ableP±2122JI j 3-10mE- 9P41 BI 00 00 Each peptide is a portion of SEQ ID NO: 21; 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 os 12 3456 7890f s=co re 78] E LG-T SD V VT V1F-_ []JRPAGLGT 11 F4CPAG-ELGT-SD1119 TabIeXXXVII-VI11-HLA- AO311mers-191 P412B3 Each peptide Is a portion of SEQ (D NO: 23; each start position is specified, the length of peptide is 10 amino acd s, and the end position for each peptide Is the start position plus nine.

rPos 1245780 R~ [D RLR LR VM VPP, [g RLRVVPPL 18 -8 RVM=VPPLPSL EN] M -PLPSLNP F16 [-A3]LRLRVMV 131, of SEQ ID NO: 27; 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.

PosJ 1234567890 sej D LADPQEDs3KF 16 )j3]QVTVDVLADP 15 E jDVaD ES 1 DI~VL-ADPQEDSG LI:A DOWV~LADPQE 131 fTableXXXVII-V 4.H LA- A03-1 Omers-1 91 P4DI 2B Each peptide is a portion of SEQ ID NO: 29; each start position is specified, the length of peptide is amino acids, and the end Iposition for each peptide is the start position pius nine.I [Posl 1234567890 iscore D SSLAT F-4NPA1 ALV 1 10 A S V GT3]L

[IDJSSPPSAL

[I~1NPPAASL 8 GSNPPA6] TableXXXVIII-V1 -HLA-A26-1 l0mers-191P4D12B 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.

Eo1234567890score) :38]1 DVVTVVLGQDI27 r51 ETSOVD W 27 KJ WWVGVIAAL 1) 2-71 GVIAALLFCL 26 JVVWVLM-SRYI 251 j ]J[TWVLGQDAKLj 2[ 4 :131EAWLLLLLLL ]F-231 IfableXXXVIII-V1 -HILA-A26- I Omers-1 911P401 2B3 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 plus nine.

[gos 1234567890 ]Fc~r 144 RVLVPPLPSL -23 455 ETQTELLSP-G]Eg~ 351 WVVGVALL]Eg~ [792 LTRENSIR7RL. 22 RM EGKQMHF 22 11861 EVKGTTSSRS 121) 369 ITIL FL il21 128 RVSTFPAGSF1 331 DVLD)PQEDSG F439 EPEGRSYSTL[

JD

9 91 EQPPRP I 9 24-91 SVRGLEDQNL F191 EN EMWGPEAWLL) 18) 129811 DTLGFPPL7TT [18~ J-2 FTGRCPAGEL Liz) 223 OWSPGLLQ 17 11231 GEYECVSTF[16 224 WTSHPGL 16 2-96 TLG FPP 161 472 EDQDGI KQ-A 16 10 WGPEAWLLLL F33 ELETSDVVTVJF F60EQVGQAWA ~jQVA WAR VDAG 116 NAVQADEGEyr 1l30 STFPAGSFQA 1EGQGLTL s 1 29)SGVRVDGDTL 1 294 1RVDGDTLGFP][ 327 QVVDVLDPQ( Th E95 421 EGHDSKDN 00 00 Tab~eXXXVIII-VI -HLA-A26- 1 Omers-1 91P40128 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 1 1234567890 1 sco-re 4531 EIETQTELLS JI 1-5] 2041 VTSEFHLVPS 1[ 141 222 TCWSHPGLL II 14 12351 RITHILHVSF 174 F24 1FLAEASVRGL I 141 247 IEASVRGLED)Q] 14 2-59 1WHIGREGAML IF14 293 VRVDGDTLGF] 14 3081 EHSGIYVCHV .1-4 [328] VTDVLDPQE JF -1 I 337] EDSGKQ VOL 14 13451 LVSASVVVVG III Th4 13-66 IWVVLMSRYH [14 13-67 VVVLMSRYHR 14 141 ESVGLRAEGH[ TF4( 14291DNSSCSVMSEjF 14 (4-36 IMSEEPEGRSY f714 44811 LTTVREIETQ IF141 F44-] TTVREIETQT IF17 450 TVREIETQTE 1l 4 452 REIETQTELL JF14.

4831 NHFVQENGTLI. 14 [-1-11 GPEAWLLLLLIF 7 121 PEAWLLLLLL J[1l-3 [16LLLLLLLASF jIF713 F4-01 VRVVLGQ9DAK]Fi131 1131 riS581 ALEEGQGLTL It Thi3 1-8011 SVTWDTEVK Fi131 1-11 VTWDTEVKGTII 131 203 AVTSEFHLVP F13 2-331 0QRITHILHV 1F13 DQNLWHGR 13 13-05 LTTEHSGIY Fj173 TTEHSGIYVC 17 F4381 EEPEGRSYSTJJ 131 441] EGRSYSTLTT F131 1471 EEQEGK L3 TableXXXVIII-V1 -HLA-A26-1 I0mers-191P4D12B3 Each peptide is a portion of SEQ ID NO: 3; eac; tr 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 11234567890 ]s-core 485 FVQENGTLRAIF 3 500 GIYINGRGHL J13 TableXXXVIII-V2-HLA- A26-10mers-191MP412B Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is amino acids, and the end position for each peptide isl the start position plus nine.1 [P05 12456890Iscore TableXXXVIII VW-HLA- 1 A26-l0mers-191 P40128B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[Po 124680score] [D HTDPRSQ-SEE o jTab~eXXXVIII-V9-HLA-A26- I Omers-1 91P401283 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

1234567890 score (13 ITFNFFLFFF lFi24 28 LWFFIYFYFY Lz24 [80 ESFTKRKKKL 123 215 (TableXXVIII-V9-HLA-A26jliners-I 91P4D128 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

[Pos 1234567890 []Fsoe F271 LWVFFIYFYF 1D 46j YVAQAGLELL 221 S9 FQLLL 17 4E EHYAQ( 16] 121 FRITFNFFLFF] [14 45 HYAA LE 19FLFPW 13 LI~I ILLRTFNF 12 F11 LRTNFF 12 11 11FLPP F 22 1 FLFLF f l 29!FYYY I121 00 00 Efa-ch peptide is a portion of SEQ ID NO: 21; 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.

jFos 123-456789-0 score] GT-SD V-VTVVL Q I ELGTSDVVT-V 15] TableXXXVIII-V1 1-HLA- A26-I0mers-1 91 P4121 Each peptide is a portion of SEQ ID NO: 23; 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.

RVMVPLPS 23 TableXXXVIII-V1 2-H L1A-1 A26-1 Omers-191 P4D12B Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide Is the tart osition plus nine.

[PosI 1234567890 re EPEGCSYSTLF20 [j3 1MSEEPEGCSY 14 8 EPGCSYST1 -1 E SYSTLTT 13N TabIeXXXVIII-V1 3-HLA- A26-1 Omers-19I1 413 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 1234567890scr 7 DVLADPQEDS 18' 3I~ QVTVDVLADP [1

STVOVLADPQELIZ

F-2 SQVTVDVLAD F- l D I DSQ VTVD VIA TableXXXVIII-V14-HLA- A26-l0mers-191P4D1B Each peptide is a portion of SEQ ID NO: 29; 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 1234567890o- (score] [FSNPAAS3m 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.

[PoRs 12345679 score] 132 FAGSFQARL 24 [150 LPSLNPGPAL j4 [ii GPEAWLLLLL Iii [439] EPEGRSYSTL 23 156 GPALEEGQGL IEE2~ 1I78 APSVTWDTEV [21!l 276 PPP-YN-wTrRL 21 176 SPAPSVTWDTI. 19 103 PPRNPLDGSV L18 407 DPRSQPEESVI 18 411 QPEESVGLRAIE18 35 ETSDVVTWL 17 72 AGEGAQELAL 117 AGSFARLRL F17 227 HPGLLQDQRI 1 3PPLTTEHSGI 16 DPQEDSGKQV 16 289 LPSGVRVDGD 15 H34 DSQVTDV -75 1 TabeXXXIX-V1 -HLA- 8B0702-1 Omers-1 91 P4012 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 ~E1 F-71 AEMWGPEAWLI 14 D MPEAWLLL 14 [99_EQPPPPRNPL] 1114 FI 581 ALEEGQGLTL 14 249 SVRGLE QNLI_ 296 DGDTLG-F-PP-L fi1 36 1 FCLLVVVV )1 409 RSQP-EESVGL] 14! D EMWGPEAW-LL 13 M121 PEAWLLLLL E13 E13 EAWLILLLLL J[13j 70 VOAGEGAQEL][13j [23j GEGAQELALL 13 [11 PPPPRNPLDG 13 11061 NPLDGSVL-R] 13 141 LRLRVLVPPL [:1j 212 PSRSMNGQPL]1 13 236 I _THILHVSFL 1 13 29 H gREGAL 1 277l PPSYNWTRLD 13j 287 GPPSVRD9 131 336 QEDSGKQVDL][ Th 1351j VVVGVIAALL ][1j 355 VIAALLFCLL ][13j 495 KPTGNGlYINI[13~ 10!1 WGPEAWLLLL Eg~ 1-00 QPPPPRN PLO 12 104 PRNLDGSVL IF12 137 FQARLRLRVL 12~j 144 RVLVPPLPSL I121 114881 PPLSL-NPG-P]II12 15 NPGPALEEGQ jiD 160 EEGQGLTLAA j12 E [VPSRSM NGQP 12 (231 LITIL 2 00 00 TableXXXIX-V1 -HLA- 80702-I Omers-191PD12 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, [Posl 1234567890 ]ER~ 1244 [LEAVGLI ].121 1262 IGREGAMLKCL] 72 13081 EHSGIYVCHV ][12] 1337jEDSGKQ VOLV 12[1~ 13-50 WVVGVIAAL][2 383 [QKYEEELTL][ 121 i3921 LTRENSIRRL][ 12, 14-41 EGRSYSTLTT][ 12 145211 REIETQTELL 11 12 FTGRCPAGEL][F 171 F 411 TVVLGQDAKL] [i1i F5-61 GDSGEQVGQVF1 Lii 138 QARLRLRVLV 11ll 2011~ sAAvTSEFHL 2-191 QPLTCWVSHP.F- ]I 2211 LTCWVSHPGLJ. 11 2575QPPPSYNWTR F111 2-80 YNWTRLDGPLF Liii 354 GVIAALLFCL J[F-Ti 357 AALLFCLLWV IF 171 13581 ALLFCLLVV\I IF j1ji 14181 LRAEGHPDSL j- 1i1 1423 HPDSLKDNSS [j i 14-51 VREIETQTEL l111r 1462 SPGSGRAEEE] 1I v1 1500 GIYINGRGHLD TableXXXIX-V2-HLA- B0702-1 Omers- 191 P40128 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amn acids, and the end position for each peptide is the start position plus nine.

Pos1 1234567890 1score L GQDAKLPCLj 11- TableXXXIX-V2-HLA- 80702-l0mers- 191 P4D128 Each peptide is a portion of SEQ ID NO: 5; 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 I LPCLYRGDsGF [71] 1LYRGDSGEQV 1010 TabeXXXIX-V7-HLA- 830702-l0mers- 191 P4D1 28 Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Iposi 135 789 scorel TableXXXIX-V9-HLA- B0702-l0mers- 191 P4D1 2B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

[Po 124679 score F59 NPA2S0A7 23 LPPLFF 25 FPV IY JZ 92 AFFQ1L 6~fj F94 RFQLLG 3 1100lLGLVPL] 3 117RLQQVS] 3 IZ MRE1AI 72~ 1-4 T FF FFFL 72~ I 43 ESHYVAQAGL F172 F57SNPSS 2 [TableXXXIX-V9-HLA- B0702-1 Omers- I 91P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptIde is amino acids, and the end position for each peptide is the start position plus ine [Po 1234567890 1 s~r E@90 KKAFRFIQCL 1jDl DI RRELLAGILL 1[j11 E12 RITIFNFLFF Il...ii E18 FF FLPFL] Ii E[f 3111FFFL [46 F1QGLL] li 53 EL SPA[11! 19 PAALAG] I- JASLVAGTLSV] iijl [E FKKK] ii [9 KfRFQLL] [87 KKKARI F95 FQLLLL]..1 [ios~ F- KRLHV] 01 EA 20 FFL L]91 E33 YFYFYFFLEM][--9 [41 EMESH 1AA]91 Ess GSNPS]9I [70 TLVHAC]9 F83 TKKK K]9 F84 KK K F]9 [12 ERYFGIF][ [123YFQGIFMQM][F9 TabIeXXXIX-V1O-HLA- B 0702-1 Omers- 191P4D128 00 00 Each peptide is a portion of SEQ ID NO: 21; 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., IPosl 1234567890 1scorel GTSDVVTVVL 1 F-4]CPAGELGTSD [14 77GLTSV~1 F7E8 TDVT l TableXXXIX.V1 1-HLA- 80702-1 Omers- 191P40128 Each pepde is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is 10 amino acids, and the end ipositon for each peptide is the start position plus nine.

JPos]13479 0 screl L RL R VMVPPL F13 -8 R VM VP-PL-P SL 13 F- QARLRLRVMV F11] F lRL RLRVM F TableXXXIX-VI 2-11-A.

80702-1 Omers- 191 P4D12B Each peptide is a portion of SEQ ID NO: 25; 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 D EPEGCSYSTL 2 TableXXXIX-Vi 3-H LA- B0702-1 Omers- 191 P4012B Each peptide is a portion of SEQ ID NO: 27; each start osition is spcfied, tihe length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[posI 123-45-67890 score

DDSQVTVDVLA

D J QVVVLD Z TableXXIV-V1 1-HLA- A0203-9mers- 191 P4D1 28 IFNoResultsFoun. j TableXXIV-V1 2-H LA- A0203-9mers- 191P4D12B Posl 1234678 NoResultsFound.

rableXXIV-V13-HLA- A0203-9mers- 191 P4D12B [Pos 12345678 NoResultsFound.

TableXXIV-V14-HLA- A0203-9mers- 191 P40128 IPos 4679score [NoResults Found.

TableXLl-V1 -HLA- 81510-1 Omers- 191 P401 28 NoResultsFound.

TableXLI-V2-HLA- 831510OA0mers- 191 P4D12B [Es 1.245677890cr NoResults =ound.

TableXLI-W7-HLA- 191 P4D128 [PsI1234567890 Iscore NoResultsFound, TableXL-V9-HLA.

B15lO-l0mers- 191 P40128 7Pos 45780score No ZesultsFoud TableXLl-VI 0-H LA- 191 P40128 TableXXIV-V7-HLA- A0203-9mers- 191 P40128 PH 1g 23456789 score TableXXIV-V9-HL IFA0203-9mers- 191P4D12B Pos 123456789cr NoesultsFound.

TableXXIV-V10H

LA-

AO203-9mers- 191 P4D1 28 Pos 123456789cr Noeuts0n 00 00 TableXLI-V1 0-H LA- Bi 510-1 Omers- 191P4DI2B 12456790 sc-r el [NoResultsFound.

TableXLI-V1 1-HLA- 81510-j1iners- 191 P4DI2B E 124680score ,[NoResultsFound.

TableXLI-V1 2-HLA- 831510-1 Omers- 191 P4D128 jEs 124679icore IFNoResultsFound.

TableXLI-VI 3-HLA- B15IO-l0mers- 191 P401 28 [P-osJ 124680icorel ,t NoResultsFound.j TableXLI-Vl4-HLA- I 831510-j1iners- J 191P4D12B 23456890 soore ,FNoResultsFound.

IITableXLII-V1-HLA- 1 B 2705-1 Omers- 191P4012B I[gI 124679score NoResultsFound.

TableXLII-V2-HLA- B2705-1 Oiners- 191 P4D12B Pos 124680scoe N oR es-ul-t s-Fou-nd.7 TableXLII-W-HLA-

B

27 05-l0mers- 191 P4D1 28 Po 1346790scre TableXLI-V9.HL1A-

B

2 705-l0mers- 191 P4DI128 Pot 135680 cr TableXLII-V9-HLA- B2705-1 Omers- 191P40128 I 7s12467890sore NoResultou.

TableXLII-VI 0-H LA- B2705-1 Omers- 191 P4012B [Pos 124679icore] ,FNoResultsFound.j TableX~LI-Vi 1-HLA- 82705-1 Omers- 191 P4D1 28 7Pos 124679 core IFNoResultsFound.

[TableXLII-V1 2-H LA- i F 2705-10Oiers- S 191P4D12B13579 cr1 NoResultsFound.] [TableXLII-VI 3-HLA1 B2705-1 Omers- 191 P401 28 No~esultound.

TableXLII-V14-HLA- B2705-1 Omers- 191 P401 2B Pos 124680score NoResultsFound.

TableXLIII-V1 -HLA- B2709-1 Omers- 191P4D12B Pos 123457890sco re N[-oResultsFound.

TableXLlIl-V2-HLA- 832709-1 Omers- 1911P401 28 Pos 123-456789-0 score FTableXLIll-W-HLA- B2709-l0iners- 191 P4D1 28 PoN 235780score TableXLIII-V7-HLA- B2709-1 Omers- 191P4D12B [Pos score790 FNoResultsFound.

JTableXLIII-V9-HLA- 191 P401 28 NoResultsEound.j TableXLll-V1 0-H LA- F82709-1 Omers- 191 P401 28 NoResultsFound.

TableXLIII-V1 1-HLA- 82709-1 Omers- 191 P4D1 28 1PsJ12345678901 jscor NoResultsFound.

TableXLI-1 42-H11A- B 2709- 1 mers- 191P4D128 Pos 12345 7890 Noeut ud j [TableXLIII-V1 3-H LA- FB2709-1 Omers- 1 o 9 lP 4 Dl 2

B

TableXLIlI-V1 4-HLA- 832709-1 Omers- 191 P40128 Pos 234567890 Fsor] FNoResultsFound.

[TableXLIV-V1 -HLA-84402- 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.

TableXLIV-V1 -HLA-B4402- Ta bIe X L V- V1 HLA.8- B4 402 Tabi1eXLiV.VI-HLA-844( l0mers-1 91 P40128 l0mers-1 91 P40128 l0mers.1911P40128 Each peptide is a portion of Each peptide is a portion of Each peptide Is a portion 00 STEQ ID NO: 3; each start SEQ ID NO: 3; each start SEQ ID NO: 3; each sta position Is specified, the position is specified, the position Is specified, th( length of peptide Is 10 length of peptide is 10 length of peptide Is amino acids, and the end amino acids, and the end amino acids, and the en position for each peptido is position for each peptide is position for each peptide the start position plus nine, the start position plus nine, the start position plus nin Pos 1234567890 score Pos 1234567890 scr os 1234567890 452 REIETQTELL F25 259 WHGEGM~7

VDAGEGAQELK

F-7 AEMWGPEWL24 262 GREGAMLKCL 794 E LALLHSKYGL 12 PEAWLLLLLL 23 319 NEFSSRDSQV 1412 DEGEYEORVS

E:

F_ 7 EAQLL 2354 GVIAALLFCL 1125 YECRVSTFPA

E:

7i77 QELALLHSKY 2 392 LTRESRRL] 14 E4 RVLVPPLPSL 1-23 GEYEORVSTF F22 [409 RSQPEESVGL 1F87 VKGTTSSRSFK [336 QEDSGKQVDL 22 412 PES=VGL=RAE1422T SHGL l 00 469 EEEE000EGI 20 41 EESVGLRAEG 14 20 LLQDQRITHI F1 99 EQPPPPRNp F 18 439 EPEGRSYSTL 1424IAAVG 174 EGPPVT 8483 NHFVQENGTL 14249 SVRGLEDQNL

E:

ETSDvvTVVL 17 4941 AKPTGNGIyi 14 2-53 LEDQNLWHIG

E

r72 AGEGAQELAL 17 jDJ GAEMWGPEAW 113 27 1 LSEGQPPPSY

E

13 EAWLLLLLLL 16l1 GPEAWLLLLL 1322 EQPPy 134GSQGLL 16 E9 LLASF 13347 SASVVVVGVI E 16 EEQGT02 6 GELETSDvvr 13 355 VIAALLFCLLEl 476 EGIKQAMNHF 16 128RV STFPAGSF 1337RKAQQMTQY 81 EMGFA 1-5 141 LRLRVLVPPL 1 383 TQKEEELTL E1 K-9I MWGPEAWLU.. 15 159 LEEGQGLTLA 1389 ELTLTRENSI 1j; 98 VEQPPPN 15 199 SRSMVTSEF 13 394 RENSIRRLHS 11 158 ALEEGQGLTL 15 231l LQDQRITHIL 13 440 PEGRSYSTLT 1r, 173 AEGSPAPSVT I 15 250 VRGLEDQNLw 1 5 IETQTELLSp 1r.

2773 EGQPPPSYNW 15 29 1 SGVRVDGDTL 145TELPGr 1 [3-50 W GVIMAL 15 1293 VRVDGDTLGF F13 361] FCLLVVVVVL 15 296 DGDTLGFPPL 1 TableXLIV-V2HLA- I38-7 EEELTLTREN 1 15 8 24RSVTDLF13 44 02-l0mers- 1388 EELTLTRENS 1535 VVVGVIAALL 13 191P4D126 peptide is a portion 1420 AEGHPSLK0 15 [352 WGVIAALLF 13 of SEQ ID NO: 5; each 437 SEEEGSys 15 438 EEPEGRSYST 13 start position is specified, R EDQE KQ]EN 468 EEEEQDE 13the length of peptide is 471 EE D Q D E G K 1 5 4 8EEEEDQ DEG 1 3 amino acids, and the end 10o WGEW l1 7 EDDGK1 position for each peptide is 158 SGEQVGQVAW 14 487 QENTLRAKP 13 the start position plus nine.

