AU2008200920A1 - Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer - Google Patents

Description

1

AUSTRALIA

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

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer The following statement is a full description of this invention including the best method of performing it known to us:- 00 1A NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 193P1E1B USEFUL IN TREATMENT AND DETECTION OF CANCER This is a divisional of AU 2002362105, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The invention described herein relates to a gene and its encoded protein, termed 00 193P1E1B, expressed in certain cancers, and to diagnostic and therapeutic methods and O 10 compositions useful in the management of cancers that express 193P1EB.

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

Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year.

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

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

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 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.

00 1B Unfortunately, these treatments are ineffective for many and are often associated with Sundesirable consequences.

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

Progress in identifying additional specific markers for prostate cancer has been 00 improved by the generation of prostate cancer xenografts that can recapitulate different 00 stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are ,IC 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.

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

SWhile 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 C' of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma 0 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 CK carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

00 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 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 00 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence Srate in women was 42.3 per 100,000.

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

SDuring 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

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cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

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

$O Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed 0 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 expen'ence remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were *expected to occur among women in the United States during.2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men In 2000; After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

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

Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were, probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy.

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

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen.may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer.

Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

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

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

0 There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past S years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the C1 past 20 years, there has been a slight but significant decrease in mortality rates among men (about per year) while rates have increased slightly among women.

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

00 SUMMARY OF THE INVENTION The present invention relates to a gene, designated 193P1E1 B, that has now been found to be over-expressed in C the cancer(s) listed in Table I. Northern blot expression analysis of 193P1E1B 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.193P1E1B are provided.. The lissue-related profile of 193P1E1B in normal adult tissues, combined with the overexpression observed in the tissues listed in Table I, shows that 193P1E18 is aberrantly over-expressed in at least some Scancers, 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 193P1E1B genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleolides encoding 193P1E1B-related proteins and fragments of 4, 5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 193P1E1B-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 193P1E1B genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 193P1E1B genes, SmRNAs, or to 193P1E1B-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 193P1E1B. Recombinant.DNA molecules containing 193P1E1B polynucleolides, cells transformed or transduced with such Smolecules, and host-vector systems for theexpression of 193P1E1B gene products are also provided. The invention further provides antibodies that bind to 193P1E1B 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 therapeulic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of Figure 2 is not encoded andlor the entire amino acid sequence of Figure 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of Figure 2 is encoded and/or the entire amino acid sequence of Figure 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 193P1E1B polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 193P1 E1B. A typical embodiment of this invention provides methods for monitoring 193P1E1B gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulalion such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 193P1 E1B such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 193P1E1B as well as cancer vaccines. In one aspect, the invention provides 00 compositions, and methods comprising them, for treating a cancer that expresses 193P1E1B in a human subject wherein the Scomposition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits S the production or function of 193P1 E1B. Preferably, the carrier is a uniquely human carrier. In another aspect of the S invention, the agent is a moiely that is immunoreaclive with 193P1E18 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human Santibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as Q defined herein.

r In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte S(CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 193P1E1B and/or one or more than Sone peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit 00 an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In Sa further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or r C 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 nudeic acid molecule may express a moiety that is immunologically reactive with 193P1E1B as described above. The one or more than one nuceic acid molecule may also be,:or encodes, a molecule that inhibits production of 193P1E1B. Non-limiting examples of such molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of 193P1E18 anlisense sequences or molecules that form a triple helix with a nudeotide double helix essential for 193P1E1B production) or a ribozyme effective to lyse 193P1E1B 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 VIlI-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 Sto 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 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure ii) a peplide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathiciy profile of Figure 6; 00 CKl 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 LC~ v) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the 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.

00 10 In one aspect the present invention provides an isolated or purified 193P1E1 B protein at least 90% identical to SEQ ID NO:3.

Lq The present invention also provides a composition comprising an immunoreactive epitope of the isolated or recombinant 193P1E1B protein, wherein the epitope comprises a CTL polypeptide epitope, with a proviso that the epitope is not the entire amino acid sequence of the 193P1E1B protein.