KYGLHVSPAY 14 4-93 RAKPTGNG1Y 13 PR 123467890 score 104 PRNPLDGSL 14 E MPLSLGAEMW 12 GQDAK LPCLY LII 14 25 FTGRCPAGEL FI LGQDAKLPCL [I-]l 7FQARLRLRVL 14 4 LETSDVVTVV 12 L~ AKLPCLYRGD 1L SLNP.PAL 14 4 TVLQDAKL 12 206] 4LPR 14 LGQDAKLPCF 1f2l iTableXLlv. HLA2I

I

246 AEASVRGL ED 1A~ 45 0 G-QDAKLPCF 121 L2~-9Po3 00 00 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 scorel 2 SHHTDPRSQS 4 HTDPRSQSEE [JHSHHTOPRSQ 22 STDPRSQSEEP 2I [9 SQSEEPEGRS 2 HHTDPRSQSE 1 [7 PRSQSEEPEG [2 [8 RSQSEEPEGR [2 TableXLIV-V9-HLA-4402.

I 0mers-191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 11 CERGYFQGIF 21 8ESFTKRKKKL 18 RELLAGILLR 17 21 FFLPFPLWF 17 11 LRITFNFFLF 16 16 NFFLFFFLPF 16 62 ASASLVAGTL 15 SFESFTKRKKK 15 [i KRKKKLKKAF I 15 S91 KAFRFQCLL FI S92 AFRFIQCLLL 94 RFIQCLLLGL 1 92 ILLRITFNFF 13 13 ITFNFFLFFF 114 23 LPFPLVVFFI 114 1301 FFIYFFYFF 1T LEMESHYVAQj[ 141 42 MESHYVAQAG][ 141 I 57 SSNPPASASL[ 141 KKAFRFQCL II j4i j125 QGIFMQAAPW 14 2 RRELLAGILL 13 4 ELLAGILLRI 13 TableXLIV-V9-HLA-B4402- 10mers-191P4D128 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score I LAGILLRITFl 13 8 GILLRITFNF I 118 FLFFFLPFPL 13 22 FLPFPLVVFF 13 24 PFPLWFFIY 11131 S25 FPLWFFIYF 131 26 PLVVFFYFY 113 28 WFFYFYFY 13 I37 YFFLEMESHY 13 S52 LELLGSSNPP 13 I 86 KKKLKKAFRFII 131 100 LLGLLKVRPL J[131 115 NSCDCERGYF[ 13 12 RITFNFFLFF 29 VFFIYFYFYF E 12 43 ESHYVAQAGL 12 I46! YVAQAGLELL 12 87 KKLKKAFRFI 12 95 FQCLLLGLL 12 11E VNSCDCERGYI 121 1 MRRELLAGILL 11I .14 TFNFFLFFFL 11 45 HYVAQAGLEL 11] 70 TLSVHHCACF 11 73 VHHCACFESF 11 SAGILLRITFN 10 1 LLRITFNFFL 10 1 271 LWFFIYFYF 10 31 FIYFYFYFFL 10 [18DCERGYFQGI][ 101 TableXLIV-V10 HLA- 84402-1 Omers- 191P4D12B Each peptide is a portion of SEQ ID NO: 21; 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.

1234567890 s SGTSDVVTVL 1 ID GELGTSDVWT 1 TableXLIV-V11-HLA- B4402-10Dmers- 191P4D128 Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of peptide is amino acids, and the end posion for each peptide is the start position plus nine.

1234567890 scoe LRLRVMVPPL 1 8 RVMVPPLPSL 12 [ARLRLRVMVP [2 TableXLIV-V1 2-H LA- 84402-1 Omers- 191P4D128 Each peptide is a portion o SEQ ID NO: 25; 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.

1234567890 scor SEEPEGCSYS 14 EPEGCSYSTL 14 EEPEGCSYST 3 PEGCSYSTLT 11 L11MSEEPEGCSY TableXLIV-V1 3-HLA- B4402-10mers- 191P4D12B Each peptide Is a portion of SEQ ID NO: 27; 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.

Posi 1234567890 s 2 SQVTVDVLAD 6 10 ADPQEDSGKQ S 1 LADPQEDSGK 4 N DSQVTVDVLA F-41 2~ F51TVDVLADPQE 7 [D y0PE]~ [Is 112345678flO score1 EI -MeltsFoundjl 00 00 TableXLV-V1 3H LA- 83510 1-j1iners- 191 P4D12B Pos 23457890score] NoResujitsFound.

TableXLV-V14-HLA- 85101-1 Omers- 191 P4D1 28 fNoResultsFound. TableXLV-V1-HA 85101-l0mers 191 PD128 F 1345789 score LNoReusoud TableXLV-V2-HLA- B5101-l0mers- 191 P4DI2B [Po s 12345678901 s INoResultsFound-.

TableXLV-W-HLA- B51O01 -1Omers- 191 P4D1 28 NoRs-ultspound.

FTab-e XV-94LA- SB5101-l0mers- 191 P41)1 28 P123457890or NoResultsFound.

TableXLV-V1O-HLA- 851 01-l0mers- 191 P40128 FPos 34689E[R INoResults Found.

TableXKLV-V1 1-H LA- 851 01-l0mers- 1 91P4012B L TableXLVI-Vl -HLA-DRB1 -0101l5mers-191P412 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 12456789012345 [Fc~oe 1279] SYNWTRLDGPLPSGV

L...N

140 RLL3LP2SL 205 TSEFHLVPSRSMNGQ 32 12991 TLGFPPLTTEHSGIY 32 3SDVVVLGQDAKLP F 371 141VTVVLGQDAKPCYJ 31j [3401 GKQVDLVSASWW 31 349 SVVVVGVIAALFCL31] 144 RVLVPPLPSLNPGPA 30g 147 VPPLPSLNPGPALEE 30 350] WWVGVMlLFCL 3 51 I PCFYRGDSGEQVGQV 2 12 PEA WLLLLLLLASFT 2 247 EASVRGLEDQNLWHI F27 358ALLFCLLVVVVVLMS] 27 371l MSRYHRRSQQMTQ-K 26 GAEMWGPEAWLLLLL 2 13 EAWL-LLLIIASFTG[25 14] AWLLLLLLLASFTGR[25 LLLLLASFTGR F25]1 TableXLVI-V1-HLA-DRB1-01 01 l5mers-19 P D 2 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.

Eos 123456789012345 19j LLLLASFTGRCPAGE 10-2 PPPRNPLDGSVLLRN 1109! DGSVLLRNAVQADEG 1122 1193 SRSFKHSRsAAvTSE 239 ILHVSFLAEASVRGL J1 251 255 DQNLWHIGREGAML 12651 GAMLKCLSEGQPpps j[ i 310 [SGIYVCHVSNE~S 454 IETQTELL-PGSGRA] 2[ [64QVAWARVD-AGEGAQEJ[ 24J [76 AQLLLHSKY GLHV 1f 24 [79 LALLHSKYGLHVSPA 1 [12 ECRVSTFPAGSFQAR JF24 [1-56 I GPALEEGQLAS iDi 162 GQGLTLAASCTAE-GS ][24 181VWDTEVKGTTSSRS] 24 210 LVPSRSMNGQLTV] 24j tJSRSMNGQPLTws [241 282 WTLDPLSGRD 4 1347 SASVVVVGVIAALLF U241 353 VGVIAALLFCLvJjiI 357 AALLFCLLVVLII R LVVVVVLMSRYHRRK[24 395 ENSIRRLHSHHTDPR F-24 442 GRSYSTL1 MEET 24 1LLLLLLLASFTGC 23 8RCPAGELETSDVTV 23 184 EKTSR FF(-I23 228] PGLDQIHLH 9 123IQRITHILHVSFLAE 1 23 J289 LPSGVRVDGDLGFP 23 3391 SGKQVDLVSASVVWV 23 SVSASWVVGVIMLL 23 3FCLLVVVWLMS 23 PIDSLKDNSSCVMSE 23 8 LTTV REIETQTELLS 2 457 QTLSPGSGRAEEE j3 43 NHFVQENGTLRAKPTD23 00 00 TableXLVI-V1 -HLA-DRB1 -0101-1 l5mers-191P4D12B 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 o'sI 1234667-89012346- [score] LSLGAEMGPAL F72 1 551RGDSGEQVQAA F72 F59 jGEQVGQVAAVA 7-12 [-1411 LRLRVLVPLPLN 72~ 12-041 VTSEFHLV PSRSMNG J[ 22 [2501 VRGLEDQNLWHIGRE][221 12-68 ILKCLSEGQP-P-PSYNW][ 2 [3111 GIYVCHVSN FSR [22 3271 QvTVDVLDPQ)EDSGK 1[[2-2] [3601 LFCLLVVVVVLMSRY ][22 [451_ VREIETQTESG ]22 [2-18L GQPLTCWSPGL [21 1256_QNLWHIGREGAMK] F21 27-7] PPSYNTRDPS]21 F33[ELETSDVTVQD[20 IVAWARVAEAE] F20 [1-23 GEYECRVFPGF20 NPGPALEEQ L ][270 1321] FSRSVVVD [20 1429 DNSSCSVMEPG] F2 1482 MN H FV Q ENGTL RA KP] F201 1490 GTLRAKPTGNGIYIN ][20 LASFTGRCPAGELET I[]19 [391 vvVVLGQD)AKLPCF III 9 [-38 QALL VPLPSIF191 QRITHILHVSFLAEA IF___ 2-42 VSFLAEA VRGLEDQ iF19[ 14-121 PEESVGLRAGHDS F19] [4151 SVGLRAEGHDLDU 71 AEMWGPEAWLLLLLL ]JF18 [911 SPAYEGRVEQPPPPRj 178 [134J AGSFQARLRLRVLVP ]r181 11651 LTLMSCESA 1181 [26J rGMKCS1QP 8]Th 2661 AMLKCLSEG3QPPPy1T8] 18 YNWTRLDGPLPSGVR F1 8 368 1WVLMSRYR QM j[ 18 3871 EEELTLTRENIR F[ a81 :11]1 GPEAWLLLLS DJz Ta leXL VI-Vl .HLA-DRBl-01 01- I S5mers-1 91 P4DI 28 Each peptide is a portion of SEQ I0 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.

[Fos 11234567890124 soe 67l WAR VDAGEGAQELAL ]j17 [681' ARDGGQEAL1171 [78-3 HSKYGLHVSPAYEGR II 17 [gI [RNAVQADEGEOR Jj17 [125[YECRVSTFPAGFQA117 135 GSFQARLRLR P E1117 [1481 PP L PS LN PGPA LEEG ]..F17 11501 LPSLNPGPALEEGQOEE17 1167 LAASCTAEG-SPAPSV l[-177 120 SAAVTSEFEHLVPSRS [[171 221] LTCWSHPGLQQ [17 122 VSHPGLLQDQII [[17 12381 HILHVSFLAASR l[ 2h 12-57 _NLWHIGREG L KCL j[17 [25 LWHIGREGAMKL [11 [2&I RLDGPLPSGVRVDGD][ i 12911 SGVRVDGDTL-GFP-PL iF 1294RVDGDThGFPPLTTE

]I

1303 PPLTTEHSGIYVCHVl 7 1330VDVLDPQEDSGKVJ Eii [33 VLDPQEDSGKV V] F171 1342 QVDLVSASV GV 177 [348 ASWGI LFC J[ 17 g3s GVAALLFCLLWW F-7 356 ALLCLWW J9~ 1407DPRSQPEESVGLRAE][ 17 4321 SCSVMSEEPEGRSYS if171 1458TELLS3GSGRAEEEE IF1-7[ R DEGIKQAMNHFVQEN IF 17 TableXLVI-V2-HLA-DRB1.0i 01l5mers-1 91 P4DI 28 Each peptide is a portion of SEQ ID NO: 5; 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 soe FI21 VTVVLGQDAKLPCLY 113 PCLYRGDSGEQVGQV1Z E21DAKLPCLYRGDGQ

J

DI~ VVTVVLGQDAKLPCL Tab-1eXLVI-V7-HLA-DRB1- 010'1- 17 l5mers-191P4D12B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the -satposition plus fourteen.

[Pos 123456789012345 sor Dj IRRLHSHHTDPRSQ-S 14 I HHTDPRSQSEEER[ I F1 RSQSEEPEGRSYSTI[101 DII SIRRLHSHHTOPRSQ [1 [ii ]DPRSQSEEPEGRSYSI7 F14 SQSEEPEGRY STLT 119 [D RRLHSHHTDPSQEf2 D I LHSHHTDPRSQSEE-P 18 [q HTDPRSQSEEPEGRSLI [12 PRSQSEEPEGRSYST 11-8I [-4]RLHSHHTDPRSSE~ L[ HSHHTDPRSQSEEPE L2-7 TableXLVI-V9-HLA-DRBI-0101-1 5mers-191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide Is the start position plus fourteen.

123456789012345 scoeJ [43 ESHYVAQAGLELLGS] IF--3 [49 QAGLELLGSSNPPAS][32 [36 FYFFLEMESHYVAQA][ 311 [10-3] LLKVRPLQHQGVNSC[ 28~ F17 FFLFFFLPFPLWFF r[27 90g KKAFRFIQCLLLGLL] F27] 98[ CLLLGLLKVRPLQHQ [26 18 FLFFFLPFPLWFFI 2 60 PPASASLVAGTLSVH 2 61 PASASLVAGTLSVHH- 24 93 FRFIQCLLLGLLKVR 2 L 97j QCLLLGLLKVRPLQH 2 00 00 TableXLVI-V9-HLA-DRB1 -01 01.l5mers-191P4D1B Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is amino acids, and the end position for each peptide is the start position plus fourteen.

[Pos 1234567890124 1W] ER [61 LAGILLRITFNFFLF lIF72 116 JNFFLFFFL-PFPLWVF IF72J AGILLRI FNFLFF[22 F521 LELLGSSNPPAS-ASL ]22 [1001 LLGLLKVRPLQHQGv] F22i GILLRITFNFFLFFF 1[211 L-2ZI LWFFIY-FYFYFFLE 21 121 RITFNFFLFFFLI PPff0 341 FYFYFFLEMES HYVA FI 20 S92 1FFQLLGLv 20f 2]1 ELLAGILLFNFF 119 f14 1TFNFFLFFFLPFPLV[[1l9! 11 FNFFLFFFLPFPWI[] F31 FIYFYFYFLEMESH J-g F33 YFYFYFFLEMESHYV][ 19 W4 YVAQAGLELLGSSNP] 19I FIQLLLKRPL 3[ 191 LLRITFNFFLFFFLP ][18 19] LFFFLPFPLWFFIy

F~

F225]__FPLWFFIF FYF 181 F 28 WFYYYFLM] 841 KRKKKLKKAFRFIQC ]F 18 [120 ERGYFQGIFMQA~] iiI [j13 ITFNFFLFFFLPFPL [17 FFFLPFPLWVFFIyF 17 I22! FLPFPLVVFFI YFF 1 29 VFFIYFYFYFFLEME 17 1371 YFFLEMESHYVQAG [17 441 SHYVAQAGLELLGSS [7 94 RFIQCLLLGLL VRP[17 2] RRELLAGILLRF [1 21! FFLPFPLVVFFIYFY ri-1I 39 FLEMESHYVAQAGLEF16 41 EMESHYVAQAGLELL 16 48 AQAGLELLGSSNPPA I 51! GLELLGSSNPP ss]Th 541 LLGSSNPPASASLVA

EE

56 GSSNPPASASLVAGT j[16 68IIV CC S J[ 161 TableXLVI-V9-HLA-DRBI-01 01- 15mers-191P4D12B3 Each peptidle is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Mjo 123456789012345 [sor TLVHAFSFTK E1 15 5]J KV RP LQH Q GVNSC DC 1 E16 TableXLVI-VI 0-HLA-DRB1-01 01 l5mers-1 91P4D128 Each peptide is a portion of SEQ ID NO: 21; 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.

Flo-s1l 12345678901235I~~ Each peptide Is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each pepfide is the start position plus fourteen.

[Pos 123456789012345 seg [14! GCSYSTLTTREE I24 12]IDNSSCSVMSEEPEGC [:I 12I CSVMSEE PEG CSYST [7-6 [EFCSYSTL1TVREIETQ 1[]71 abIeXLVI-V13-HLA-DRB1..0101-1 15mers-191P4D12BJ Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptidle is 15 amino acids, and the end position for each peptidle is the 1 start position plus fourteen.

l 123456789012345 scr FSSRDSQVTVDVLAD [I DSQVTVDFA 1ED 7 14 ILAD)PQEDSGKQVDLvIF17 [2]QVTVDVAPES 16 101 TVOVLADPQEDSGKQ Lji6i [SQVTVDVLADPQEDS [2]SSRDSQVVDVLDP 141 [12 DVLADPQEDSGKQVD [j [TableXLVl-V14-HLA-DRB1 -01 'I--1 Each peptide is a portion of SEQ ID NO: 29; each start position Is specified, the length of peptide is 15 amino acids, and the end position for each peptidle is the tart position plus fourteen.

[PosI 123456789012345 score 11i PPASASLVAGTLSVH

D

2] PASASLVAGTLSVH-H Eg~ L E LL G SSNp-pASA S-L 2 2 [j GLELLGSSNPPASAS 16 EN~2 LLGSSNPPASASLVA 16 E2] GSSNPPASASLVAGT 16 2]AGLELLGSSNPPASAF15 21LGSSNPPASASLVAG i13 ASSLVAGTLSVHHO ELLG SNppAS ASLV 14 L:8] SSNPPASASLVAGTL 14 TabIeXLVI-V1 1-HLA-DRBI-0101..

l5mers-191P4D12B Each peptide is a portion of SEQ ID NO: 23; 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.

[PosI 123456789012345 Fs~r [JRLRLRVMVPPLPSLN 30 [11RVMVPPLPSLNPGPA L9 [0LRLRVMVPPLPSLNP QARLRL RVMVPp~S~~ []AQARLRVMVP 18E IIGSFQARLRLRMVP1 GS1FQARLRLRVMVPP I17 Ejfl RLRVMVPPLPSLNPG 16g I2 FPAGSFQARLRLRVMF15 2LRVMVPPLPSLNPGP 1 [278RRMVpPL1 I TableXLVI-V12-HLADRBI-0101-1 II 1 Smers-1 91 P401 2B 00 00 FTableXLVI-V14-HL11A-DRBI-o101-] Each peptide Is a portion of SEQ ID NO: 29; 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.

[Posl 123456789012345 sce] A ASLVAG T-L S-VHHC A C r4 TableXLVII-V1 -HLA-DRBI-0301- 1 Smers-191 P4DI 28 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 plus fourteen.

Posl 12345678024 [scorel 178 1APSVTWDTEvKG1TS] 29I~ 227 HPGLLQ RTHL 1281 411TVVLGQDALP FJ[27 73791 AQQMTQ E TL] 7251 141 AWLLLLLLLASFTGR] 231 290 PSGVRVDGDTLGFPP J 231 3 9 VVTVLQD LCFIiI 1703PPRNPLDGVLN F 221 247 EASVRGLEDQNLWHI If-221 117 RNAVQADEGEYECRV][ 21J 142 RLRVLVPPPLP ]211 233 DQRITHILHVSF-LAE 11211 325 1DSQVTV0VDED] 211 738] ASVVWGVIAALLFC]21 F3491 SVVWVGVIMLLFOL]21 Z--16GAEMWGPEAWLLLL]L 20 17561 GPALEEGQGLTLAAS 201 2421 VSFLAESR ED ]20 249 SVIRGLEDQLHG] 20 2921 GVRV0DTL FPP 2] 0 135 VVVGVIMLFCL] 201 3521 WGVIALF LW] 201 13531 VGVIAALLFCLLVW 20~ 363 LLWWVLMSRYHRR] 20i 1126 ECRVSTFPAGSFQAR] 19 13021 FPPLTEHSGIYVC] 9 1328 VTVDVLDPQEDSGKQ][ 19~ 136511VVWVLMSRYHRRKA][ 19 1387 EEELTLTRENSIRRL E]19l I 77 QELALLHSKYGLHVS .j 18] TableXLVII-V1-HLA-DRBI-0301- 11Smers-1 911P40D1 2B 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 posilion for each peptide is the start Lposition plus fourteen. I E~ 1234568024 1Isr F1III SVLLRNAVQA0E 1265 IGAMLKOLSEGQPP F7i8 1286 DGPLPSGVRV DT] 78 13-191 NEFSSRDSQVTDVL] 7Dj 1329TVDVLDPQDKV] ThI- 1433CSVMSEEPEGRSYST] F 1I VREIETQTELLPG ]18 [7]JGLHVSPAYEGRVQ Liz-7 [97] R'/EQPPPPRNLG 17B 1 ILHVSFILAEASG 12-55jDQNLWHIGREGAMLKj 171 1311 GIYVCHVSNEFSRD] F71 IE DPQEDSGKQV-DLVS-A][ E36 BVVLMVSRYHRRAQ[ 7j I381 IQMTQKYEEETLR[ F 17 E4011LHSHHTDPRSQPEES1[ 1 l1 EESVGLRAEGPL I[ 17 H~I YSTLTTVREIETQTE F[ 17 K~jDEG1KQAMNHFVQEN [17 F479] K QA MN H FVQ ENGTI[ 171 471 TLRAKPTGNGYN [17 DI LGAEMWGPALL I[ 161 EAWLLLLLLLST nt16 Ej DAKLPCFYRG DSGQItZ E~j VDAGEGAQELHS l[:16 F1i-4 AGSFQARLRLRVLVP I[16 FEI~LRNAVQADEGEC j[15 F130] STFPAGSFQALL j 15 gi~j FPAGSFQARLRLRVL 1[]15 9 SRsAAvSEFHLVPS I15~ 94 LC HPLQDQR I15~ F2-36 ITHILHVSFLAEASV 1[15 [JAMNHF VQENGT LRAK I 5 F15]_WLLLLLLLASF GRC j 4 LLLLg4 ASFTGRCPA I 4 I1ELAIIHSKYGLHVSP [4 r1709 DGSVLILRNAVQADEG1 41 1ll~ GSVLRNVQAGE 14 TableXLVII-V1 -HLA-DRB1 -0301l5mers-1 91 P4012 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.

IPos F123456789012345 ]scorel 144 RVLVPPLPSLNPGPA F14 280 1YNWTRLDGPLPSGVR Jt.141 1342 QVDLVSASVV V [14 13-56 IMLLFCLLVVL [14 1360 LFCLLVVVVVLMS-RY 1[14 F48] LTTVREIETQTELLS It 14 4I49 TTVREIETQTLS It 57 Q-TELLSPGSGRAEEE I[ 141 Tab~eXLVII-V2-HLA-DRBI -0301-] l5mers-191P4D12B J Each peptide is a portion of SEQ ID NO: 5; 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.

[Posj 123456789012345 score] [D TVVLGQDAKLPCYR [:jA []jJVVIVLGQDAKLPCLI 2i72 [DIDAKLPCLYRGDSGEQ -1-6 D I VTWLGQDAKLPCLY F7J [TableXLVII-W-HLA-DRBI -0301l5mers-191P4D12B Each peptide Is a portion of SEQ I0 NO: 15; 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.

[Ps123456789012345 scorel [~LHSHHT0PRSQSEEP 17 [IRRLHSHHTDP SOS Il1l LI1RSQSEEPEGRSYSTL 9~ HTDPRSQSEEPEGRSF19

SHHTDPRSQSEEPEG

12 PRS-QSEEPEGRSYST138J 1SQSEEPEGRSYSTLT EII81 Tab~eXLVII-Vg9HLA..DRBI -0301- 1 Smers-1 91 P40128 00 00 Each peptide is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide Is the start position plus fourteen.

[81 GILLRITFNF FLFF251 M121 QGVNSCDCERGYFQG 24i YFYFFLEESYA 23~ [61 LAGILLRITFNFFLF7 22 [27] AGILLRITNFF [211 [91 LFFFLPFPLWFFIY )[211 LLRITFNFF2F0L 201 FFF-PFPLW F 20 z [74 SHYVAQAGLELL-GSS F20 L-931 FRFIQCLLLGLLVR )20 9[7 QCLLLGLLVPQ CLLLGLLVPQQ i[Z2G [161 NFFLFFFLPFPL 1 9[s [241 PFPLWFFIYFY-FYF ][191 IFPLWFFYYFFF F7[ 9h 751] GLELLGSS-NPPASAS [19 [681 AGTLSVHHAEFIfI [901 KKAFRFIQCLLLGLL] [19 9[2 AFRFIQCLLLGLLKV [19[ 1 4 TFNFFLFFPPL [181 [26 PLVFYY F -[18.

[29 VFFFFFENE [18) [12RITNFFFFFLPFP [7 221 FLPFPLVVFFIYFYF [171 28 WFIFYYFL EM- 1[ 7 [7911 FESFTKRKKKLKKAF 17) [82 FTKRKKKLKKFF 17) 86 KKKLKKAFRFIQGCLL [17) 27 LWFFIYFYF FFL 16) F76] CACFESFTKRKKL [16 15AILITNFI9 1 33 15FFLEEHV M41 IEMESHYVAQAGLEL-L 15s I78 C-FESFTKRKKKLKKA[ L-891 LKKAFRFIQCLLLGL )[15) [l11 GVNSCDCEGFI [15 11-] FCDCRYQIMA7h F96 IQCLLLGLLVPL [14) RRLADLITN) a [49 QALLLS9PA 1101 LGLK RPLQHQGV[[13 TabieXLVII-V9-HLA-DRB1 -0301. l5mers-191P4D12B Each peptide is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide Is 15 amino acids, and the end.

position for each peptide is the start position plus fourteen.

[Po 12345678901 2347 [scorel [101 LGLLKVRPLOQGV j[ 13 [103 1LLKVRPLQHQGVN-SC]113 36j FYFFLEMVESHYVQA 112 F37jYFFLEMESHY-VAQAG)[ 12 39 FLEMESHYVAAG E l12 52j LELLGSSNPPASA7SL 12 [674 ASLVAGTLSVHHCAC J[ 12 [106VRPLQHQGVNSCDCE2 1TableXL VII-VI0HADB-31 1l5mers-191P4D12B Each peptide Is a portion of SEQ ID NO: 21; 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.

[Posj 123456789012345] soe 112 GELGTSMVVVVLG-Q 12~ [I iAGELGTSDWTL i LASFTGRCPAGELG 170 12]ASFTGRCPA GELGTS 9 12] FTGROPAGELTD E11A 113l ELGTSD-VVTV-vLGQ-D I [TabIeXLVII-V1 I-HLA-DRBI-0301- 1 5mers-191P34D12B Each peptide is a portion of SEQ ID NO: 23; each start position is specified, the length of pepfide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

[Po 12457801345 Fscr [1 -l]RLRVMVPPLPSLNGLP [2]AGSFQARLRLMp

LIP)

[2]FPAGSFQARLRLkV-M 7Th [1 2 LRVMVPPLPSLNPGPf141 I 13) RVMVPPLPSLNPGPA 14 [2]RQARLRLRvmvPPLPS. 13 2]RLRLRVMVPPLPSLN L_12 12I SFQARLRLRVMPPL 8 8IALLVVPPLPSLI iMVPPLPSLNPGPA&lEF1 F TableXLVII V1 2-HLA-DRB1-0301-1 l15mers-191P4D12B3 Each peptide Is a portion of SEQ ID NO: 25; each start position Is~l specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

[Ps123456789012-34S scorel [2 CSVMIS E E PEGqCSYST 181 [2]SCS VMSEEPEGCSYS1-2 SVMSEEPEGCSYSTL 5505VMSEEPEGCSY El

SEEPEGCSYSTLTTV

TabieXLVII-VI 3-HLA-DRB1-0301-) 5mers-191P4D12B Each peptide Is a portion of SEQ ID NO: 27; 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 123456789012345 scr [10 TVDVLA0PQEDSK ZIP) [2]DSQV1VDVLADPQE 2 2 [ii VDVLADPQEDSGKQV[1j 6 TabeXLVII-V1 4-HLA-DRBI- 0301-1 Smers-1 91 P401 2B Each peptide Is a portion of SEQ ID NO: 29; 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. g 1I 123456789012345 scor 2] GLELL GSSNPPASA 19) 12]LELLGSSNPPA SASL 12) 15 JASLVAGTLSVHHCAC 12 [14 ISASLVAGTLSVHHCAil 2] LGSSN PPASASLVAG IMiP-PASASLVAGTLSVH TableXLVIII-VI-HLA-DRB1-0401 1 Smers-1 91 P4D12B 00 00 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.

[Posl 123456789012345 jIsc~i 1205 TSEFHLVSRM Q 28z~ 12-99 ITLGFPPLTTEHSGY ]281 f 47 DAKLPCFY RGDSGEQ[ 261 11621 GQGLTLMASCTAEGS 1L26I 2-55 DQNLWHIGREG-AMLK][ 26~ 3-11 IGIYVCHVSNEFSSRD 1126 1395 ENSIRRLHHTP [261 14-151 SVGLRAE GHPDS LKD 26Z~ F4 5 DEGIKQAMNHFVQ-E-N 1261 F-71AEMWGPEALLL 1221 1 PEAWLLLLLAF ]221 FI501 LPCFYRGDSGEVG] 22I F511 PCFYRGDSEVQ] F221 rl180 I SVTWDTEVKGT SSR 22 193 SRSFKHSRS TE] 221 241 HVSFLAEASVRGLED 1[22 1358 ALLFCLLVVVM 1122 3781 TQKYEEELTLTEN F[221 441GRSYSTTVET 1221 [13 EAWULLLLST 1201 [151 WLLLLLLAFRCI 201 11 LLLLLLLAFGC I201 I 371 SDVTVVLGQDAKL][7 F.591I G EQ VG Q VAWAR-VDAG][ 20 76 iAQELALLHSKYGLHV][F 201 871GLHVSPAYGVQ1 201 FI1-111 SVLLRNAVQADEE [20 1I44 RVLVPPLPLPP [20 F1-] VPPLPSLN AE [201 1184 DTEVKGTfSSRSK- 1201 211 SMAVTSEFHLVSR 20i~ M281 GQPLTCWSHPGLLQ] 201 1227 HPGLLQQTHL F201 1233 5,5THIHVFLA 12-391 ILHVSFLAVRL]20 1242 VSFLAESVRGLEIJQ [L20 247 EASVRGLEDQLH .[20 251LWHIGREGAMLKCS ]2o l26] EGAMLKCLSEGQPPP J[ 20 3021 FPPLTTEHSGIYVCH I[ 20 TableXLVlll-V1-HLA-DRBI-41 15mers-191P4D12B 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.

Igos 1234567890124 [oVel 1325DSQVTVDVLDPQD 20 1340 GKQVDLVSASVWG l[ 20 342 QVDLVSASVVVV 201 E34 SASVVVVGVIAALF j[201 1R941 SVVWGVIM O 1201 352 WGVIAALLFC LLWV Fl20j I53 VGVIMALLFCL LVWV FI 20 1357 MALLFCLLWVLM 20Z~ 13-601 LFCLLVVWVVLMSRY]1201 361] FCLLVVVVVLMSRYH][ 201 1364 LWWVVLMSRY HRRK] 201 13-68 IVVLMSRYHRRK AQQM[ E~ 13891 ELTLTRENSIRRLI-S 1 201 1424 PDSLKDNSSCSVMSE 27 4-33 ICSVMSEEPEGRSYST]1[: 1445 YSTLTTVREIEQT ]20] 44-8] LTTVREIETQEL II 20 IRj QTELLSPGSGAE 1120 1479 KQAMNHFVQENGTLR Fl 20 4-831 NHFVQENGTLRAKPTEIL29 1 28 1RCPAGELETSDVV1781 i~ [29 OPAGELETSDVVT ]181 331 ELETSDVVT:-VLGQD I[ A [38j DVTWLGQDAKLPC ]18 [j HVSPAYEGRVEQPPP Ill j.8 F1 3 PPRNPLDGSVLRNA j If LDVLLRNAVQj[ F-18 I 8 LDGSVLLRNA VQAE I[Zi Ig1 ADEGEYECRVSTFPA IDi L IN1 GEYECRVSTFPAGS7FF 118 1i RVSTFPAGSFQA RLR 1118 PGPALEEGQGLTLAA i [i§90 -TfSS FHSAAV II 18 M291 QPLTOWVSHPGQ =1 18 13081 EH-SGIYVCHVSES]1 Eg CHVSN EFSSR SQVT 18 131 N-EFSSRDSQVTVDVL ][18 1 328 VTVDVLDPQEDSGKQF][ 8 1331 DLQESQV L 8 TableXLVI -1 -HLA-DRB1-0401l5mers-191P4D12B 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 pep tide Is the start position plus fourteen.

Posl 123456789012345 re 3391 SGKQVDLVSAS-VVVV I[ 1-81 33 RYHRRKAQQMTQKYE[ -IF 7 386] YEEELTLTRENSIRR 1[181 392 LTRENSIRRLHSHHT][ 7 407 DPRSQPEESVLE] F1 81 4231 HPDSLKDNSSVS] 18i 1435 VMSEEPEGRSYSTLT 1 181 449 TTVREIETQTLS I[Z8 I441 IETQTELLSPGRAIii 1472 EDQDEGIKQAMNHFV 1118 11341 AGSFQARLRLVV F1171 13181 SNEFSSRDSQVTVDV 1171 [64 QVAWARVDAGEGAE]I 83I HSKYGLHVSP AYEGR ]f16 2561 QNLWHIGREGMK 16~ 1279 SYNWTRLDGPLPSGV[ IF161 13101 SGIYVCHVSNEFSS 1[]6 1482 MNHFVQENGTRK] jj176 367 WVVLMSRYHRRKQ] PLSLGAEMWGPAW] 74 j76] GAEMWGPAWLLLLL [14 [14 AWLLLLLLLAS~fTGR 14.

[17 LLLLLLASFTRP F1141 [18 LLLLLASFTGRCPA 114 [19 LLLLASFTGRAE114 31j AGELETSDVVTWVLG I~ 36 T -SDVVTVVLGQDAKL] F14 39q VVTWVLGQDAKLPCF ]IF-14 411 TWLGQDAKLPCFYR 3[ 7 62 jVGQVAWARVDAGEGA][ 14 95 EGRVEQPPPPRNPLD[ JF141 11051 RNPLDGSVLL-RNAVQ][ 14 LI 1RNAVQADEGEYECRV[ F17 [i 261 ECRVSTFPAGSFQARI 1[ 141 [140 RLRLRVLVPPLPSLN I. 74 [142 RLRVLVPPLPSLNPG [F141 [143 LRVLVPPLPSLNPGP I1141 [1-56 GPALEEGQLLA IL 4 [-164fLTLAASCTAEGSPA 1114 00 00 TableXLVIII-VI -HLA-DRBI .0401 15mers-191 P4D12B Each peptide Is a portion of SEQ ID NO: 3; each start po 'sition is specified, theilength of peptide Is 15 amino acids, and the end position Ifor each peptide Is the start position pius fourteen.

[Pos 1234567890124 score I17 8APSVTWDTEVKGTTS F7I 1207 EFHLVPSRSM-NGQPL] 74~ 213 SRSMNGQ-PLTCW-SH][ 17 21 LTOWSHPLQQ 1[4~ 228 PGLLQD-RITHILHV] [14 237 LTHILHVSFA~V ]F 141 250 VIRGLEDQNWR] r1 4! 265 GALCLRQP 14 268 LKCLSEGQPPPS-YN-W][ i 1282 I GPLPGVRVD]7i DG LPSGVIRVDDTL]j F290 PSGVRVDGDTLGFPP[14 [Y92GRDDLFPL [4 F327 QVTV0VLDPQEDSK[4 [3301 VDVLDPQEDs-GKQ-VDJ[ 14 [i 8]ASVVWGVIAALLFC ][14i 350 VVVVGVIM-ALLFCLL -]1 1356 IAALLFCLLV-VVVVL ][j41 1362LLVVVVVLMS-RYHR[]14 363 LLWWVSYR [14 1365VVVVLSYRK 387 EEELTLTRENSIRRL Df14 398 IRRLHSHHTPSP] 141 142SCSVMSEEPEGRSYS ]141 4RITTLS~ TalXVIII-V2-HLA-DRB1 -0401- [Tab e rs 9P4D12B Each peptide is a portion of SEQ ID NO: 5; 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. IEos 12345678-9012345 scoej I DAKLPCLRGSE 26~~ [1-3PCLYRGOSGEQVGQV FT2 [l1 LPCLYRGDSGEQVGQJ[ 20 Li1] VVTVVLGQDAKLPCL E D3 TVLGQDAKLCY 14i TableXLVIII-V2-HLA-DRB1-0401- 1 Smers-1 91 P4D12B Each peptide is a portion of SEQ 10 NO: 5; 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 124568902345 score [15 LYRGDSGEQVGQvAW 71j2] [TableXLVIlk-v7.HLADRBI-0401-] Each peptide Is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 15 amino acids, and the end postion for each peptidle Is the start position plus fourteen.

[E[I 123456789012345-]s~r [D LHSHHTDPRSQSE1p8] I S~ SQ SEE P EG RSYSTLT LF71! [-2]IRLHSHHTDPRQ 14 [12PRSSEEEGSYST12 [TbeXLVI I kV9HLADRB1-0401. J Each peptide is a portion of SEQ ID) NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the endi position for each peptide Is the start position plus fourtee.

[Po 123456789012345 scr [37 YFFLEMVESHYVAQAG [F 26 [86 KKKLKKAFRFIQCLL 1I 26 [103 LLKVRPLQHQG-vNS-cJ 26 [2 RITFNFFLFFFLPFP I22 117 FFLFFFLPFPLWFF 221 [Al YFYFYFFLEME-SHYV][ 221 36I FYFFLEMESHYVAQA] IF22 [7NJ CACFESFTKRKKKLK]IF 22! Lg1 KKAFRFIQCLLLGLL ]22 r i1RGYFQDGIFMVQMAAP7WEf 22f [~RELLAGJLLRITFNF 1120 [GILLRITFNFFLFFF [201 161 [NFFLF FFLPFPL VVF [-20] 4 SHYVAQAGLELLGSS

D

:49 QAGLELLGSSNPPAS 20 51_LELSNIAA L2a l TableXLVIII-V9-HLA-DRB1 -0401l15mers-191124D1213 Each peptide is a portion of SEQ ID~ NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

IPos 123456789012345 1FSC70] F931 FRFIQOLLLGLLKvR jF 598 CLLLGLLKVRLQHQ][ [41! EMESHYVAQAGLELL J[ T 8 62J ASASLVAGTLSVHHC IF-181 I 73VHHCACFESFTK-RKK 1181 891 LKKAFRFIQCLLLGL 11-18 [141 TFNFFLFFFLPFPLV [[F16 [15 FNFFLFFFLPFPLW 1L 16! [18j FLFFFLPFPLWFFI j-1i6] [22j FLPFPLWFFYFF [161 [28 FFIYFYFYFFLEM J[16 3011 FFIYFYFYFFLES ][16 [311 FIYFYFYFFLEMESH 196 I 321 IYFYFYFFLEMESHY 1[ 161 3YFYFFLEMESHYVAQ]j[ 16~ [43 ESHYVAQAGLELLG-S] [i6 AFRFIQLLLG-L-L-KV J 120 ERYFGIMQPW][ 17 [AGILLRITFNFFL-F7FE]D.i [24j PFPLWFFIYFYFYF 14 [25j FPLWFFIYFYFYFF 1 14~ LFFIYFYFYFFL 4 129j VFFIYFYFYFFLEME [14 E439 FLEMESHYVAQAGLE ]~141 52g LELLGSNPSS 14 64l ASLVAGTLSVHHCACII.I 70I TLSVHHCACFESFTK J[14 [97 QCLLLGLVLQ II 1i4I [100 LLGLLKVRPLQHQGV JIL Ej LLAGILLRITFNFFL I[ 172 El21 FFLPFPLWFFIYFY 12 461 YVAQAGLELLGSSNP][ 121 47 VAQAGLELLGSSNPP] IF12! 48jAQAGLELLGSSNPPAj[ 55 LGS S ASL:VAG] 00 00 TableXLVlIl-V9-HLA-DRBI -0401- 1 5ners-1 91 P4012B rEach peptide Is a portion of SEQ ID NO: 19; each start position Is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus fourteen. I [Pos 1234567890124 [scorel [5-61 GSSNPPASASL VAGT I[ 121 [57 SSNPPASASLAGLj12 PPASASLVAGTLSVH F-1 j [611 PASASLVAGTLSVH]-iI1-21 [661 LVAGTLS-VHHCACFE Jj 12 67 VAGTLSV HHCAC FESF 121 751 HCACFE-SFTKRKK-KL 11121 7 ACFESFTKK K [12 F941 RFIQCLLLLLVR 112 951 FIQCLLLGLVP 1121 1704] LKVRPLQ GNSDj F-1 11081PLQHQGVNCD E[ i 1714VNSCDCERGYFQGF [12 111881 DCERGYF-QGIFMQAA L121 1122GYFQGIFMQMPWEG 1112 TabeXLVIII-V1 0-HLA-DRB1 -1 T401-15mers-1iM1i 1 Each peptide is a portion of SEQ ID NO: 21; 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 112345678901-2345 sre RCPAGELTDT r-1-8 [11 AGELGTSVTL 14~ I 2] FTGRCPAGELGTSDAF 1j2, CPAGELG-TSDV'vVV L 112 GELGTSDW-MVVLGQ EZ I TabIeXLVIII-VI 1-HLA-DRB1-1 0401-l5mers-191 P4D1 28 Each peptide Is a portion of SEQ ID NO: 23; 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. [Po0s51 1123456789012345 1score] TableXLVIII-V1 1-HLA-DRB1- 0401-1 Smers-1 91 P401 28 Each peptide Is a portion of SEQ ID NO: 23; 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.j [EIj 123456789012345 so1- [2]AGSFQARLRL MV 171 [11 IRLRVMVPPLPLG F71 [7jAJLRVMVPPLPSNG 14 [2]FPAGSFQARRL MZii [2]GSFQARLRL-RVMVPP 172 ARLRLRVMVPPLS EI12 [jI LRLRVMVPPLPSLN FTableXLIl-V1 2-HLA-DRB1 -0401- 5mers-191P4D 128 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide Is 15 amino acids, and the end position for each peptide is the Lstart position plus fourteen.

E l12,34567890123457][cr [RI GCSYSTLTTVRE:lET 2I2 CSVMSEEPEGCSS F20

SCSVMSEEPEOSYS

[21 IDNSSCSVMSEEPEGC 2 [27 VMSEEPEGCSYSTLT2 [2]MSEEPEGCSYSTLTT][12 [7J EEPEGCSYSTTTR 1 [El EPEGCSYSTLTTVE][12 TabIeXLV1II-V1 3-HLA-DRBI-1 0401-1 5mers-1 91 P401 2B Each peptide is a portion of SEQ ID NO: 27; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the Istart position plus fourteen.