The present invention also provides a method of inhibiting growth of cancer cells that express 193P1 E1B, comprising: administering to said cells an antibody or fragment thereof, either of which specifically binds to a 193P1 El B protein of SEQ ID NO:3, or a variant thereof.

The present invention also provides a method of generating a mammalian immune response directed to a 193P1E1B protein, comprising: exposing cells of the mammal's immune system to an immunogenic portion of a) a 193P1E1B protein of SEQ ID NO:3, or a variant thereof and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to 193P1E1B.

The present invention also provides an assay for detecting the presence of a 193P1 E1 B protein in a biological sample obtained from a patient who has or who is suspected of having cancer, comprising: contacting the sample with the antibody or fragment thereof that specifically binds to a 193P1E1B protein of SEQ ID NO:3, or a variant thereof; and, detecting the formation and/or presence of a complex comprising the antibody or fragment thereof and the 193P1E1B protein.

The present invention also provides an assay for detecting the presence of a 193P1 E1 B polynucleotide in a biological sample, comprising: contacting the sample with a polynucleotide probe that specifically hybridizes under stringent conditions to a polynucleotide that is at least 90% identical to the entire polynucleotide of SEQ ID NO:1; and, detecting the presence of a hybridization complex formed by the hybridization of the probe with the 193P1 E1 B polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of 193P1E1B polynucleotide within the sample.

00 6A The present invention also provides a method for monitoring expression levels Sof a 193P1E1B gene product in a biological sample obtained from a subject who has or who is suspected of having cancer versus a normal sample, comprising: N, determining the level of a 193PIE1B gene product expressed by cells in a biological sample obtained from a subject who has or who is suspected of having O cancer; \determining the level of a 193P1E1B gene product expressed by cells in a corresponding normal sample; 00 comparing the level of 193P1E1B gene product determined in the biological O 10 sample to the level of 193P1E1B gene product in the corresponding normal sample, NI wherein the 193P1E1B gene product comprises 193P1E1B mRNA or a 193P1ElB protein that is at least 90% identical to SEQ ID NO:3.

The present invention also provides a 193P1E1B protein comprising an isolated or recombinant protein that is at least 90% homologous to the entire sequence of SEQ ID NO:3.

The present invention also provides a recombinant expression vector comprising a polynucleotide that encodes the polypeptide sequence at least 90% identical to SEQ ID NO:3.

The present invention also provides a host cell that contains an expression vector of the invention.

The present invention also provides a process for producing a protein comprising culturing the host cell of the invention under conditions sufficient for the production of the protein, wherein the amino acid sequence of the protein is selected from the group consisting of SEQ ID NO:3, 11, and 13.

The present invention also provides a composition comprising a pharmaceutically acceptable carrier and the viral vector of the invention.

The present invention also provides an antibody or fragment thereof that immunospecifically binds to an epitope on a protein comprising the amino acid sequence of SEQ ID NO: 1, or 13.

The present invention also provides a hybridoma that produces an antibody of the invention.

The present invention also provides a vector comprising a polynucleotide encoding a monoclonal antibody according to the invention.

The present invention also provides an in vitro method for detecting the presence of a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13 00 6B or a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2, 10, or S12, in a test sample comprising: contacting the sample with an antibody or polynucleotide, respectively, that ,IC specifically binds to the protein or polynucleotide, respectively; and detecting binding of protein or polynucleotide, respectively, in the sample thereto.

The present invention also provides an in vitro method of inhibiting growth of a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 00 13, comprising providing an effective amount of an antibody according to the invention 0O O 10 to the cell, whereby the growth of the cell is inhibited.

,IC The present invention also provides an in vitro method of delivering a cytotoxic agent to a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, comprising providing an effective amount of an antibody according to the invention to the cell.

The present invention also provides use of a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, for the preparation of a medicament to induce an immune response from a subject, whereby a T cell or B cell is activated.