1234567890124 Fsor [gI~TVDVLADPQEDSGKQ F 276 VLADPQEDSGK VO 18i 12] DSQVTVDVLADPQED 14 QVTVDVLADPQEDSG 14 [D FSSRDSQVTVDVLAD F12 [23 ISSRDsQVTVDVLADpI 12 [2]SQVTVDVLADPQDS DJ2 14 LADPQDEDSGKVD)LV 12 TabIeXLVIII-V14-HLA-DRB1 -1 I 0401-1 Smers-191 P4DI2B J Each peptide Is a portion of SEQ ID NO: 29; 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 ]scorel [2]GLELLGSSNPPASAS [EN [1-3ASASLVAGTLSVHH 8 LELLGSSNPPASS 14 [15 ASLVAGTLSVHHCAC [2]LGSSNPPASASLVAG 2 [2]GSSNPPASASLVAGT 12j [2]SSNPPASASLVAGTL 1 71 PPASASLVAGTLS-VH] 71 ,[12 PASASLVAGTLSVHHjF12 TableXLIX-V1-HLA-DRB1-1 101-1 15Smers-1 91NP412B3 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 I 123456789012345 Iscore 1255 NLHIGRE-MLKI 26 279 SYNWTRLDGLSG ]F [12 PEAWLLLLLLA F I2-31 20-1]1 SAAVTSEFHLVPSRS 1L.23~ 164 QVAWARVAEGQI 22 [140 RLRLRVLVPPSL 1122 E218 GQPLTCWVSHPGLLQ ll2g 123 DQRITH1LHVSFLAE- 122 1286 DGPLPSGVRVDGDTL Jf221 29TLGFPPT SI F1221 138 LMSRYHRRA J F221 I 37l SDVVTVVLGQDKPf 211 12611 IGREGAMLKCLSEGQ 1[21E 13FCLLVVWVLMSRYH 1121l I DAKLPCFYRGDSGEQI1 201 113 AGSFQARLRLRVV 11801 SVWDTEVKGTTSSR IL720 365 WWVLMSRYHRRKA fl201 13-861 YEEELTLTRENSIRR 1120 [39 LRENSIRRLHSHHT II 00 00 TableXLIX-V1 -HLA-DRBl -1 101l5mers-191P4D12B 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 s 11234567890123451 s ore 14F1 SGLRAEGHPDSLKD [20 (3471 SASVWVVGVIMALLF [19 [3581 ALLFCLLVVVVVLMS IF 9 [13 EAWLLLLLLLASFTG ]F718~ [161 LLLLLLLASfT GRCP] I8~ F76 A Q ELA LL HS KYGL H-V II j18 91jSPAYEGRV EQPPPPRI F181 1122 EGEYECRVST-FPAGS] 1l8 [T11RVLVPPLPSLNPGPA 11 18] Fj147 VPPLPSLN-P-GPA-LEE 1181 2411 HVSFLAEAS-VRGLED 118] 1265 GAMLKCLSEGQPPPS ]jil 111 GIYVCHVSNEFSSRD] 118 442 GRSYSTLTTVREIET IF18( 274 VTSEFHLVPSRSMNG ]7i1- 205 TSEFHLVPSRSMNGQ]F17 3671 VVVLMSRYHRRKAQQ][-171 1190O TTSSRSFKH-SRSAAV I16 277 PPSYNWTRLD-G-PLPS 16 71 346 VSASVVVVGVIMALL ][76 360 LFCLLVVVVVLMSRYJF1 4871 QENGTLRAKP-TGNGI J[ 161 GAQELALLHSKYGLH]F151 Il-0h7] PLOGSVLLRNAVQAD] IF71 F17-81 APSVTWDEVGT] F 17 11921 SSRSFKHSSAT [151 219 QPLTCWSPGLQ 157 (23-01 LLQDQRITLHF 15i 343 VDLVSASWVA j-1i5 362 CLLWVVWMRH IF- ii F36] LLVVVVVLMSRYHRR [15 1411 QPEESVGLRAEGHPD 1-5 F4- J EGIKQAMNFEG [15 1VEGTRKT 5 i LLFTCAGEL I[14 j LETSDVVTVVLGQ DA l[-14 M61 TSDVVTVVLGQDAKL I[ 14 F-411 TVVLGQDAKLPCFYR l[ 14 1 59 GEQVGQVAARDG j TableXLIX-V1-HLA-DRBI-1 101- 1 5mers-1 91 P4D1 2B 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 1 1234567890124 F Re [61I QVG QVAWA R VDAGEG L-1h] [81LLHSKYGLHVSPAYE IF 141 [1381 QARLRLRVL VPPLPSI .1 N16 GQGLTLMASCTAEGS114 [181 VTWDTEVKGTTSSRS ]D14 [184 DTEVKGTTSSRSKH ][14 [2271 HPGLLQDQRITHILH 141 (2521 GLEDQNLWHIGREG-A][ 1 2-761 PPPSYNWTR LDGPLP ]Iij (290 PSGVRVDGDTLGFPP EE (3081 EHSGIYVCHVSNE:FS ][14 1350 WWVGVIMLLFC7LL ][14 135 MLLFCLLVVVVVLM 141 1364 LVVVVVLMSRYHRR] 141 397 SIRRLHSHHT DPRSQ ]Li 4 F40] LHSHHTDPRS QPEESF 74 (420 AEGHPDSLKDNSSCS] 141 433 CSVMSEEPEGR-SYST][ 1 I 143 VMSEEPEGRSYSTLT][ 141 445 YSTLTTVREI1ETQTE:][ 14 454 IETQTELLSPGSRA] 14 1457 QTELLSPGSG EE[14 1479KQAMNHFVENTL] 74 4831 NHFVQENGTLRAKT i 141 11 LLLLASFTGRCPG [13 I q VTVVLGQDAKLPCFY DI13 85 JKYGLHVSPAYEGRVE f[ 13 1 06 NPLDGSVLLRNAVQA Jj[ 3 13-711 FQARLRLRVLVPLP 13 215j SMNGQPLTCVVSHPG [131 237 THILHVSFLAEASVR [_13 327 QVTVDVLDPQ DSG 23 3740 GKQVDLVSASWV [13 I~SVVVVGVFMALLFCL I 411VREIETQTELLS-PGS 11 13 DI LSLGAEMWGPEAWLLIF1712 TableXLIX-VI-HLA-DRB1-1 101- I 5mers-1 91 P4D1 2B 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.

[PoI 1234567890124 e [22 LASFTGRCPAGELET F 7 [62 VGQVAWARVDAGEGA 7h [73 GEGAQELALHYG] 12 [82 LHSKYGLHVSA E12 [83 HSKYGLHVSPAYEGR IF 12 [92 PAYEGRVEQPPPPRN 1 121 [109 DGSVLLRNAVQADEG ]1 12 [11 VLLRNAVQ EEE112 [123 GEYEORVSTFPG F 112 [141 LRLRVLVPPLSN 12 [153 LNIPGPALEEGQ FT ]12 [1591 LEEGQGLTLAASCTA 11121 1764 GLTLAASCTAGP 11121 120 EFHLVPSRSMNGQPL I112] jITHILHVSFLAEASV Jj12 129ILHVSFLAEASVRGL 1_2 12EASVRGLEDQNLW-HI 12 (26 LKCLSEGQPPPS-YNW 12g (2-92 GVRVDGDTLGFPL 112 (3SGIYVCHVSNEFS 1LJ2I 13RDSQVTVDVLPQD] 12 1329 TVDVLD)PQEDSGKQV][ Eg 1337 EDSGKQVDLVSASW][ 12 13-95 ENSIRRLHSHH TDPR ][12 14131 EESVGLRAEGHPDSLI][ j2I 421l EGHPDSLKDNSFV] 12 1429 DNSSCSVMSEP F-1-2 14481 LTTVREIETQT-E-LLS ][F121 1455 ETQTELLSIPG SGRAE][ 12 (489 NGTLRAKPTGN-GIYI ][12 TableXL1-11V2-HLA-DRB1-1 101l5mers-191P4D12B Each peptide is a portion of SEQ ID NO: 5; 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~o 12345678901 2345-]Scrl 9 DAKLPCLYRGDSE 00 00 1VLGDKLPCLYRI 14 vi-wi-VVVLGQAKLPCLYFj ITabIeXLIX-W7-HLA-DRBI-1 101 l5mers-191 P4012B Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide Is amino acids, and the end position for each peptide is the start position plus fourteen.

[Pos 1123456789012345 scorel IiI1 SIRRLNSHHTDPRS -1-4 171 jLH-SHHTOPRSQSEEP 14 [14 ISQSEEPEGRSYSTLT 14~ IRRLHSHHTDPRSQSE 8I~ [12 PRSQSEEPGSS F78 F21IRRLHSNHTDPRSQS 61 F_ 81HTDIPIRSQISEEPE 61 110 TOPRSQSEEPEGRSY Mfj TableXLIX-V9-HLA-DRBI-1 101l5mers-191P4012Bj Each peptide is a portion of SEQ ID NO: 19; 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.

7PosI 12345678024 scorel 97 QCLLLGLLKVRP LQH 28 [1_211RGYFQG:F MQMAPWE 22~ [37 YFFLEMESHYVAQAG I] 21! [79 FESFTKRKLKFI 21 [76CCEFKKKK1 201 [1_03 ILLKVRPLQHQG-VNS-C] 20 221 FLPFPLVFIFF] 19 [17j FFLFFFLPFPLWFF F1_i.

491 QAGLELLGSSNPPAS [Ir 7.66I LVAGTLSVHAF [j F51F FYFYFFLE MESHYVAF1117! FKKFRFIQCLLLGLL 7 F[120 ER GY F QGIFMQAAP-W][ 171 'FNFFLFFFLPFPLWV ][16 33 YFYFYFFLEMESHYV lE 161 [36 FYF FLEMESHYVAQA] 16 161KKKLKKFICL] 15 32 RELLAGILIFF]T 41ELLAGILLRITFNFF ][14 Tab~eXLIX-V9-HLA-DRB1 -1101 -1 1 Smers-191 P40128 Each peptide Is a portion of SEQ ID NO: 19; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start I position plus fourteen.

[Pos [123456789012345- [coreI [7VAGTLSVHHCACFES[14 [3TKRI<KKLKKAFRFIQ][ 141 IiHQGVNSCDCERGYFQ][ 14 [26j PLWFFIYFYFYFFL] 13] F61] PASASLVAGTLSV7HH 13 I93 FRFIQCLLLGLLKVR 13 98 CLLLGLLKVRPLQQ D__13 TbIeXLIX-VI 0-HLA-DRBI-1 101- [T 15mers-191P4D128 Each peptide Is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 1amino acids, and the end position for each peptide Is the start position plus fourteen.j EgF 123456789012345 I cr jj1IjJLGTSDVVTWLGQDA 4 72LASFTGRCPAGELGT 1T2 [gELGTSDVVTVWLG FZ9I [11 LLASFTGRCPAGELGiii7 S-FT GR C P A GELG TSD 137 [3 TGRCPAGELGTSDW 16 R RCP A GE LG TSDVVT VL I ]BAGELGTSDYVVTWLG E96 [TableXLIX-V1 1 -HLA-DR1-1101l5mners-191P4D12B Each peptide is a portion of SEQ ID NO: 23; 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 12345 6789012 345 Fs-o-e [3RLRLRVMVPPLPSLN Ii [3AGSFQARLRLRVMVP LI DI 3VVPPLPP 18 7[QRLRLRV MVPPLPSE]I I~ F FQRLRLRVMVPPLP L-ii3 7FFPAGSFQARLRLV Zi1P TabIeXLIX-V12-HLA-DRB1-1 101-1 l5mers-191P4012B37 Each peptide is a porton of SEQ ID NO: 25; 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.

Posj 123456789012345 I score [14 GCSYSTLT1VREE ZIP] [3 DNSSCSVMSEEPEGCI 172~ [31CSVMSEEPEGCSYST 1 NSSCSVMSEEPEGCS Lii LTabIeXLIX-V13-HLA-DRB1-1 101 1 Smers-1 91 P02 Each peptide is a portion of SEQ ID NO: 27; 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.

[Ps 23456789012345 e [3 jDSQVTVDVLADPQED F-7 I QVTVDVLAD)PQEDSG II3 [10 TVDVLADPQEDSK 12I [illVDVLADPQEDSGKV i [3 SRDSQVTVDVADPQ IF 17 [m jADPQEDSGKQVDLVS

DI

STabIeXLIX-V14-HLA-DRB1-1 101- 1 Smers-1 91P4031 2B 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 pepide is the start position plus fourteen.

[Pos 123456789012345 Iscre [12 PASASLVAGTLSVHHjF_ 1I3 I 71GLELLGSSNPPASAS I LELLGSSNPPASASLF-121[ [EIiiPPASASLV TLH 11 F3 8SSNPPASASLVAGTL I 14 SASLVAGTLSVIIO 13 fI AGLELLGSSNPP-ASA E31 E3 ELLGSSNPPAAL 116 [3 LLGSSNPPASASLA 1 [3 jSNPPASASLVAGTS161 [Is ASLVAGTLSVH=HCAC 136 2008202217 20 May 2008 Table L: Properties of 191P4D12(b) 00 00 Ig1P4D12(b)B v.1

ORF

Protein length Transmembrane region Signal Peptide

PI

Molecular weight Localization Motifs Bioinformatic Program ORF finder TM Pred HMMTop Sosul

TMHMM

Signal P p1/MW tool p1/MW tool

PSORT

PSORTI11 Pfam Prints Blocks http:Ilwww.ch.embnet.org/ hftp://www.enzim.hulhmmtop/ http:Ilwww.genome.ad.jp/SOSuiI hftp://www.cbs.dtu.dk/servicesTMHMM http:Ilwww.cbs.dtu.dkservices/SignaIP/ hftp://www.expas y.ch/tools/ http://www.expasy.ch/tools/ http:/Ipsort.nibb.ac.jp/ httpJ/psor~nibb.ac.jp/ http://www.sanger.ac.uk/Pfam/ http:llwww.biochem.ucl.ac.uk/ http:/Iwww.blocks.fhcrc.org/ Outcome 264-1796 2 TM, aa 14-30, 351 -370 1 TM, aa 347-371 2 TM, aa 14-31, 347-369 1 TM, aa 350-372 yes, cleaved aa 31-32 pl 5.27 55.4 kDa 46% plasma membrane 39.1 cytoplasmic, 21 nuclear Immunoglobulin domain Cadherin signature Ig domain, Herpesviwus glycoprotein D Bioinformatic v.6 Program URL Outcome ORF ORE finder Protein length Transmembrane region Signal Peptide pI Molecular weight Localization Motifs TM Pred HMMTop Sosui

TMHMM

Signal P p1/MW tool p1/MW tool

PSORT

PSORT 11 Pfam Prints Blocks http://www.ch.embnetorg/ http:/lwww.enzim.hulhmmtop/ http://www.genome.ad.jp/SOSuV/ http://www.cbs.dtu.dk/servicesrrMHMM httpJ/www.cbs.dtu.dk/services/SignaIP/ http://www.expasy.ch/toolsI httpJ/www.expasy.ch/tools/ http://psort.nibb.ac.jp/ http://psort.nibb.ac.jp/ http://www.sanger. ac.uk/Pfam/ http://www.biochem.ucl.ac.ukl http:f/www.blocks fiicrc-org/ 295 as I TM, aa 135-156 1 TMV, aa 132-156 1 TM, aa 132-154 1 TM, aa 135-157 none pl 5.28 32.6 k~a 70% plasma membrane, 20% endoplasmic reticulum 39% cytoplasmic, 21% nuclear Immunoglobulin domain none Herpesvirus glycoprotein D 00 00 Table LI: Exon boundaries of transcript 191 P4D12(b) v.1 Exon Number Start End Length 1 2 342 341 2 343 702 360 3 703 993 291 4 994 1114 1211 1115 1263 149 6 1264 1420 157 7 1421 1496 76 8 1497 1571 9 1572 3459 1888 Table UlI(a). Nucleotide sequence of transcript variant 191 P4DI 2(b) v.6 (SEQ ID NO: 105) ggccgtcgtt acggcttctt tcccctagtg cagttcctta agctggagac t ctaccgagg gcgaaggcgc cttacgaggg tcctgcgcaa .ccgccggcag tgaatcctgg ctgagggcag gccgttcctt gccgcagcat accaaaggat ttgaagacca aagggcagcc tacgagtgga acgtctgcca ttgaccccca tgggtgtgat gataccatcg ccagggagaa aggagagtgt gctctgtgat agatagaaac atcaggatga gggccaagcc ggcctgcctc ttgggggcct cttgaccttt caccatgcat tgtgtgtgtg ctgtcatatc gggcaacact aaagcaggta ggtggagact ggtgtgaggg gtCCCtgggt tgggcctgCt aatactgctC tgtatttttt tcaggctggc gttggccaca gggggtagct gagacccaag ttcaagtctg ctcagacgtg ggactccggc ccaggaacta ccgcgtggag cgcagtgcag cttccaggcg tccagcacta cccagccccc caagcactcc gaatgggcag cacccacatc aaatctgtgg ccctccctca tggggacact tgtcagcaat ggaagactct cgccgcactc gcgcaaggcc ctccatccgg agggctgaga gagtgaagag ac agac tgaa aggcatcaaa cacgggcaat ccttccctag ccttaaacac acctccaacc gcaggtcact gaggggtgac agagtcaagt gtcagggttt ttttctcaga gtggctcaga aacctgtctc cagccagagg gcatgtacat cgaatcactt atttattttt cttgaactcc gcgtgggaag acggctgggt tgcgagaggc ctactgctgg gtaactgtgg gagcaagtgg gcgctactgc cagccgccgc gcggatgagg cggctgcggc gaagagggcc agcgtgacct cgctctgctg ccactgactt ctccacgtgt caca ttggca tacaactgga ttgggctttc gagttctcct gggaagcagg ttgttctgcc cagcagatga aggctgcatt gc cgagggc c cccgagggcc ctgctgtctc caggc catga ggcatctaca gcctggctcc ccccatttct cttctgttca gtgtgtgtgc tgtccgtgga gaactgtggt ggcgtgtgtg ccccagagca cccaggtgtg ctaccacttc ct tgaactgt attttctgta ttaatttttt atttttattt tgggCtcaag cagctctggg gtgtagaacg aagaactctg catcatttac tgctgggcca ggcaagtggc actccaaata ccccacgcaa gcgagtacga tccgagtgct agggcctgac gggacacgga ccgtcacctc gtgtggtgtc ccttccttgc gagaaggagc cacggctgga ccccactgac caagggattc tggacctagt ttCtggtggt cccagaaata cccatcacac accctgatag gcagttactc caggctctgg accattttgt tcaatgggcg ttctgttgac tgcggaagat t cgggagggc atgtgtgcct ggggtgactg gtatgtgcca tcatgtggct gtattaatga cgggcatagc ggagccatgg tacagaagcc aatatacatg tctttttttt ttttttagag caatcctcct ggagctcgga gggccggggc cagcttcctg aggccggtgc ggacgcaaaa atgggctcgg cgggcttcat ccccctggac gtgccgggtc ggtgCCtCCC CC tggCagcCC ggtcaaaggc agagttccac c cat c ctgg c tgaggcctct tatgctcaag tgggcctctg cac tgagcac tcaggtcact gtcagcctcg ggtggtggtg tgaggaggag ggaccccagg tctcaaggac cacgctgacc gcgggccgag t caggagaat gggacacctg atgggagatt gctccccatc tccaccaatt gtgtgagtgt tgtccgtggt cgggatttga gtgtgtgacc tgcagaggt t tggagctgga gggcaagtgt CtCtgCCCtC CgCCgggagc ttcttgccct atggagtctc cCt cagocct gctcccgatc tggggctggg ccttctgggt c ccgcgggtg C tgcC Ctgc t gtggacgcgg gtgagcccgg ggCtcagtgc agcaccttcc ctgccctcac tcctgcacag acaacgtcca ttggtgccta ctgctccagg gtgaggggcc tgcctgagtg cccagtgggg agcggcatct gtggatgttc gtggtggtgg ctcatgtccc ctgaccctga agccagccgg aacagtagct acggtgaggg gaggaggaag gggaccctac gtctgaccca ttagctcatc ccactgactg gagtctctcc tgactgactg gtgtattatg gtggttgcgt tctgcctgaa ggaggagaga atctgcctcc gaagcagcca tggtggcctc ttcttgcagg ttccattagt actatgttgc ccctagtagc 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 2340 2400 2460 2520 2580 tgggacttta agtgtacacc actgtgcctg ctttgaatcc tttacgaaga gaaaaaaaaa 2640 attaaagaaa gcctttagat ttatccaatg tttactactg ggattgctta aagtgaggCC 2700 cctccaacac cagggggtta attcctgtga ttgtgaaagg ggCtacttcc aaggcatctt 2760 00 catgcaggca gccccttggg agggcacctg agagctggta gagtctgaaa ttagggatgt 2820 C) gagcctcgtg gttactgagt aaggtaaaat tgcatccacc attgtttgtg ataccttagg 2880 C) gaattgcttg gacctggtga caagggctcc tgttcaatag tggtgttggg gagagagaga 2940 gcagtgatta tagaccgaga gagtaggagt tgaggtgagg tgaaggaggt gctgggggtg 3000 Sagaatgtcgc ctttccccct gggttttgga tcactaattc aaggctcttc tggatgtttc 3060 tctgggttgg ggctggagtt caatgaggtt tatttttagc tggcccaccc agatacactc 3120 agccagaata cctagattta gtacccaaac tcttcttagt ctgaaatctg ctggatttct 3180 ggcctaaggg agaggctccc atccttcgtt ccccagccag cctaggactt cgaatgtgga 3240 gcctgaagat ctaagatcct aacatgtaca ttttatgtaa atatgtgcat atttgtacat 3300 aaaatgatat tctgttttta aataaacaga caaaacttga aaaa 3344 Table 1.lll(a). Nucleotide sequence alignment of 191P4D12(b) M. (SEQ ID NO: 106) and 19113012(b) v.6 (SEQ ID NO: 107).

ci V.1 1 gGCCGTCGTTGTTGGCCACAGCGTGGGAAGCAGCTCTGGGGGAGCTCGGA ci V.6 1 ggccgtcgttgttggccacagcgtggaagcagctctgggggagctcgga 00 V.1 51 GCTCCCGATCACGGCTTCTTGCGGGTAGCTACGGCTGGGTGTGTAGAACG 100 ci V.6 51 gctcccgatcacggcttcttgggggtagctacggctgggtgtgtagaacg 100 V.1 101 GGGCCGGGGCTGGGGCTGGGTCCCCTAGTGGAGACCCAAGTGCGAGAGGC 150 V.6 101 gggccggggctggggctgggtcccctagtggagacccaagtgcgagaggc 150 V.1 151 AAGAACTCTGCAGCTTCCTGCCTTCTGGGTCAGTTCCTTATTCAAGTCTG 200 V.6 151 aagaactctgcagcttcctgccttctgggtcagttccttattcaagt-- 197 V.1 201 CAGCCGGCTCCCAGGGAGATCTCGGTGGAACTTCAGAAACGCTGGGCAGT 250 V.6 197 V.1 251 CTGCCTTTCAACCATGCCCCTGTCCCTGGGAGCCGAGATGTGGGGGCCTG 300 V.6 197 V.1 301 AGGCCTGGCTGCTGCTGCTGCTACTGCTGGCATCATTTACAGGCCGGTGC 350 V.6 ctgctactgctggcatcatttacaggccggtgc 230 V. 1 351 CCCGCGGGTGAGCTGGAGACCTCAGACGTGGTAACTGTGGTGCTGGGCCA 400 V. 6 231 cccgcgggtgagctggagacctcagacgtggtaactgtggtgctgggcca 280 V. 1 401 GGACGCAAAACTGCCCTGCTTCTACCGAGGGGACTCCGGCGAGCAAGTGG 450 V. 6 281 ggacgcaaaactgccctgcttctaccgaggggactccggcgagcaagtgg 330 V. 1 451 GGCAAGTGGCATGGGCTCGGGTGGACGCGGGCGAAGGCGCCCAGGAACTA 500 V.6 331 ggcaagtggcatgggctcgggtggacgcgggcgaaggcgcccaggaacta 380 v.1 501 GCGCTACTGCACTCCAAATACGGGCTTCATGTGAGCCCGGCTTACGAGGG 550 V.6 381 gcgctactgcactccaaatacgggcttcatgtgagcccggcttacgaggg 430 V.1 551 CCGCGTGGAGCAGCCGCCGCCCCCACGCAACCCCCTGGACGGCTCAGTGC 600 235 V.6 431 ccgcgtggagcagccgccgcccccacgcaaccccctggacggctcagtgc 480 00 V.1 601 TCCTGCGCAACGCAGTGCAGGCGGATGAGGGCGAGTACGAGTGCCGGGTC 650 V.6 481 tcctgcgcaacgcagtgcaggcggatgagggcgagtacgagtgccgggtc 530 V.1 651 AGCACCTTCCCCGCCGGCAGCTTCCAGGCGCGGCTGCGGCTCCGAGTGCT 700 S V.6 531 agcaccttccccgccggcagcttccaggcgcggctgcggctccgagtgct 580 V.1 701 GGTGCCTCCCCTGCCCTCACTGAATCCTGGTCCAGCACTAGAAGAGGGCC 750 V.6 581 ggtgcctcccctgccctcactgaatcctggtccagcactagaagagggcc 630 V.1 751 AGGGCCTGACCCTGGCAGCCTCCTGCACAGCTGAGGGCAGCCCAGCCCCC 800 ri V.6 631 agggcctgaccctggcagcctcctgcacagctgagggcagcccagccccc 680 V.1 801 AGCGTGACCTGGGACACGGAGGTCAAAGGCACAACGTCCAGCCGTTCCTT 850 00 V.6 681 agcgtgacctgggacacggaggtcaaaggcacaacgtccagccgttcctt 730 V. 1 851 CAAGCACTCCCGCTCTGCTGCCGTCACCTCAGAGTTCCACTTGGTGCCTA 900 V.6 731 caagcactcccgctctgctgccgtcacctcagagttccacttggtgccta 780 V.1 901 GCCGCAGCATGAATGGGCAGCCACTGACTTGTGTGGTGTCCCATCCTGGC 950 V.6 781 gccgcagcatgaatgggcagccactgacttgtgtggtgtcccatcctggc 830 V. 1 951 CTGCTCCAGGACCAAAGGATCACCCACATCCTCCACGTGTCCTTCCTTGC 1000 V.6 831 ctgctccaggaccaaaggatcacccacatcctccacgtgtccttccttgc 880 V.1 1001 TGAGGCCTCTGTGAGGGGCCTTGAAGACCA-AAATCTGTGGCACATTGGCA 1050 'V.6 881 tgaggcctctgtgaggggccttgaagaccaaaatctgtggcacatcggca 930 V.1 1051 GAGAAGGAGCTATGCTCAAGTGCCTGAGTGAAGGGCAGCCCCCTCCCTCA 1100 V.6 931 gagaaggagctatgctcaagtgcctgagtgaagggcagccccctccctca 980 V.1 1101 TACAACTGGACACGGCTGGATGGGCCTCTGCCCAGTGGGGTACGAGTGGA 1150 V.6 981 tacaactggacacggctggatgggcctctgcccagtggggtacgagtgga 1030 V.1 1151 TGGGGACACTTTGGGCTTTCCCCCACTGACCACTGAGCACAGCGGCATCT 1200 V.6 1031 tggggacactttgggctttcccccactgaccactgagcacagcggcatct 1080 V.1 1201 ACGTCTGCCATGTCAGCAATGAGTTCTCCTCAAGGGATTCTCAGGTCACT 1250 V.6 1081 acgtctgccatgtcagcaatgagttctcctcaagggattctcaggtcact 1130 V.1 1251 GTGGATGTTCTTGACCCCCAGGAAGACTCTGGGAAGCAGGTGGACCTAGT 1300 V.6 1131 gtggatgttcttgacccccaggaagactctgggaagcaggtggacctagt 1180 V. 1 1301 GTCAGCCTCGGTGGTGGTGGTGGGTGTGATCGCCGCACTCTTGTTCTGCC 1350 V.6 1181 gtcagcctcggtggtggtggtgggtgtgatcgccgcactcttgttctgcc 1230 V.1 1351 TTCTGGTGGTGGTGGTGGTGCTCATGTCCCGATACCATCGGCGCAAGGCC 1400 V.6 1231 ttctggtggtggtggtggtgctcatgtcccgataccatcggcgcaaggcc 1280 00 V.1 1401 CAGCAGATGACCCAGAAATATGAGGAGTGAGCTGACCCTGACCAGGGAGAA 1450 S V.6 1281 cagcagatgacccagaaatatgaggaggagctgaccctgaccagggagaa 1330 V.1 1451 CTCCATCCGGAGGCTGCATTCCCATCACACGGACCCCAGGAGCCAGCCGG 1500 VA6 1331 ctccatccggaggctgcattcccatcacacggaccccaggagccagccgg 1380 V.1 1501 AGGAGAGTGTAGGGCTGAGAGCCGAGGGCCACCCTGATAGTCTCAAGGAC 1550 V.6 1381 aggagagtgtagggctgagagccgagggccaccctgatagtctcaaggac 1430 V.1 1551 AACAGTAGCTGCTCTGTGATGAGTGAAGAGCCCGAGGGCCGCAGTTACTC 1600 V.6 1431 aacagtagctgctctgtgatgagtgaagagcccgagggccgcagttactc 1480 V.1 1601 CACGCTGACCACGGTGAGGGAGATAGAAACACAGACTGAACTGCTGTCTC 1650 S V.6 1481 cacgctgaccacggtgagggagatagaaacacagactgaactgctgtctc 1530 (1 V.1 1651 CAGGCTCTGGGCGGGCCGAGGAGGAGGAAGATCAGGATGAAGGCATCA 1700 V.6 1531 caggctctgggcgggccgaggaggaggaagatcaggatgaaggcatcaaa 1580 V.1 1701 CAGGCCATGAACCATTTTGTTCAGGAGAATGGGACCCTACGGGCCAAGCC 1750 V.6; 1581 caggccatgaaccattttgttcaggagaatgggaccctacgggccaagcc 1630 V.1 1751 CACGGGCAATGGCATCTACATCAATGG7GCGGGGACACCTGGTCTGACCA 1800 V.6 1631 cacgggcaatggcatctacatcaatgggcggggacacctggtctgaccca 1680 V.1 1801 GGCCTGCCTCCCTTCCCTAGGCCTGGCTCCTTCTGTTGACATGGGAGATT 1850 V.6 1681 ggcctgcctcccttccctaggcctggctccttctgttgacatgggagatt 1730 V.1 1851 TTAGCTCATCTTGGGGGCCTCCTTA-AACACCCCCATTCTTGCGGAAGAT 1900 V.6 1731 ttagctcatcttgggggcctccttaaacacccccatttcttgcggaagat 1780 V.1 1901 GCTCCCCATCCCACTGACTGCTTGACCTTTACCTCCAACCCTTCTGTTCA 1950 V.6 1781 gctccccatcccactgactgcttgacctttacctccaacccttctgttca 1830 V.1 1951 TCGGGAGGGCTCCACCAATTGAGTCTCTCCCACCATGCATGCAGGTCACT 2000 V.6 1831 tcgggagggctccaccaattgagtctctcccaccatgcatgcaggtcact 1880 V.1 2001 GTGTGTGTGCATGTGTGCCTGTGTGAGTGTTrGACTGACTGTGTGTGTGTG 2050 V.6 1881 gtgtgtgtgcatgtgtgcctgtgtgagtgttgactgactgtgtgtgtgtg 1930 V.1 2051 GAGGGGTGACTGTCCGTGGAGGGGTGACTGTGTCCGTGGTGTGTATTATG 2100 VA6 1931 gaggggtgactgtccgtggaggggtgactgtgtccgtggtgtgtattatg 1980 V.1 2101 CTGTCATATCAGAGTCAAGTGAACTGTGGTGTATGTGCCACGGGATTTGA 2150 V.6 1981 ctgtcatatcagagtcaagtgaactgtggtgtatgtgccacgggatttga 2030 V.1 2151 GTGGTTGCGTGGGCAACACTGTCAGGGTTTGGCGTGTGTGTCATGTGiGCT 2200 00 V.6 2031 gtggttgcgtgggcaacactgtcagggtttggcgtgtgtgtcatgtggct 2080 V.1 2201 GTGTGTGACCTCTGCCTGAAAAAGCAGGTATTTTCTCAGACCCCAGAGCA 2250 V. 6 2081 gtgtgtgacctctgcctgaaaaagcaggtattttctcagaccccagagca 2130 V.1 2251 GTATTAATGATGCAGAGGTTGGAGGAGAGAGGTGGAGACTGTGGCTCAGA 2300 V. 2131 gttatagaagtggaaagga acggtaa 2180 V.1 230 CCCAGGGTGCGGCATAGCGGAGCTII ATIIIIIICGIITGIIIG 235 V.6 2181 cccaggtgtgcgggcatagctggagctggaatctgcctccggtgtgaggg 2230 CI V.1 2351 AACCTGTCTCCTACCACTTCGGAGCCATGGGGGGCAAGTGTGAAGCAGCCA 2400 V.6 2231 aacctgtctcctaccacttcggagccatgggggcaagtgtgaagcagcca 2280 00 V.1 2401 GTCCCTGGGTCAGCCAGAGGCTTGAACTGTTACAGAAGCCCTCTGCCCTC 2450 V.6 2281 gtccctgggtcagccagaggcttgaactgttacagaagccctctgccctc 2330 V.1 2451 TGGTGGCCTCTGGGCCTGCTGCATGTACATATTTTCTGTAAATATACATG 2500 V.6 2331 tggtggcctctgggcctgctgcatgtacatattttctgtaaatatacatg 2380 V.1 2501 CGCCGGGAGCTTCTTGCAGGAATACTGCTCCGAATCACTTTTAATTTTTT 2550 V.6 2381 cgccgggagcttcttgcaggaatactgctccgaatcacttttaatttttt 2430 V.1 2551 TCTTTTTTTTTTCTTGCCCTTTCCATTAGTTGTATTTTTTATTTATTTTT 2600 V.6 2431 tcttttttttttcttgccctttccattagttgtattttttatttattttt 2480 V.1 2601 ATTTTTATTTTTTTTTAGAGATGGAGTCTCACTATGTTGCTCAGGCTGGC 2650 V.6 2481 atttttatttttttttagagatggagtctcactatgttgctcaggctggc 2530 V.1 2651 CTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTCAGCCTCCCTAGTAGC 2700 V.6 2531 cttgaactcctgggctcaagcaatcctcctgcctcagcctccctagtagc 2580 V.1 2701 TGGGACTTTAAGTGTACACCACTGTGCCTGCTTTGAATCCTTTACGAAGA 2750 V.6 2581 tgggactttaagtgtacaccactgtgcctgctttgaatcctttacgaaga 2630 V.1 2751 GAAAAAAATTAAAGAAAGCCTTTAGATTTATCCAATGTTTACTACTG 2800 V.6 2631 gaaaaaaaaaattaaagaaagcctttagatttatccaatgtttactactg 2680 V.1 2801 GGATTGCTTAAAGTGAGGCCCCTCCAACACCAGGGGGTTAATTCCTGTGA 2850 V.6 2681 ggattgcttaaagtgaggcccctccaacaccagggggttaattcctgtga 2730 V. 1 2851 TTGTGAAAGGGGCTACTTCCAAGGCATCTTCATGCAGGCAGCCCCTTGGG 2900 V.6 2731 ttgtgaaaggggctacttccaaggcatcttcatgcaggcagccccttggg 2780 V.1 2901 AGGGCACCTGAGAGCTGGTAGAGTCTGAAATTAGGGATGTGAGCCTCGTG 2950 V.6 2781 agggcacctgagagctggtagagtctgaaattagggatgtgagcctcgtg 2830 238 V.1 2951 GTTACTGAGTAAGGTAAAATTGCATCCACCATTGTTTGTGATACCTTAGG 3000 00 V.6 2831 gttactgagtaaggtaaaattgcatccaccattgtttgtgataccttagg 2880 V.1 3001 GAATTGCTTGGACCTGGTGACAAGGGCTCCTGTTCAATAGTGGTGTTGGG 3050 V.6 2881 gaattgcttggacctggtgacaagggctcctgttcaatagtggtgttggg 2930 V.1 3051. GAGAGAGAGAGCAGTGATTATAGACCGAGAGAGTAGGAGTTGAGGTGAGG 3100 S V.6 291gagagagagagcagtgattatagaccgagagagtaggagttgaggtgagg 2980 V.1 30 TGAAGGAGGTGCTGGGGGTGAGAATGTCGCCTTTCCCCCTGGGTTTTGGA 3150 V.6 981tgaaggaggtgctgggggtgagaatgtcgcctttccccctgggttttgga 3030 V.1 151TCACTAATTCAAGGCTCTTCTGGATGTTTCTCTGGGTTGGGGCTGGAGTT 3200 V6 3031 tcactaattcaaggctcttctggatgtttctctgggttggggctggagtt 3080 00 S V.1 3201 CAATGAGGTTTATTTTTAGCTGGCCCACCCAGATACACTCAGCCAGAATA 3250 C] V.6 3081 caatgaggtttatttttagctggcccacccagatacactcagccagaata 3130 V.1 3251 CCTAGATTTAGTACCCAAACTCTTCTTAGTCTGAAATCTGCTGGATTTCT 3300 V.6 3131 cctagatttagtacccaaactcttcttagtctgaaatctgctggatttct 3180 V.1 3301 GGCCTAAGGGAGAGGCTCCCATCCTTCGTTCCCCAGCCAGCCTAGGACTT 3350 V.6 3181 ggcctaagggagaggctcccatccttcgttccccagccagcctaggactt 3230 V.1 3351 CGAATGTGGAGCCTGAAGATCTAAGATCCTAACATGTACATTTTATGTAA 3400 V.6 3231 cgaatgtggagcctgaagatctaagatcctaacatgtacattttatgtaa 3280 V.1 3401 ATATGTGCATATTTGTACATAAAATGATATTCTGTTTTTAAATAAACAGA 3450 V.6 3281 atatgtgcatatttgtacataaaatgatattctgtttttaaataaacaga 3330 V.1 3451 CAAAACTTGaaaaa 3464 V.6 3331 caaaacttgaaaaa 3344 Table LiV(a). Peptide sequences of protein coded by 191P4012(b) v.6 (SEQ ID NO: 108) NNGQPLTCVV SHPGLLQDQR ITHILHVSFL AEASVRGLED QNLjWHIGREG AMLKCLSEGQ PPPSYNWTRL DGPLPSGVRV DGDTLGFPPL TTEHSGIY'VC IiVSNEFSSRfl SQVTVDVLDP 120 QEDSGKQVDL VSASVVVVGV IAALLFCLLV VVVVLMSRYH RRKAQQMTQK YEEELTLTRE 180 NSIRRLHSHH TDPRSQPEES VGLRAEGHPD SLKDNSSCSV MSEEPEGRSY STLTTVREIE 240 TQTELLSPGS GRAEEEEDQD EGIKQAMNHF VQE1NGTLRAK PTGNGIYING RGHLV 295 Table LV(a). Amino acid sequence alignment of 19113012(b) M. (SEQ ID NO: 109) and 191P4D12(b) v.6 (SEQ ID NO: 110) V.1 216 MNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREG 265 V.6 1 MNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREG V.1 266 AMLKCLSEGQPPPSYNWTRILDGPLPSGVRVDGDTLGFPPLTTEHSGIYVC 315 V. 6 51 AMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVC 100 V. 1 316 1VSNESSRflQVTVDVLDPQEDSGKQVDLVSASvvvVGVIAALLFCLLV 365 239 I 1 1 I1 1 1 1 I1 1 11 1 Ii I1 11 1 IIIIIIIIIIIIIIIII II II I II V.6 101 IIVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAAIJLFCLLV V.1 366 VVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEES V.6 151 VVVVLMSRYHRRKAQQMTQKYEEELTIJTRENSIRpTRSHHJTDPRSQPEES V. 1 416 VGLRAEGH-PDSLKNSSCSVMSEEPEGRSYSTLTT=REIETQTELLSPGS V.6 201 VGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTI'VREIETQTELLSPGS V.1 466 GRAEEEEDQDEGIKQAHFVQENGTLRAkKPTOGIYINGRGHLV V.6 251 GRAEEEEDQDEGIKQAVNHFVQENGTLRAJCPTGNGIYINGRGHLV 150 415 200U 465 250 Table 111(b). Nucleotide sequence of transcript variant 191 P4DI 2(b) v.7 (SEQ ID NO: ggccgtcgtt acggcttctt tcccctagtg cagttcctta gc tgggcagt aggcctggct agctggagac tctaccgagg gcgaaggcgc cttacgaggg tcctgcgcaa ccgccggcag tgaatcctgg ctgagggcag gccgttcctt gccgcagcat a cc aaagga t ttgaagacca aagggcagcc t acgagtgga acgtctgcca ttgaccccca tgggtgtgat gataccatcg ccagggagaa aagagcccga ctgaactgct tcaaacaggc gcaatggcat cc taggccty aacaccCCCa caacccttct tcactgtgtg gtgactgtcc caagtgaact ggtttggcgt tcagacccca tcagacccag gtctcctacc agaggcttga tacatatttt cacttttaat tttttatttt actcctgggc acaccactgt gttggccaca gggggt agc t gagacccaag ttcaagtctg ctgcctttca gctgctgctg ctcagacgtg ggact ccggc ccaggaacta ccgcgtggag cgcagtgcag cttccaggcg tccagcacta cccagccccc caagcactcc gaatgggcag cacccacatc aaatctgtgg ccctccctca tggggacact tgtcagcaat ggaagactct cgccgcactc gcgcaaggcc ctccatccgg gggccgcagt gtctccaggc catgaaccat ctacatcaat gctccttctg tttcttgcgg gttcatcggg tgtgcatgtg gtggaggggt gtggtgtatg gtgtgtcatg gagcagtatt gtgtgcgggc acttcggagc actgttacag Ctgtaaatat ttttttcttt tatttttttt tcaagcaatc gcctgctttg gcgtgggaag acggctgggt tgcgagaggc cagccggctc accatgcccc ctactgctgg gtaactgtgg gagcaagtgg gcgctactgc cagccgccgc gcggatgagg cggctgcggc gaagagggcc agcgtgacct cgctctgctg ccactgactt Ctccacgtgt cacattggca tacaactgga ttgggctttc gagttctcct gggaagcagg ttgttctgcc cagcagatga aggctgcatt tactccacgc tctgggcggg tttgttcagg gggcggggac ttgacatggg aagatgctcc agggctccac tgcctgtgtg gactgtgtcc tgccacggga tggctgtgtg aatgatgcag atagctggag Catgggggca aagccctctg acatgcgccg tttttttctt tagagatgga Ctcctgcctc aatcctttac cagctctggg gtgtagaacg aagaactctg ccagggagat tgtccctggg catcatttac tgctgggcca ggcaagtggc actccaaata ccccacgcaa gcgagtacga tccgagtgct agggcctgac gggacacgga ccgtcacctc gtgtggtgtc ccttccttgc gagaaggagc cacggctgga ccccactgac caagggattc tggacctagt ttctggtggt cccagaaata cccatcacac tgaccacggt ccgaggagga agaatgggac acctggtctg agattttagc ccatcccact caattgagtc agtgttgact gtggtgtgta tttgagtggt tgacctctgc aggt tggagg ctggaatctg agtgtgaagc ccctctggtg ggagcttctt gccCtttcca gtctcactat agcctcccta gaagagaaaa ggagctcgga gggccggggc cagcttcctg ctcggtggaa agccgagatg aggccggtgc ggacgcaaaa atgggctcgg c ggg ct t cat ccccctggac gtgccgggtc ggtgCCtCCC cctggcagcc ggtcaaaggc agagttccac ccatcctggc tgaggcctct tatgctcaag tgggcctctg cac tgagcac tcaggtcact gtcagcctcg ggtggtggtg tgaggaggag ggaccccagg gagggagata ggaagatcag cctacgggcc acccaggcct tcatcttggg gactgcttga tctcccacca gactgtgtgt ttatgctgtc tgcgtgggca Ctgaaaaagc agagaggtgg cctccggtgt agccagtccc gcctctgggc gcaggaatac ttagttgtat gttgctcagg gtagctggga aaaaaattaa 111) gctcccgatc tggggctggg cc tt ctgggt cttcagaaac tgggggcctg CCCgCgggtg ctgccctgct gtggacgcgg gtgagcccgg ggctcagtgc agcaccttcc ctgccctcac tcctgcacag acaacgtcca ttggtgccta c tgctccagg gtgaggggcc tgcctgagtg cccagtgggg agcggcatct gtggatgttc gtggtggtgg ctcatgtccc ctgaccctga agccagagtg g~aacacaga gatgaaggca aagcccacgg gCCtCCCttC ggcctcctta CCtttacctc tgcatgcagg gtgtggaggg a tat cagag t acactgtcag aggtattttc agactgtggc gagggaacct tgggtcagcc ctgctgcatg tgctccgaat tttttattta Ctggcct tga ctttaagtgt agaaagcctt 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 2340 2400 2460 2520 2580 2640 2700 tagatttatc caatgtttac tactgggatt gcttaaagtg aggcccctcc aacaccaggg 2760 ggttaattCc tgtgattgtg aaaggggcta cttccaaggc atcttcatgc aggcagcccc 2820 ttggagggc acctgagagc tggtaga~tc tgaaattagg gatgtgagcc tcgtggttac 2880 00 tgagtaaggt aaaattgcat ccaccattgt ttgtgatacc ttagggaatt gcttggacct 2940 ggtgacaagg gctcctgttc aatagtggtg ttggggagag agagagcagt gattatagac 3000 S cgagagagta ggagttgagg tgaggtgaag gaggtgctgg gggtgagaat gtcgcctttc 3060 S cccctgggtt ttggatcact aattcaaggc tcttctggat gtttctctgg gttggggctg 3120 gagttcaatg aggtttattt ttagctggcc cacccagata cactcagcca gaatacctag 3180 S atttagtacc caaactcttc ttagtctgaa atctgctgga tttctggcct aagggagagg 3240 S ctcccatcct tcgttcccca gccagcctag gacttcgaat gtggagcctg aagatctaag 3300 atcctaacat gtacatttta tgtaaatatg tgcatatttg tacataaaat gatattctgt 3360 S ttttaaataa acagacaaaa cttgaaaaa 3389 Table 1-11(b). Nucleotide sequence alignment of 191P4D12(b) v.1 (SEQ ID NO: 112) and 191P4D12(b) v.7 (SEQ ID NO: 113) V.1 1 gGCCGTCGTTGTTGGCCACAGCGTGGGAAGCAGCTCTGGGGGAGCTCGGA s0 V.7 1 1111111111111111ggaacacttgggagtcga 00 .1 51 GCTCCCGATCACGGCTTCTTGGGGGTAGCTACGGCT(GQTGTGTAGAACG 100 S V.7 51 gctcccgatcacggcttcttgggggtagctacggctgggtgtgtagaacg 100 V. 1 2.01 GGGCCGGGGCTGGGGCTGOGTCCCCTAGTGGAGACCCAAGTGCGAGAGGC 150 V.7 101 gggccggggctggggctgggtcccctagtggagacccaagtgcgagaggc 150 V.1 151 AAGAACTCTGCAGCTTCCTGCCTTCTGGGTCAGTTCCTTATTCAAGTCTG 200 V.7 151 aagaactctgcagcttcctgccttctgggtcagttccttattcaagtctg 200 V.1 201 CAGCCGGCTCCCAGGGAGATCTCGGTGGAACTTCAGAAACGCTGGGCAGT 250 V.7 201 cagccggctcccagggagatctcggtggaacttcagaaacgctgggcagt 250 V.1 251 CTGCCTTTCAACCATGCCCCTGTCCCTGGGAGCCGAGATGTGGGGGCCTG 300 V.7 251 ctgcctttcaaccatgcccctgtccctgggagccgagatgtgggggcctg 300 V.1 301 AGGCCTGGCTGCTGCTGCTGCTACTGCTGGCATCATTTACAGGCCGGTGC 350 V.7 301 aggcctggctgctgctgctgctactgctggcatcatttacaggccggtgc 350 V.1 351 CCCGCGGGTGAGCTGGAGACCTCAGACGTGGTAACTGTGGTGCTGGGCCA 400 V.7 351 cccgcgggtgagctggagacctcagacgtggtaactgtggtgctgggcca 400 V.1 401 GGACGCAAAACTGCCCTGCTTCTACCGAGGGGACTCCGGCGAGCAAGTGG 450 V.7 401 ggacgcaaaactgccctgcttctaccgaggggactccggcgagcaagtgg 450 V. 1 451 GGCAAGTGGCATGGGCTCGGGTGGACGCGGGCGAAGGCGCCCAGGAACTA 500 V.7 451 ggcaagtggcatgggctcgggtggacgcgggcgaaggcgcccaggaacta 500 V.1 501 GCGCTACTGCACTCCAAATACGGGCTTCATGTGAGCCCGGCTTACGAGGG 550 V.7 501 gcgctactgcactccaaatacgggcttcatgtgagcccggcttacgaggg 550 V.1 551. CCGCGTGGAGCAGCCGCCGCCCCCACGCAACCCCCTGGACGGCTCAGTGC 600 V.7 551 ccgcgtggagcagccgccgcccccacgcaaccccctggacggctcagtgc 600 V.1 601 TCCTGCGCAACGCAGTGCAGGCGGATGAGGGCGAGTACGAGTGCCGGGTC 650 00 V.7 601 tcctgcgcaacgcagtgcaggcggatgagggcgagtacgagtgccgggtc 650 S V.1 651 AGCACCTTCCCCGCCGGCAGCTTCCAGGCGCGGCGCGGCTCCGAGTGCT 700 S V.7 651 agcaccttccccgccggcagcttccaggcgcggctgcggctccgagtgct 700 V.1 701 GGTGCCTCCCCTGCCCTCACTGAATCCTGGTCCAGCACTAGAAGAGGGCC 750 V.7 701 ggtgcctcccctgccctcactgaatcctggtccagcactagaagagggdc 750 V.1 751 AGGGCCTGACCCTGGCAGCCTCCTGCACAGCTGAGGGCAGCCCAGCCCCC 800 V.7 751 aggcgccgcgcctcaacgggaccgcc 800 V.1 801 AGCGTGACCTGGGACACGGAGGTCAGGCACACGTCCAGCCGTTCCTT 850 S V.7 801 agggctgaagagcaagaactcgctct 850 00 S V.1 851 CAAGCACTCCCGCTCTGCTGCCGTCACCTCAGAGTTCCACTTGGTGCCTA 900 V.7 8351 cagatcgttcgctactaatcatgtct 900 V.1 901 GCCGCAGCATGAATGGGCAGCCACTGACTTGTGTGGTGTCCCATCCTGGC 950 V.7 901 gcgactatgcgccgatgggttcactg 950 V.1 951 CTGCTCCAGGACCAAGGATCACCCACATCCTCCACGTGTCCTTCCTTGC 1000 V.7 951 ctccagcaagtacactccaggctctg 2000 V.1 1001 TGAGGCCTCTGTGAGGGGCCTTGAAGACCAAATCTGTGGCACATTGGCA 1050 V.7 1001 tggctttaggctagacaacggcctgc 1050 V.1 1051 GAAGACAGTAGGCGATAGGACCCCCC 1100 V7 1051 gaagacagtaggcgataggacccccc 1100 V.1 1101 TACACTGGACACGGCTGGATGGGCCTCTGCCCGTGGGGTACGAGTGGA 1150 V.7 1101 taacgaagcgaggcttccgggtcatg 1150 V.1 1251 TGGGGACACTTTGGGCTTTCCCCCACTGACCACTGAGCACAGCGGCATCT 1200 V.7 1151 tggaattgctcccctaccggaacgac 1200 V.1 1201 ACGTCTGCCATGTCAGCAATGAGTTCTCCTCAGGGATTCTCAGGTCACT 1250 V.7 1201 acttcagcgcaggttccaggtccgtat 1250 V.2 1252 GTGGATGTTCTTGACCCCCAGGAGACTCTGGGJGCAGGTGGACCTAGT 1300 V.7 1251 gtgttctaccagaaccggacgtgctg 1300 V.1 1301 GTCAGCCTCGGTGGTGGTGGTGGTGTGATCGCCGCACTCTTGTTCTGCC 1350 V.7 1301 gtactgtggtggggtaccgattgttc 1350 V.1 1351 TTCTGGTGGTGGTGGTGGTGCTCATGTCCCGATACATCGGCGCAAGGC 1400 242 V.7 1351 ttctggtggtggtggtggtgctcatgtcccgataccatcggcgcaaggcc 1400 V.1 1401 CAGCAGATGACCCAGAAATATGAGGAGGAGCTGACCCTGACCAGGGAGAA 1450 00 V.7 1402. cagcagatgacccagaaatatgaggaggagctgaccctgaccagggagaa 1450 V.1 1451 CTCCATCCGGAGGCTGCATTCCCATCACACGGACCCCAGGAGCCAGCCGG 1500 V.7 1451 ctccatccggaggctgeattcccatcacacggaccccaggagcca 1495 S V.1 1501 AGGAGAGTGTAGGGCTGAGAGCCGAGGGCCACCCTGATAGTCTCAAGGAC 1550 C1 V.7 1495 V.1 1551 AACAGTAGCTGCTCTGTGATGAGTGAAGAGCCCGAGGGCCGCAGTTACTC 1600 V.'7 gagtgaagagcccgagggccgcagttactc 1525 V.1 1601 CACGCTGACCACGGTGAGGGAGATAGAAACACAGACTGAACTGCTGTCTC 1650 00 V.7 1526 cacgctgaccacggtgagggagatagaaacacagactgaactgctgtctc 1575 V.1 1651 CAGGCTCTGGGCGGGCCGAGGAGGAGGAAGATCAGGATGAAGGCATCAAA 1700 V.7 1576 caggctctgggcgggccgaggaggaggaagatcaggatgaaggcatcaaa 1625 V.1 1701 CAGGCCATGAACCATTTTGTTCAGGAGAATGGGACCCTACGGGCCAAQCC 1750 V.7 1626 caggccatgaaccattttgttcaggagaatgggaccctacgggccaagcc 1675 V.1 1751 CACGGGCAATGGCATCTACATCAATGGGCGGGGACACCTGGTCTGACCCA 1800 V.7 1676 cacgggcaatggcatctacatcaatgggcggggacacctggtctgaccca 1725 V.1. 1801 GGCCTGCCTCCCTTCCCTAGGCCTGGCTCCTTCTGTTGACATGGGAGATT 1850 V. 7 1726 ggcctgcctcccttccctaggcctggctccttctgttgacatgggagatt 1775 V.1 1851 TTAGCTCATCTTGGGGGCCTCCTTAAACACCCCCATTTCTTGCGGAAGAT 1900 V.7 1776 ttagctcatcttgggggcctccttaaacacccccatttcttgcggaagat 182S V.1 1901 GCTCCCCATCCCACTGACTGCTTGACCTTTACCTCCAACCCTTCTGTTCA 1950 V.7 1826 gctccccatcccactgactgcttgacctttacctccaacccttctgttca 1875 V.1 1951 TCGGGAGGGCTCCACCAATTGAGTCTCTCCCACCATGCATGCAGGTCACT 2000 V.7 1876 tcgggayggctccaccaattgagtctctcccaccatgcatgcaggtcact 1925 V.1 2001 GTGTGTGTGCATGTGTGCCTGTGTGAGTGTTGACTGACTGTGTGTGTGTG 2050 V.7 1926 gtgtgtgtgcatgtgtgcctgtgtgagtgttgaCtgactgtgtgtgtgtg 1975 V.1 2051 GAGGGGTGACTGTCCGTGGAGGGGTGACTGTGTCCGTGGTGTGTATTATG 2100 V.7 1976 gaggggtgactgtccgtggaggggtgactgtgtccgtggtgtgtattatg 2025 V.1 2101 CTGTCATATCAGAGTCAAGTGAACTGTGGTGTATGTGCCACGGGATTTGA 2150 V.7 2026 ctgtcatatcagagtcaagtgaactgtggtgtatgtgccacgggatttga 2075 V.2 2151 OTGGTTGCGTGGOCAACACTGTCAGGGTTTGGCGTGTGTGTCATGTGGCT 2200 I II I111111111111 111111 IIIII1111 IIIII IIIII IIIII 1 V.7 20*76 gtggttgCgtgggcaacactgtcagggtttggcgtgtgtgtcatgtggct 2125 00 V.1 2201 GTTTACCGCGAAGCGT= CCGCCAAC 2250 V.7 2126 gtgtgtgacctctgcctgaaaaagcaggtattttctcagaccccagagca 2175 S V.1L 2251. GTATTAATGATGCAGAGGTTGGAGGAGAGAGGTGGAGACTGTGGCTCAGA 2300 V.7 2176 gtattaatgatgcagaggttggaggagagaggtggagactgtggctcaga 2225 V.1 2301 CCCAGGTGTGCGGGCATAGCTGGAGCTGGAATCTGCCTCCGGTGTGAGGG 2350 V.7 2226 cccaggtgtgcgggcatagctggagctggaatctgcctccggtgtgaggg 2275 V.1 2351 AACCTGTCTCCTACCACTTCGGAGCCATGGGGGCAAGTGTGAAGCAGCCA 2400 V.7 2276 aacctgtctcctaccacttcggagccatgggggcaagtgtgaagcagcca 2325 CI V.1 2401 GTCCCTGGGTCAGCCAGAGGCTTGAACTGTTACAGAAGCCCTCTGCCCTC 2450 V.7 2326 gtccctgggtcagccagaggcttgaactgttacagaagccctctgccctc 2375 V.1 2451 TGGTGGCCTCTGGGCCTGCTGCATGTACATATTTCTGTATATACATG 2500 V. 7 2376 tgtgccggcgtcttaaattttattct 2425 V.1 2501 CGCGACTTGAGAATGTCATATTATTT 2550 V. 7 2426 cgcgacttgagaatgtcatattattt 2475 V. 1 2551 TCTTTTTTTTTTCTTGCCCTTTCCATTAGTTGTATTTTTTATTTATTTTT 2600 V.7 2476 tcttttttgcttcttgtttttatatt 2525 V.1 2601 ATTTTTATTTTTTTTTAGAGATGGAGTCTCACTATGTTGCTCAGGCTGGC 2650 V.7 2526 atttttatttttttttagagatggagtctcactatgttgctcaggctggc 2575 V.1 2651 CTTGAACTCCTGGGCTCAAGCATCCTCCTGCCTCAGCCTCCCTAGTAGC 2700 V.7 2576 cttgaactcctgggctcaagcaatcctcctgcctcagcctccctagtagc 2625 V.1 2702. TGGGACTTTAAGTGTACACCACTGTGCCTGCTrGTCCTTTACGAAGA 2750 V. 7 2626 tggsactttaagtgtacaccactgtgcctgctttgaatcctttacgaaga 2675 V.1 2751 GAAAAAATTAAAGAAAGCCTTTAGATTTATCCAATGTTTACTACTG 2800 V. 7 2676 gaaaaataaaacttaatacatttcat 2725 V.1 2801 GGATTGCTTAAAGTGAGGCCCCTCCACACCAGGGGGTTAATTCCTGTGA 2850 V.7 2726 ggattgcttaaagtgaggcccctzccaacaccagggggttaattcctgtga 2775 V.1 2851 TTGTGAAAGGGGCTACTTCCGGCATC~TAGCAGGCAGCCCCTTGGG 2900 V.7 2776 tttaaggtctcagattctcgcgcctg 2825 V.1 2901 AGGGCACCTGAGAGCTGGTAGAGTCTGAAATTAGGGATGTGAGCCTCGTG 2950 V.7 2826 aggacgggtgaattgatagagggcct 2875 V.1 2951. GTTACTGAGTAAGGTAAAATTGCATCCACCATTGTTTGTGATACCTTAGG 3000 V.7 2876 gttactgagtaaggtaaaattgcatccaccattgtttgtgataccttagg 2925 00) V.1 3001 GAATTGCTTGGACCTGGTGACAAGGGCTCCTGTTCAATAGTGGTGTTGGG 3050 V.7 2926 gaattgcttggacctggtgacaagggctcctgttcaatagtggtgttggg 2975 V.1 3051 GAGAGAGAGAGCAGTGATTATAGACCGAGAGAGTAGGAGTTGAGGTGAGG 3100 V.7 2976 gagagagagagcagtgattatagaccgagagagtaggagttgaggtgagg 3025 V.1 3101 TGAAGGAGGTGCTGGGGGTGAGAATGTCGCCTTTCCCCCTGGGTTTTGGA 3150 V.7 3026 tgaaggaggtgctgggggtgagaatgtcgcctttccccctgggttttgga 3075 V.1 3151. TCACTAATTCAAGGCTCTTCTGGATGTTTCTCTGGGTTGGGGCTGGAGTT 3200 C] V.7 3076 tcactaattcaaggctcttctggatgtttctctgggttggggctggagtt 3125 V. 1 3201 CAATGAGGTTTATTTTTAGCTGGCCCACCCAGATACACTCAGCCAGAAA 3250 00 V.7 3126 caatgaggtttatttttagctggcccacccagatacactcagccagaata 3175 V.1 3251 CCTAGATTTAGTACCCAAACTCTTCTTAGTCTGAAATCTGCTGGATTTCT 3300 V.7 3176 cctagatttagtacccaaactcttcttagtctgaaatctgctggatttct 3225 V.1 3301 GGCCTAAGGGAGAGGCTCCCATCCTTCGTTCCCCAGCCAGCCTAGGACTT 3350 V.7 3226 ggcctaagggagaggctcccatccttcgttccccagccagcctaggactt 3275 V.1 3351 CGAATGTGGAGCCTGAAGATCTAAGATCCTAACATGTACATTTTATGTAA 3400 V.7 3276 cgaatgtggagcctgaagatctaagatcctaacatgtacattttatgtaa 3325 V.1 3401 ATATGTGCATATTTGTACATAAAATGATATTCTGTTTTTAAATAAACAGA 3450 V.7 3326 atatgtgcatatttgtacataaaatgatattctgtttttaaataaacaga 3375 V.1 3451 CAAAACTTGaaaaa 3464 V.7 3376 caaaacttgaaaaa 3389 Table LIV(b). Peptide sequences of protein coded by 191P4DI2(b) v.7 (SEQ ID NO: 114) MPIJSLGAEMW GPEAWLLLLL LLASFTGRCP AGELETSDVV TVVLGQDAKL PCFYRGDSGE QVGQVAWARV DAGEGAQELA LLHSKYGLHV SPAYEGRVEQ PPPPIUNPLDG SVLLRNAVQA 120 DEGEYECRVS TFPAGSFQAR LRLjRVLVPPL PSLNPGPALE EGQGLTLAAS CTAEGSPAPS 180 VTWflTEVKGT TSSRSFKHSR SAAVTSEFHL VPSRSMNGQP LTCVVSHPGL LQDQRITHIL 240 IIVSFLAEASV RGLEDQNLWH IGREGAMLKC LSEGQPPPSY NWTRLDGPLP SGVRVDGDTL 300 GFPPLTTEHS GIYVCHVSNE FSSRDSQVTV DVLDPQEDSG KQVD)LVSASV VVVGVIAALL 360 FCLLVVVVVL MSRYHRRKAQ QMTQKYEEEL TLTRENSIRR LHSRHTDPRS QSEEPEGRSY 420 STLTTVREIE TQTELLSPGS GRAEEEEDQD EGIKQAmNHp VQEUGTLRAK PTGNGIYING 480 RGHLV 485 Table LV(b). Amino acid sequence alignment of 191 P4DI2(b) vM (SEQ ID NO: 115) and 191P4D12(b) v.7 (SEQ ID NO: 116).