The present invention also provides use of an effective amount of antibody or antigen binding fragment thereof for the preparation of a medicament which delivers a cytotoxic agent to a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, wherein the antibody or antigen binding fragment thereof comprises the antibody according to the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The 193P1E1B SSH sequence of 227 nucleotides.

00 6C Figure 2. A) The cDNA and amino acid sequence of 193P1E1B variant 1 (also Scalled "193P1E1B v.l" or "193P1E1B variant is shown in Figure 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 C including the stop codon.

B) The cDNA and amino acid sequence of 193P1E1B variant 2 (also called S"193P1E1B is shown in Figure 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

C) The cDNA and amino acid sequence of 193P1E1B variant 3 (also called "193P1E1B is shown in Figure 2C. The codon for the start methionine is CNI underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

D) The cDNA and amino acid sequence of 193P1E1B variant 4 (also called "193P1E1B is shown in Figure 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

E) The cDNA and amino acid sequence of 193P1E1B variant 5 (also called "193P1E1B is shown in Figure 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

F) The cDNA and amino acid sequence of 193P1E1B variant 6 (also called "193P1E1B is shown in Figure 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

G) The cDNA and amino acid sequence of 193P1E1B variant 7 (also called "193P1E1B is shown in Figure 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

H) The cDNA and amino acid sequence of 193P1E1B variant 8 (also called "193P1E1B is shown in Figure 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.

I) The cDNA and amino acid sequence of 193P1E1B variant 9 (also called "193P1E1B is shown in Figure 21. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 989-1981 including the stop codon.

00 J) The cDNA and amino acid sequence of 193P1E1B variant 10 (also called "193P1E18 v.10') is shown in Figure 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-1971 including the stop codon.

0) K) The cDNA and amino acid sequence of 193P1E1B variant 11 (also called '193P1E1B v.11") is shown in Figure 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 989-1909 including C the stop codon.

L) The cDNA and amino acid sequence of 193P1E1B variant 12 (also called "193P1E1B v.12") is shown in Figure 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-1026 including the stop codon.

0 M) The cDNA and amino acid sequence of 193P1E1B variant 13 (also called "193P1E1B v.13") is shown in Figure 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 952-2070 including 00 the stop codon.

0Figure 3.

A) Amino acid sequence of 193P1E1B v.1 is shown in Figure 3A; it has 412 amino acids.

B) The amino acid sequence of 193P1E1B v.5 is shown in Figure 3B; It has 412 amino acids.

C) The amino acid sequence of 193P1E1B v.6 is shown in Figure 3C; it has 412 amino adds.

D) The amino acid sequence-of 193P1E18 v.9 is shown in Figure 3D; it has 330 amino adds.

E) The amino acid sequence of 193P1E1B v.10 is shown in Figure 3E; it has 388 amino acids.

F) The amino acid sequence of 193P1E1B v.11 is shown in Figure 3F; it has 308 amino acids.

G) The amino acid sequence of 193P1E1B v.12 is shown in Figure 3G; it has 73 amino acids.

H) The amino acid sequence of 193P1E1 B v.13 is shown in Figure 3H; it has 372 amino acids. As used herein, a reference to 193P1E1 B includes all variants thereof, including those shown in Figures 2, 3, 10, and 11, unless the context dearly indicates otherwise.

Figure 4. Figure 4A shows the alignment of 193P1E1B v.1 with gi 2178775. Figure 48 shows the alignment of 193P1E1B v.5 with gi 2178775. Figure 4C shows the alignment of 193P1E1B v.11 with gi 2178775. Figure 4D shows the alignment of 193P1E1B v.12 with gi 2178775. Figure 4E shows the alignment of 193P1E1B v.1 with Ecoliarginine repressor. Figure 4F shows the Alignment of 193P1E1B v.1 with human adenosine deaminase. Figure 4G shows the Clustal alignment of 193P1E18 protein variants.