V.1 1 MPLSLGAEMWGPEAWLLLLLLLASPTGRCPAGELETSDVVTVVLGQDAXKL V.7 1 MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKcL V.1 51 PCFYRGDSGEQVGQVAWARVDAGEGAQELALL1.SKYCLHVSPAYEGRVEQ 100 00 V.7 51 PCFYRGDSGEQVGQVWAVDAGEGAQELLHMS1GCLHSPAYEGRVEQ V.1 101 PPPNLGVLNVAEEECVTPGFALLVVP V.7 101 PPPPRPLDGSVLLRAVQADEGEYECRVSTPAGSFQARJLRLRVLVPPL V.1 151 PSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSRSFHSR V.7 151 PSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVJXGTTSSRSFKHSR V.1 201 SAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILVSFMASV V.7 201 SAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASV V.1 251 RGIJEDQNLWHIGREGALKCLSEGQPPPSYWTRLDGPLPSGVRVDGDTL V.7 251 RGLEDQNLWHIGREGAMLKCLSEGQPPPSWTRLDGPLPSGVVGDTL V.1 301 GFPPLTTEHSGIYVCHVSNEFSSRDSQVVDVLDPEDSGKQIDLVSASV V.7 301 GFPPLTTEHSGIYVCHVSNEFSSPJJSQVTVDVIDPQEDSGKQVJDLVSASV V.1 351 VVGIALCLVVLSYRRAQTKBETTESR V.7 351 VVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRR V.1 401 LHSHHTDPRSQPEESVGLRAEGI{PDSLKDNSSCSVMSEEPEGRSYSTLTT V.7 401

SEEPEGRSYSTLTT

V.1 451 VRITTLSGGAEEQEIQMHVEGLA

TW

V.7 426 VRITTLSGGAEEQEGKANFQNTRKTN 100 150 150 200 200 250 250 300 300 350 350 400 400 450 425 Soo 475 V.1 501 IYINGRGHLV IllIhIiI V.7 476 IYINGRGHLV Table LII(c). Nucleotide sequence of transcript variant 191 P4DI2(b) v.8 (SEQ ID NO: ggc cgtcgtt acggcttctt tCCCCtagtg cagttcctta gctgggcagt aggcctggct agc tggagac tctaccgagg gcgaaggcgc cttacgaggg tcctgcgcaa c cgccggcag tgaatcctgg ctgagggcag gccgttcctt gccgcagcat accaaaggat ttgaagacca aagggcagcc tacgagtgga acgtctgcca ttgaccccca tgggtgtgat 9ttggccaca gggggtagct gagacccaag ttcaagtctg ctgcctttca gctgctgctg Ctcagacgtg ggactccggc ccaggaacta ccgcgtggag cgcagtgcag cttccaggcg tccagcacta Cccagccccc caagcactcc gaatgggcag cacccacatc aaatctgtgg ccCtcCctca tggggacacfr tgtcagcaat ggaagactct CgCC9Cactc gcgtgggaag acggctgggt tgcgagaggc c agc cggct c accatgcccc c tactgctgg gtaactgtgg gagcaagtgg gcgctactgc Cagccgccgc gcggatgagg cggctgcggc gaagagggcc agcgtgacct cgctctgctg ccactgactt CtCCaCgtgt cacattggca tacaactgga ttgggctttc gagttctcct gggaagcagg ttgttctgcc cagctctggg gtgtagaacg aagaactctg ccagggagat tgtccctggg catcatttac tgctgggcca ggcaagtggc actccaaata ccccacgcaa gcgagtacga tccgagtgct agggcctgac gggacacgga ccgtcacctc gtgtggtgtc ccttccttgc gagaaggagc cacggctgga ccccactgac Caagggattc tggacctagt ttctggtggt ggagctcgga gggCCggggC cagcttcctg Ctcggtggaa agccgagatg aggccggtgc ggacgcaaaa atgggctcgg cgggcttcat CCCcctggac gtgccgggtc ggtSCCtccc cctggcagcc ggtcaaaggc agagttccac ccatcctggc tgaggcctct tatgctcaag tgggcctctg cactgagcac tcaggtcact gt Cagcctcg ggtggtggtg 117) gCtCCCgatc tggggctggg cct tctgggt cttcagaaac tgggggcctg Cccgcgggtg ctgccctgct gtggacgcgg gtgagcccgg ggctcagtgc agcaccttcc CtgccctCaC tcctgcacag acaacgtcca ttggtgccta ctgctccagg gtgaggggcc tgcctgagtg Cccagtgggg agcggcatct gtggatgttc gtggtggtgg ctcatgtccc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 00 00 gataccatcg ccagggagaa aggagagtgt gctctgtgat agatagaaac atcaggatga gggccaagcc ggcctgcctc ttgggggCCt cttgaccttt caccatgcat tgtgtgtgtg ctgtcatatc gggcaacact aaagcaggta ggtggagact ggtgtgagg gtccctgggt tgggcctgct aatactgctc tgtattttt~t tcaggctggc tgggacttta attaaagaaa cctccaacac catgcaggca gagCCtCgtg gtgattatag atgtcgcctt gggttggggc cagaatacct ctaagggaga tgaagatcta atgatattct gcgcaaggcc ctccatccgg agggctgaga gagtgaagag acagactgaa aggcatcaaa cacgggcaat cct tcc ctag ccttaaacac acctccaacc gcaggtcact gaggggtgac agagtcaagt gtcagggttt ttttctcaga gtggctcaga aacctgtctc c agccagagg gcatgtacat cgaatcactt atttattttt cttgaactcc agtgtacacc gcctttagat cagggggtta gccccttggg ctggtgacaa accgagagag tccccctggg tggagttcaa agatttagta ggctcccatc agatcctaac gtttttaaat cagcagatga aggctgcatt gccgagggcc cccgagggcc ctgctgtctc caggccatga ggcatctaca gcctggctcc ccccatttct cttctgttca gtgtgtgtgc tgtccgtgga gaactgtggt ggcgtgtgtg ccccagagca cccaggtgtg ctaccacttc cttgaactgt attttctgta ttaatttttt atttttattt tgggc tcaag actgtgcctg ttatccaatg attcctgtga agggcacctg gggctcctgt taggagt tga ttttggatca tgaggtttat cccaaactct CttcgttCCC atgtacattt cccagaaata cccatcacac acc ctgatag gcagt tactc caggctctgg accattttgt tcaatgggg ttctgttgac tgcggaagat tcgggagggc atgtgtgcct ggggtgactg gtatgtgcca tcatgtggct qtattaatga cgggcatagc ggagccatgg tacagaagcc aatatacatg tctttttttt ttttttagag caatcctcct ctttgaatcc tttactactg ttgtgaaagg agagctggta tcaatagtgg ggtgaggtga ctaattcaag ttttagctgg tcttagtctg cagccagcct tatgtaaata tgaggaggag gyac cc cagy tctcaaggac cacgctgacc gcgggccgag tcaggagaat gggacacctg atgggagatt gctccccatc tccaccaatt gtgtgagtgt tgtccgtggt cgggatttga gtgtgtgacc tgcagaggtt tggagctgga gggcaagtgt CtCtgCCCtC cgccgggagc ttcttgccct atggagtctc gcctcagcct tt tacgaaga ggattgctta ggctacttcc gagtctgaaa tgttggggag aggaggtgct gctcttctgg cccacccaga aaatctgctg aggacttcga tgtgcatatt ctgaccctga agccagccgg aacagtagct acggtgaggg gaggaggaag g ggaccc tac gtctgaccca ttagctcatc ccactgactg gagtCtCtcc tgactgactg gtgtattatg gtggttgcgt tctgcctgaa ggaggagaga atctgcctcc gaagcagcca tgg tggCc t c ttcttgcagg ttccattagt actatgttgc ccctagtagc gaaaaaaaaa aagtgaggcc aaggcatctt ttagggatgt agagagagca gggggtgaga atgtttctct tacactcagc gatttctggc atgtggagcc tgtacataaa 1440 1500 1.560 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 3401 aaacagacaa aacttgaaaa a Table 1.1II(c). Nucleotlde sequence alignment of 191P4D12(b) v.1 (SEQ ID NO: 118) and 191P4D12Q,) v.8 (SEQ ID NO: 119) V.1 1 gGCCGTCGTTGTTGGCCACAGCGTGGGAAGCAGCTCTGGGGGAGCTCGGA V.8 1 ggccgtcgttgttggccacagcgtgggaagcagctctgggggagctcgga s0 V.1 51 GCTCCCGATCACGGCTTCTTGGGGGTAGCTACGGCTGGGTGTGTAGAACG 100 V.8 51 gctcccgatcacggcttcttgggggtagctacggctgggtgtgtagaacg 100 V.1 101 GGGCCGGGGCTGGGGCTGGGTCCCCTAGTGGAGACCCAAGTGCGAGAGGC 150 V.8 101- gggccggggctggggctgggtcccctagtggagacccaagtgcgagaggc 150 V.1 151 AAGAACTCTGCAGCTTCCTG3CCTTCTGGGTCAGTTCCTTATTCAAGTCTG 200 V.8 151 aagaactctgcagcttcctgccttctgggtcagttccttattcaagtctg 200 V.1 201 CAGCCGGCTCCCAGGAGATCTCGGTGGAACTTCAGAALACGCTGGGCAGT 250 V.8 201 cagccggctcccagggagatctcggtggaacttcagaaacgctgggcagt 250 V.1 251 CTGCCTTTCAACCATGCCCCTGTCCCTGGGAGCCGAGATGTGGGGGCCTG 300 V.8 251 ctgcctttcaaccatgcccctgtccctgggagccgagatgtgggggcctg 300 V.1 301 AGGCCTGGCTGCTGCTGCTGCTACTGCTGGCATCATTTACAGGCC-GGTGC 350 V. 8 301 aggcctggctgctg'ctgctgctactgctggcatcatttacaggccggtgc 350 00 V.1 353. CCCGCGGGTGAGCTGGAGACCTCAGACGTGGTAACTGTGGTGCTGWCCA 400 V.8 351 cccgcgggtgagctggagacctcagacgtggtaactgtggtgctgggcca 400 V.1 401 GGACGCAAAACTGCCCTGCTTCTACCGAGGGGACTCCGGCGAGCAAGTGG 450 V.8 401 ggacgcaaaactgccctgcttctaccgaggggactccggcgagcaagtgg 450 451 GGCAAGTGGCATGGGCTOGGGTGGACGCGGGCGAGGCGCCCAGGAACTA 500 V.8 451 ggaggctgccgtgcggggaggcagat 500 V.3. 501 GCGCTACTGCACTCCAAATACGGGCTTCATGTGAGCCCGGCTTACGAGGG 550 501 gcgctactgcactccaaatacgggcttcatgtgagcccggcttacgaggg 550 C1 V.1 551 CCGCGTGGAGCAGCCGCCGCCCCCACGCAACCCCCTGGACGGCTCAGTGC 600 V.8 551 ccgcgtggagcagccgccgcccccacgcaaccccctggacggctcagtgc 600 V.1 601 TCCTGCGCAACGCAGTGCAGGCGGATGAGGGCGAGTACGAGTGCCGGGTC 650 V. 8 601 tcctgcgcaacgcagtgcaggcggatgagggcgagtacgagtgcc 9 gggtc 650 V. 1 651 AGCACCTTCCCCGCCGGCAGCTTCCAGCGCGGCTGCGGCTCCGAGTGCT 700 V.8 651 agcaccttccccgccggcagcttccaggcgcggctgcggctccgagtgct 700 V.1 701 GGGCCCTCCCCGATCGCACCAAGWC 750 V. 8 701 gggccctccccgaccgtcgatgaagc 750 V.*1 751 AGGGCCTGACCCTGGCAGCCTCCTGCACAGCTGAGGGCAGCCCA.GCCCCC 800 V. 8 751 aggcgccgcgcctcaacgggaccgcc 800 V.1 801 AGCGTGACCTGGGACACGGAGGTAGOCACACGTCCAGCCGTTCCTT 850 V. 8 801 agggctgaagagcaagaactcgctct 850 V.1 851 CAAGCACTCCCGCTCTGCTGCCGTCACCTCAGAGTTCCACTTGGTGCCTA 900 V-8 851 111111111 11111111111111111111111111111111111111 V.1 901 GCCGCAGCATGA.TGGGCGCCACTGACTTGTGTGGTGTCCCTCCTGG3C 950 V. 8 901 gccgcagcatgaatgggcagccactgacttgtgtggtgt~ccatcctggc 950 V.1 951 CTGCTCCAGGACCAAAGGATCACCCACTCCTCCACG3TGTCCTTCCTTGC 1000 V.8 951 ctccagcaagtacactccaggctctg 1000 V.1 1001 TGGCTTTAGGCTAGCAATTTOCCTGC 1050 V.8 1001 tggctttaggctagacaacggcctgc 1050 V.1 1051 GAGAAGGAGCTATGCTCAGTGCCTGAGTGAGG GCCCCCTCCCTC!A 1100 V. 8 1051 gaagacagtaggcgataggacccccc 1100 V.1 1101 TACAACTGGACACGGCTGGATGGGCCTCTGCCCAGTGGGGTACGAGTOGA 1150 V.8 1101 tacaactggacacggctggatgggcctctgcccagtggggtacgagtgga 1150 00 V.1 1151 TGGGGACACTTTGGGCTTTCCCCCACTGACCACTGAGCACAG.CGGCATCT 1200 C] V.8 1151 tggggacactttgggctttcccccactgaccactgagcacagcggcatct 1200 V.1 1201 ACGTCTGCCATGTCAGCAATGAGTTCTCCTCAAGGGATTCTCAGGTCACT 1250 SV. 8 1201 acgtctgccatgtcagCaatgagttctcctcaagggattctCaggtcact 1250 V.1 1251 GTGGATGTTCTTGACCCCCAGGAAGACTCTGGGAAGCAGGTGGACCTAGT 1300 V. 8 1251 gtggatgttcttgacccccaggaagactctgggaagcaggtggacctagt 1300 V.1 1301 GTCAGCCTCGGTGGTGGTGGTGGGTGTGATCGCCGCACTCTIGTTCTGCC 1350 V.8 1301 gtcagcctcggtggtggtggtgggtgtgatcgccgcactcttgttctgcc 1350 00 V.1 1351 VTCTGGTGGTGGTGGTGGTGCTCATGTCCCGATACCATCGGCGCAAGGCC 1400 0 V.8 1351 ttctggtggtggtggtggtgctcatgtcccgataccatcggcgcaaggcc 1400 V.1 1401 CAGCAGATGACCCAGAAATATGAGGAGGAGCTGACCCTGACCAGGGAGAA 1450 V.8 1401 cagcagatgacccagaaatatgaggaggagctgacctgaccagggagaa 1450 V.1 1451 CTCCATCCGGAGGCTGCATTCCCATCACACGGACCCCAGGAGCCAGCCGG 1500 V.8 1451 ctccatccggaggctgcattcccatcacacggaccccaggagccagccgg 1500 V.1 1501 AGGAGAGTGTAGGGCTGAGAGCCGAGGGOCACCCTGATAGTCTCAAGGAC 1550 V.8 1501 aggagagtgtagggctgagagccgagggccaccctgatagtctcaaggac 1550 V.1 1551 AACAGTAGCTGCTCTGTGATGATGAAGA~.GCCCGAGGGCCGCAGTTACTC 1600 V.8 1551 aacagtagctgctctgtgatgatgaagagcccgagggccgcagttactc 1600 V.1 1601 CACGCTGACCACGGTGAGGGAGATAGAAACACAGACTGAACTGCTGTCTC 1650 V.8 1601 cacgctgaccacggtgagggagatagaaacacagactgaactgctgtctc 1650 V.1 1651 CAGGCTCTGGGCGGGCCGAGGAGGAGGAAGATCAGGATGAAGGCATCAAA 1700 V. 8 1651 caggctctgggcgggccgaggaggaggaagatcaggatgaaggcatcaaa 1700 V.1 1701 CAGGCCATGAACCATTTTGTTCAGGAGAATGGGACCCTACGGGCCAAGCC 1750 V.8 1701 caggccatgaaccattttgttcaggagaatgggaccctacgggccaagcc 1750 V.1 1751 CACGGGCAATGGCATCTACATCAATGGGCGGGGACAOCTGGTCTGACCCA 1800 V.8 1751 cacgggcaatggcatctacatcaatgggcggggacacctggtctgaccca 1800 V.1 1801 GGCCTGCCTCCCTTCCCTAGGCCTGGCTCCTTCTGTTGACATGGOAGATT 1850 V.8 1801 ggcctgcctcccttccctaggcctggctccttctgttgacatgggagatt 1850 V.1 1851 TTAGCTCATCTTGGGGGCCTCCTTAAACACCCCCATTTCTTGCGGAAGAT 1900 V.8 1851 ttagctcatcttgggggcctccttaaacacccccatttcttgcggaagat 1900 249 V. .1 1901 GCTCCCCATCCCACTGACTGCTTGACCTTTACCTCCAACCCTTCTGTTCA 1950 00V.8 1901 gctccccatcccactgactgcttgacctttacctccaacccttctgttca 1950 V.1 1951 TCGGGAGGGCTCCACCAATTGAGTCTCTCCCACCATGCATGCAGGTCACT 2000 V.8 1951 tcgggagggctccaccaattgagtctctcccaccatgcatgcaggtcact 2000 SV.1. 2001 GTGTGTGTGCATGTGTGCCTGTGTGAGTGTTGACTGACTGTGTGTGTGTG 2050 VA 001gtgtgtgtgcatgtgtgcctgtgtgagtgttgactgactgtgtgtgtgtg 2050 V.1 2051 GAGGGGTGACTGTCCGTGGAGGGGTGACTGTGTCCGTGGTGTGTATTATG 2100 r-V.8 2051 gaggggtgactgtccgtggaggggtgactgtgtccgtggtgtgtattatg 2100 (iV.1. 2102. CTGTCATATCAGAGTCAAGTOAACTGTGGTGTATGTGCCACGGGATTTGA 21.50 (iV.8 2101 ctgtcatatcagagtcaagtgaactgtggtgtatgtgccacgggatttga 2150 00 V. 1 2151 GTGGTTGCGTGGGCAACACTOTCAGGGTTTGGCGTGTGTGTCATGTGGCT 2200 V. 8 2151 gtggttgcgtgggcaacact~tcagggtttggcgtgtgtgtcatgtggct 2200 V. 1 2201 GTGTGTGACCTCTGCCTGAAAAAGCAGGTATTTTCTCAGACCCCAGAGCA 2250 V. 8 2201 gtgtgtgacctctgcctgaaaaagcaggtattttctcagaccccagagca 2250 V. 1 2251 GTATTAATGATGCAGAGGTTc zGAGGAGAGAGGTGGAGACTGTGGCTCAGA 2300 V. 8 2251 gtattaatgatgcagaggtt(-gaggagagaggtggagacegtggctcaga 2300 V. 1 2301 CCCAkGGTGTGCGGGCATAGCTGGAGCTGJ JATCTGCCTCCGGTGTG;AGGG 2350 V. 8 2301 cccaggtgtgcgggcatagctggagctggaatctgcctccggtgtgaggg9 2350 V.1 2351 AACCTGTCTCCTACCACTTCGGAGCCATOGGGGCAAGTGTGAAGCAGCCA 2400 V.8 2351 aacctgtctcctaccacttcggagccatgggggcaagtgtgaagcagcca 2400 V.1 2401 GTCCCTGGGTCAGCCAGAGGCTTGACTQTTAAGAGCCCTCTGCCCTC 2450 V.8 2401 gtccctgggtcagccagaggcttgaact~ttacagaagccctctgccctc 2450 V.1 2451 TGGTGGCCTCTGGGCCTGCTGCATGTACTATTTCTGTATATACATG 2500 V.8 2451 tggtggcctctgggcctgctgcatgtacatattttctgtaaatatacatg 2500 V.1 2501 CGCCGGGAGCTTCTTGCAGGAATACTGCTCCGATCACTTTTAATTTTTT 2550 V.8 2501 cgccgggagcttcttgcaggaatactgctccgaatcacttttaattttt 2550 V.1 2551 TCTTTTTTTTTTCTTGCCCTTTCCATTAGTTGTATTTTTTATTTATTTTT 2600 V. 8 2551 tcttttttgcttcttgtttttatatt 2600 V.1 2601 ATTTTTATTTTTTTTTAGAGATGGAGTCTCACTATGTTGCTCAGGCTGGC 2650 V. 8 2601 atttttatttttttttagagatggagtctcactatgttgctcaggctggc 2650 V.1 2651 CTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTCAGCCTCCCTAGTAGC 2700 250 V.8 2651 cttgaactcctgggctcaagcaatcctcctgcctcagcctccctagtagc 2700 V.1 2701 TGGGACTTTAAGTGTACACCACTGTGCCTGCTTTGAATCCTTTACGAAGA 2750 00111 ~lIIIfI ffl 111 SV.8 2701 tgggactttaagtgtacaccactgtgcctgctttgaatcctttacgaaga 2750 ClV.1 2751 GAAAAbAAATTAAAGAAAGCCTTTAATTTATCCAATGTTTACTACTG 2800 Ct V.8 2751 Saaaaaaaaaattaaagaaagcctttagatttatccaatgtttactactg 2800 SV.1 2 801 GGATTGCTTAAAGTGAGGCCCCTCCAACACCAGGGGGTTAATTCCTGTGA 2850 CIV.8 2801 ygattgcttaaagtgaggcccctccaacaccagggggttaattcctgtga 2850 V.1 2851 TTGTGAAAGGGGCTACTTCCAAGGCATCTTCATGCAGGCAGCCCCTTGGG 2900 V.8 2851 ttgtgaaaggggctacttccaaggcatcttcatgcaggcagccccttggg 2900 V.1 2901 AGGGCACCTGAGAGCTGGTAGAGTCTGAAATTAGGGATGTGAGCCTCG;TG 2950 00 V.8 2901 agggcacctgagagctggtagagtctgaaattagggatgtgagcctcgtg 2950 SV.1 2951 GTTACTGAGTAAGGTAAAATTGCATCCACCATTGTTTGTGATACCTTAGG 3000 V.8 2951 2950 V.1 3001 GAATTGCTTGGACCTGGTGACAAGGGCTCCTGTTCAATAGTGGTGTTGGG 3050 V.8 ctggtgacaagggctcctgttcaatagtggtgttggg 2987 V.1 3051 GAGAGAGAGAGCAGTGATTATAGACCGAGAGAGTAGGAGTTGAGGTGAGG 3100 V.8 2988 gagagagagagcagtgattatagaccgagagagtaggagttgaggtgagg 3037 V.1 3101 TGAAGGAGGTGCTGGGGGTGAGAAJTGTCGCCTTTCCCCCTGGGTTTTGGA 3150 V.8 3038 tgaaggaggtgctgggggtgagaatgtcgcctttcccctgggttttgga 3087 V.1 3151 TCACTAATTCAAGGCTCTTCTGGATGTTTCTCTGGGTTGGGGCTGGAGTT 3200 V.8 3088 tcactaattcaaggctcttctggatgtttctctgggttggggctggagtt 3137 V.1 3201 CAATGAGGTTTATTTTTAGCTGGCCCPLCCCAGATACACTCAGCCAGAATA 3250 V.8 3138 caatgaggtttatttttagctggcccacccagatacactcagccagaata 3187 V.1 3251 CCTAGATTTAGTACCCAAACTCTTCTTAGTCTGAAATCTGCTGGATTTCT 3300 V.8 3188 cctagatttagtacccaaactcttcttagtctgaaatctgctggatttct 3237 V.1 3301 GGCCTAAGGGAGAGGCTCCCATCCTTCGTTCCCCAGCCAGCCTAGGACTT 3350 V.8 3238 ggcctaagggagaggctcccatccttcgttccccagccagcctaggactt 3287 V.1 3351 CGAATGTOGAGCCTGAAGATCTAAGATCCTAACATGTACATTTTATGT4A 3400 V.8 3288 cgaatgtggagcctgaagatctaagatcctaacatgtacattttatgtaa 3337 V.1 3401 ATATGTGCATATTTGTACATAAAATGATATTCTGTTTTTAAATAAACAGA 3450 V.8 3338 atatgtgcatatttgtacataaaatgatattctgtttttaaataaacaga 3387 V.1 3451 CAAAACTTGaaaaa 3464 V.8 3388 caaaacttgaaaaa 3401 00 Table LIV(c). Peptide sequences of protein coded by 1919P01 2(b) v.8 (SEQ ID NO: 120) MPLSLGAEMW GPEAWLLLLL LLASFTGRCP AGELETSDVV TVVLGQDAKL PCFYRGDSGE QVGQVAWARV DAGEGAQELA LLHSKYGLHV SPAYEGRVEQ PPPPRNPLD)G SVLjLRNAVQA 120 DEGEYECRVS TFPAGSFQAR LRLRVLVPPIL PSLNJPGPALE EGQGILTLAAS CTAEGSPAPS 180 VTWDTEVKGT TSSRSFKHSR SAAVTSEPHL VPSRSMNGQP LTCVVSHPGL LQDQRITHIL 240 SHVSFLAEASV RGLEDQNLWH IGREGAMLKC LSEGQPPPSY NWTRLDGPLP SGVRVDGDTL 300 GFPPLTTEHS GIYVCHVSNE FSSRDSQVTV DVLDPQEDSG KQVDLVSASV VVVGVIAALL 360 ciFCLLVVVVVL MSRYHRRKAQ QMTQKYEEEL TLTRENSIRR LHSHHTDPRS QPEESVGLRA 420 EGHPDSLKDbI SSCSVMSEEP EGRSYSTLTT VREIETQTEL LSPGSGRAEE EEDQDEGIKQ 480 ANNHFVQENG TLRAKPTGNG IYINGRGHLV 510 Table LV(c). Amino acid sequence alignment of 191 P401(b) v.1 (SEQ ID NO: 121) and 191 P4D12(b) v.8 (SEQ ID NO: c]122) CiV-1 1 MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKcL N V.8 1 MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKL so 00 V. 1 51 PCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSI(YGLHVSPAYEGRVEQ 100 V.8 51 PCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQ 100 V. 1 101 PPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVTJVPPL 150 V. 8 101 PPPPRNPLDGSVLLRNAVQAflEGEYECRVSTFPAGSFQARLRLRVLVPPL 150 V.1 151 PSLNPGPAIIEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSR 200 V.8 151 PSLUIPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSR 200 V.1 201 SAAVTSEPHLVPSRSMNGQPLTCVVSHPGIJLQDQRITHILHVSFLAEASV 250 V.8 201 SAAVTSEPHLiVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASV 250 V.1 251 RGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTR jDGPLPSGVRVDGDTL 300 V.8 251 RGLEDQNLWf{IGREGAM~LKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTL 300 V.1 301 GFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASV 350 V.8 301 GFPPLTTEHSGIYVCHVSbNEFSSRlSQVTVDVLDPQEDSGKQVDLVSASV 350 1 351 VVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMvTQKYEEELTLTRENSIRR 400 V.8 351 VVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRR 400 V.1 401 LHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTT 450 V. 8 401 LHSHHTDPRSQPEESVGLRABGHPDSLKDNSSCSVMSEEPEGRSYSTLTT 450 V. 1 451. VREIETQTELLSPGSGRABEEEDQDEGIKQAMNHFVQENGTLRAKPTGNG 500 V.8 451 VREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQEGTLRACPTGNG 500 V.1 501 IYINGRGHLV 510 V.8 501 IYINGRGHLV 510 Table 1-1I(d). Nucleotide sequence of transcript variant 191P4D12(b) v.9 (SEQ ID NO: 123) 252 00 00 gtCtgaCCCa ttagctcatc ccactgactg gagtctctcc tgactgactg gtgtattatg gtggttgcgt tctgcctgaa ggaggagaga atctgcctcc gaagcagcca tggtggcctc ttcttgcagg ttccattagt actatgttgc ccctagtagc gaaaaaaaaa aagtgaggcc aaggqcatctt ttagggatgt ataccttagg gagagagaga gCtgggggtg tggatgtttc ag ata cac tc ctggatttct cgaatgtgga atttgtacat ggcctgCCtC ttgggggCCt cttgaccttt caccatgcat tgtgtgtgtg ctgtcatatc gggcaacact aaagcaggta ggtggagact ggtgtgagg gtccctgggt tgggCCtgCt aatactgctc tgtatttttt tcaggctggc tgggacttta attaaagaaa cctccaacac catgcaggca, gagcctcgtg gaattgcttg gcagtgatta agaatgtcgc tCtgggttgg agccagaata ggcctaaggg gcctgaagat aaaatgatat cct tccc tag ccttaaacac acctccaacc.

gcaggtcact gaggggtgac agagtcaagt gtcagggttt ttttctcaga gtggctcaga aacctgtctc cagccagagg gcatgtacat cgaatcactt atttattttt cttgaactcc agtgtacacc gcctttagat cagggggtta gCCCCttggg gttact~agt gacctggtga tagaccyaga CtttcCCCCt ggctggagtt cctagattta agaggctccc ctaagatcct tctgttttta gCCtggCtcc ccccatttct cttctgttca gtgtgtgtgc tgtccgtgga gaactgtggt ggCgtgtgtg ccccagagca CCaggtgtg ctaccacttc cttgaactgt att~t t ctgt a ttaatttttt atttttattt tgggctcaag actgtgcctg ttatccaatg attcctgtga agggcacctg aaggtaaaat caagggct cc gagtaggagt gggttttgga caatgaggt gtacccaaac atccttcgtt aacatgtaca aataaacaga ttCtgttgaC tgcggaagat tcgggagggc atgtgtgc~t ggggtgactg gtatgtgcca tcatgtggct gtattaatga cgggcatagc ggagccatgg tacagaagcc aatatacatg tctttttttt tttttt agag caatcctcct ctttgaatcc tttactactg t tgtgaaagg agagctggta tgcatccacc tgttcaata9 tgaggtgagg tcactaattc tatttttagc tcttcttagt ccccagccag ttttatgtaa caaaacttg atgggagatt gctccccatc tccaccaatt gtgtgagtgt tgtccgtggt cgggatttga gtgtgtgacc tgcagaggtt tggagctgga gggcaagtgt CtCtgCCCtC cgccgggagc ttCttgccct atggagtctc gcctcagcct tttacgaaga ggattgctta ggctacttcc gagtctgaaa attgtttgtg tggtgttggg tgaaggaggt aaggctcttC tggcccacc ctgaaatctg cctaggactt atatgtgcat 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 1669 Table 1-11(d). Nucleotide sequence alignment of 19P4D1(b) v.1 (SEQ ID NO: 124) and 191P4D1(b) v.9 (SEQ ID NO: 125) v.1 1791 GTCTGACCCAGGCCTGCCTCCCTTCCC!TAGGCCTGGCTCCTTCTGTTGAC 1840 V. 9 1 gtctgacccaggcctgcctcccttccctaggcctggctccttctgttgac v.1 1841 ATGGGAGATTTTAGCTCATCTTGGGGGCCTCCTTAAACACCCCCATTTCT 1890 V.9 51 atgggagattttagctcatcttgggggcctccttaaacacccccatttct 100 v.1 1891 TGCOGAAGATGCTCCCCATCCCACTG1ACTGCTTGACCTTTACCTCCAACC 1940 v.9 101 tgcggaagatgctccccatcccactgactgcttgacctttacctccaacc 150 v.1 1941 CTTCTGTTCATCGGGAGGGCTCCACCAATTGAGTCTCTCCCACCATGCAT 1990 v.9 151 cttctgttcatcgggagggctccaccaattgagtctctCCCaccatgcat 200 v.1 1991 GCAGGTCACTGTGTGTGTGCATGTGTGCCTGTGTGAGTGTTGACTGACTG 2040 v.9 201 gcaggtcactgtgtgtgtgcatgtgtgcctgtgtgagtgttgactgactg 250 v.1 2041 TGTGTGTGTGGAGGGGTGACTGTCCGTGGAGGGGTGACTGTGTCCGTGGT 2090 v.9 251 tgtgtgtgtggaggggtgactgtccgtggaggggtgactgtgtccgtggt 300 v.1 2091 GTGTATTATGCTGTCATATCAGAGTCAAGTGAACTGTGGTGTATGTGCCA 2140 v.9 301 gtgtattatgctgtcatatcagagtcaagtgaactgtggtgtatgtgcca 350 v.1 2141 CGGGATTTGAGTGGTTGCGTGGGCAACACTGTCAGGGTTTGGCGTGTGTG 2190 v.9 351 cgggatttgagtggttgcgtgggcaacactgtcagggtttggcgtgtgtg 400 v.1 2191 TCATGTGGCTGTGTGTGACCTCTGCCTGAAAAAGCAGGTAT2TTCTCAGA 2240 00 v.9 401. tcatgtggctgtgtgtgacctctgcctgaaaaagcaggtattttctcaga 450 v.1 2241 CCCCAGAGCAGTATTAATGATGCAGAGGTTGGAGGAGAGAGGTGGAGACT 2290 v.9 :51 500ggatttagtcgggtggaaagtggc o Sv.1 221GTGGCTCAGACCCAGGTGTGCGGGCATAGCTGGAGCTGGAATCTGCCTCC 2340 v.9 501 gtggctcagacccaggtgtgcgggcatagctggagctggaatctgcctcc 550 v.1 2341 GGTGTGAGGGAACCTGTCTCCTACCACTTCGGAGCCATGGGGGCAAGTGT 2390 551 ggtgtgagggaacctgtctcctaccacttcggagccatgggggcaagtgt 600 2391 GAAGCAGCCAGTCCCTGGGTcAGccAG.AGGCTTGAACTGTTACAGAAGCC 2440 v.9 601 gaagcagccagtccctgggtcagccagaggcttgaactgttacagaagcc 650 00 v.1 2441 CTCTGCCCTCTGGTGGCCTCTGGGCCTGCTGCATGTACATATTTTCTGTA 2490 v.9 2651 cttcccgtgccggcgtcttctttcga 700 v. 291AATATACATGCGCCGGGAGCTTCTTGCAGGAATACTGCTCCGAATCACTT 254 0 v.9 701 aatatacatgcgccgggagcttcttgcaggaatactgctccgaatcactt 750 v.1 2541 TTAATTTTTTTCTTTTTTTTTI'CTTGCCCTTTCCATTAGTTGTATTTTTT 2590 v.9 751 ttaatttttttcttttttttttcttgccctttccattagttgtatttttt 800 v.1 2591 ATTTATTTTTATTTTrATTTTTTTTTAGAGATGGAGTCTCACTATGTTGC 2640 v.9 801 atttatttttatttttatttttttttagagatggagtctcactatgttgc 850 v. 1 2641 TCAGGCTcGGCCTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTCAGCCT 2690 v.9 851 tcaggctggccttgaactcctgggctcaagcaatcctcctgcctcagcct 900 V.1 2691 CCCTAGTAGCTGGGACTTTAAGTGTACACCACTGTGCCTGCTTTGAATCC 2740 v.9 901 ccctagtagctgggactttaagtgtacaccactgtgcctgctttgaatcc 950 v.1 2741 TTTACGAAGAGAAAAAATTAAAGAAAGCCTTTAGATTTATCCAATG 2790 v. 9 951 tttacyaagagaaaaaaaaaattaaagaaagcctttagatttatccaatg 1000 v.1 2791 TTTACTACTGGGATTGCTTAAAGTGAGGCCCCTCCAACACCAGGGGGTTA 2840 V.9 1001 tttactactgggattgcttaaagtgaggcccctccaacaccagggggtta 1050 v.1 2841 ATTCCTGTGATTGTGAAAGGGGCTACTTCCAAGGCATCTTCATGCAGGCA 2890 v-9 1051 attcctgtgattgtgaaaggggctacttccaaggcatcttcatgcaggca 1100 v.1 2891 GCCCCTTGGGAGGGCACCTGAGAGCTGGTAGAGTCTGATTAGGGATGT 2940 v. 9 1101 gccccttgggagggcacctgagagctggtagagtctgaaattagggatgt 1150 v.1 2941 GAGCCTCGTGGTTACTGAGTAAGGTAALAATTGCATCCACCATTGTTTGTG 2990 254 v.9 1151 gagcctcgtggttactgagtaa99taaaattgcatccaccattgtttgtg 120o v.1 2991 ATACCTTAGGGAATTGCTTGGACCTGGTGACAAGGGCTCCTGTTCAATAG 3040 Sv.9 1201 ataccttagggaattgcttggacctggtgacaaggctcctgttcaatag 1250 Clv.1 3041 TGGTGTTGGGGAGAGAGAGAGCAGTGATTATALGACCGAGAGAGTAGGAGT 3090 C~v.9 1-251 tggtgttggggagagagagagcagtgattatagaccgagagagtaggagt 1300 v.1 3091 TGAGGTGAGGTGAAGGAGQTGCTGGGGGTGAGAATGTCGCCTTTCCCCCT 3140 v.9 1301 tgaggtgaggtgaaggaggtgctgggggtgagaatgtcgcctttccccct 1350 v.1 3141 GGGTTTTGGATCACTAATTCAAGGCTCTTCTGGATGTTTCTCTOGGTTGG 3190 v.9 1351 gggttttggatcactaattcaaggctcttctggatgtttctctggttgg 1400 .1 3191 GGCTGGAGTTCAATGAGGTTTATTTTTAGCTGGCCCACCCAGATACACTC 3240 00 v.9 1401 ggctggagttcaatgaggtttatttttagctggCCCaccagatacactc 1450 -v.1 3241 AGCCAGAATACCTAGATTTAGTACCCAAACTCTTCTTAGTCTGATCTG 3290 v.9 1451 agccagaatacctagatttagtacccaaactcttcttagtctgaaatctg 1500 v. 1 3291 CTGGATTTCTGGCCTAAGGGAGAGGCTCCCATCCTTCGTTCCCCAGCCAG 3340 v. 9 1501 ctggatttctggcctaagggagaggctcccatccttcgttCcccagccag 1550 v.1 3341 CCTAGGACTTCGAATGTGGAGCCTGAAGATCTAAGATCCTAACATGTACA 3390 v.9 1551 cctaggacttcgaatgtggagcctgaagatctaagatcctaacatgtaca 1600 v.1 3391 TTTTATGTAAATATGTGCATATTTGTACATAAAATGATATTCTGTTTTA 3440 v.1 3441 AATAAACAGACAAAACTTG 3459 v.9 1651 aataaacagacaaaacttg 1669 Table LIV(d). Peptide sequences of protein coded by 19112012(b) v.9 (SEQ ID NO: 128) MRRELLAGIL ILRITFNFFLF FFLPFPLVVF FIYFYFYFFL EMESHYVAQA GLELLGSSNP PASASLVAGT LSVHHCACFE SFTKRKKKLK KAFRFIQCLL LGLLKVRPLQ HQGVNSCDCR 120 RGYFQGIFMQ AAPWEGT 137 Table LV(d). Amino acid sequence alignment of 1911241312(b) v.1 and 19113012(b) v.9 (NO SIGNIFICANT MATCH)

Claims (31)

1. 3. A protein of claim 2 that is at least 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% homologous to an entire =K amino acid sequence shown in Figure 2. i 4. A composition of claim 1 wherein the substance comprises a CTL polypeptide or an analog thereof, from 00 the amino acid sequence of a protein of Figure 2. A composition of claim 4 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2.
2. 6. A composition of claim 1 further limited by a proviso that the polypeptide Is not an entire amino acid sequence of a protein of Figure 2.
3. 7. A composition of claim 1 that comprises an antibody polypeptide epitope from an amino acid sequence of Figure 2.
4. 8. A composition of claim 7 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2.
5. 9. A composition of claim 7 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to the end of said peptide, wherein the epitope comprises an amino acid position selected from: a) an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure b) an amino acid position having a value less than 0.5 In the Hydropathicity profile of Figure 6; c) an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; d) an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; e) an amino acid position having a value greater than 0.5 in the Beta-tur profile of Figure 9; f) a combination of at least two of a) through e); g) a combination of at least three of a) through e); h) a combination of at least four of a) through or I) a combination of five of a) through e). A polynucleotide that encodes a protein of claim 1.
6. 11. A polynucleotide of claim 10 that comprises a nucleic acid molecule set forth in Figure 2. 00 O 12. A polynucleotide of claim 10 further limited by a proviso that the encoded protein is not an entire amino C< acid sequence of Figure 2.
7. 13. A composition of claim 11 wherein the substance comprises a polynucleotide that comprises a coding sequence of a nucleic acid sequence of Figure 2.
8. 14. A polynuceotide of claim 11 that further comprises an additional nucleotide sequence that encodes an S additional peptide of claim 1. rC 15. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 0 0 16. A method of generating a mammalian immune response directed to a protein of Figure 2, the method 0 comprising: exposing cells of the mammal's immune system to a portion of a) a 191P4D12(b)-related protein and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to said protein.
9. 17. A method of generating an immune response of claim 16, said method comprising: providing a 191P4D12(b)-related protein that comprises at least one T cell or at least one B cell epitope; and, contacting the epitope with a mammalian immune system T cell or B cell respectively, whereby the T cell or B cell is activated.
10. 18. A method of claim 17 wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the 191P4D12(b)-related protein.
11. 19. A method of claim 17 wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 191P4D12(b)-related protein. A method of claim 17 wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producing activity of a B cell.
12. 21. A method for detecting, in a sample, the presence of a 191P4D12(b)-related protein or a 191P4D12(b)- related polynucleotide, comprising steps of: contacting the sample with a substance that specifically binds to the 191P4D12(b)-related protein or to the 191P4D12(b)-related polynucleotide, respectively; and, determining that there is a complex of the substance with the 191P4D12(b)-related protein or the substance with the 191P4D12(b)-related polynucleotide, respectively.
13. 22. A method of claim 21 for detecting the presence of a 191P4D12(b)-related protein in a sample OO comprising steps of: contacting the sample with an antibody or fragment thereof either of which specifically bind to the 191P4D12(b)- 1 related protein; and, determining that there is a complex of the antibody or fragment thereof and the 191P4D12(b)-related protein.
14. 23. A method of claim 21 further comprising a step of: taking the sample from a patient who has or who is suspected of having cancer.
15. 24. A method of claim 21 for detecting the presence of a protein of Figure 2 mRNA in a sample comprising: CN producing cDNA from the sample by reverse transcription using at least one primer; Samplifying the cDNA so produced using 191P4D12(b) polynucleotides as sense and antisense primers, wherein the 191P4D12(b) polynucleotides used as the sense and antisense primers serve to amplify a 191P4D12(b) cDNA; and, 0 detecting the presence of the amplified 191P4D12(b) cDNA. A method of claim 21 for monitoring one or more 191P4D12(b) gene products In a biological sample from a patient who has or who is suspected of having cancer, the method comprising: determining the status of one or more 191P4D12(b) gene products expressed by cells in a tissue sample from an individual; comparing the status so determined to the status of one or more 191P4012(b) gene products in a corresponding normal sample; and, identifying the presence of one or more aberrant gene products of 191P4D12(b) in the sample relative to the normal sample.
16. 26. The method of claim 25 further comprising a step of determining if there are one or more elevated gene products of a 191P4D12(b) mRNA or a 191P4D12(b) protein, whereby the presence of one or more elevated gene products in the test sample relative to the normal tissue sample indicates the presence or status of a cancer.
17. 27. A method of claim 26 wherein the cancer occurs in a tissue set forth in Table I.
18. 28. A composition comprising: a substance that a) modulates the status of a protein of Figure 2, or b) a molecule that is modulated by a protein of Figure 2, whereby the status of a cell that expresses a protein of Figure 2 is modulated.
19. 29. A composition of claim 28, further comprising a physiologically acceptable carrier. A pharmaceutical composition that comprises the composition of claim 28 In a human unit dose form.
20. 31. A composition of claim 28 wherein the substance comprises an antibody or fragment thereof that specifically binds to a protein of Figure 2. 258
21. 32. An antibody or fragment thereof of claim 31, which is monoclonal.
22. 33. An antibody of claim 31, which is a human antibody, a humanized antibody or a chimeric antibody. 00
23. 34. A non-human transgenic animal that produces an antibody of claim 31. A hybridoma that produces an antibody of claim 32. LC 36. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses a protein of Figure 2, said method coriprising: providing the cytotoxic agent or the diagnostic agent conjugated to an antibody or fragment thereof of claim 4; and, exposing the cell to the antibody-agent or fragment-agent conjugate.
24. 37. A composition of claim 28 wherein the substance comprises a polynucleotide that encodes an antibody 00 or fragment thereof, either of which immunospecifically bind to a protein of Figure 2.
25. 38. A composition of claim 28 wherein the substance comprises a) a ribozyme that cleaves a polynucleotide having a 191P4D12(b) coding sequence, or b) a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier.
26. 39. A composition of claim 28 wherein the substance comprises human T cells, wherein said T cells specifically recognize a 191P4D12(b) peptide subsequence in the context of a particular HLA molecule. A method of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising: administering to the cells the composition of claim 28.
27. 41. A method of claim 40 of Inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: administering to said cells an antibody or fragment thereof, either of which specifically bind to a 191P4D12(b)- related protein.
28. 42. A method of claim 40 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: administering to said cells a 191P4012(b)-related protein.
29. 43. A method of claim 40 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: administering to said cells a polynudeotide comprising a coding sequence for a 191P4012(b)-related protein or comprising a polynucleotide complementary to a coding sequence for a 191P4D12(b)-related protein.
30. 44. A method of claim 40 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: I administering to said cells a ribozyme that cleaves a polynucleotide that encodes a protein of Figure 2. A method of claim 40 of inhibiting growth of cancer cells that express a protein of Figure 2 and a 00 particular HLA molecule, the method comprising steps of: 0administering human T cells to said cancer cells, wherein said T cells specifically recognize a peptide subsequence of a protein of Figure 2 while the subsequence is in the context of the particular HLA molecule.
31. 46. A method of claim 40, the method comprising steps of: Sadministering a vector that delivers a nucleotide that encodes a single chain monoclonal antibody, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express a protein of Figure 2. 00 CO CO 0 C