Figure 5. Hydrophilicity amino acid profile of 193P1E18 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp Woods 1981. Proc. Nail. 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. Hydropalhicity amino acid profile of 193P1E1B 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 193P1E1B determined by computer algorithm sequence analysis using the method of Janin (Janin 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 8. Average flexibility amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran and Ponnuswamy 1988. Int. J. Pept. Protein Res.

32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

00 O Figure 9. Beta-turn amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using N the method of Deleage and Roux (Deleage, Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

SFigure 10. Schematic alignment of SNP variants of 193P1E1B. Variants 193P1E1B v.2 through v.8 are variants with single nudeotide differences. Though these SNP variants are shown separately, they could also occur in any Scombinations and in any transcript variants that contains the base pairs. Numbers correspond to those of 193P1E1B v.1.

Black box shows the same sequence as 193P1E1B v.1. SNPs are indicated above the box.

SFigure 11. Schematic alignment of protein variants of 193P1E1B. Protein variants correspond to nucleotide variants. Nucleotide variants 193P1E1B v.2, v.3, v.4, v.7, and v.8 in Figure 10 code for the same protein as 193P1E1B v.1.

Nudeotide variants 193P1E1B v.9 through v.13 are splice variants of v.1. Single amino acid differences were indicated Cabove the boxes. Black boxes represent the same sequence as 193P1E1B v.1. Numbers underneath the box correspond to 00 amino acid positions in 193P1E1B v.1.

SFigure 12. Intentionally Omitted.

Figure 13. Secondary structure prediction for 193P1E1B (SEQ ID NO: 123). The secondary structure of 193P1E1B protein was predicted using the HNN Hierarchical Neural Network method (Guermeur, 1997, .http:lpbil.ibcp.fr/cgi-bin/npsa_automal.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology -server(http://www.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and Srandom coils from the primary protein sequence. The percent of the protein in a given secondary.structure is as follows: h: Alpha helix 29.13%; c: Random coil 60.92%; e: Extended strand 9.95%.

Figure 14. Expression of 193P1E1B by RT-PCR. The schematic diagram depicts the location of PCR primers Set A and set B on lhe sequences of the 3 variants of 193P1E1 B. (B and C) First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC- 4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from 2 different patients. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primer Set A or primer Set B to 193P1E1B, was performed at cycles of amplification. Strong expression of 193P1E1B was observed in prostate cancer xenograft pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis pool, pancreas cancer pool, and in the 2 different prostate cancer metastasis to lymph node. Low expression was observed in:.

prostate cancer pool, but no expression was detected in VP1 and VP2. Figure 14C shows that the transcipt encoding encoding 193PIE1B v.1 through v.8, is expressed ate higher levels that the transcript encoding 193P1E1B v.9. But both transcripts are expressed at similar proportion in all tissues tested.

Figure 15. Expression of 193P1E1B in normal human tissues. Two multiple tissue northern blots, with 2 pg of .mRNMane, were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of two 193P1E1B transcripts, approximately 3.5 kb and 2 kb, in testis and thymus.

Figure 16. Expression of 193P1E1B in prostate cancer xenografts. RNA was extracted from normal prostate, and from prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 pg of total RNA/lane was probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1 B in all 4 xenografts but not in normal prostate.

Figure 17. Expression of 193P1E1B in patient cancer specimens. RNA was extracted from a pool of three patients for each of the following, bladder cancer, colon cancer, ovary cancer and metastasis cancer, as well as from normal prostate normal bladder normal kidney normal colon Northern blots with 10 Iig of total RNAlane 00 O were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show CK expression of 193P1E1B in bladder cancer pool, colon cancer pool, ovary cancer pool and metastasis cancer pool, but not in Sany of the normal tissues tested.

Figure 18. Expression of 193P1E1B in bladder cancer patient specimens. RNA was extracted from bladder S cancer cell lines normal bladder bladder tumors and matched normal adjacent tissue (NAT) isolated from bladder cancer patients. Northern blots with 10 pg of total RNANane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1 B in the two bladder cancer O cell lines, and in 3 patient bladder tumors tested but not in normal bladder tissues.

0 Figure 19. Expression of 193P1E1B in cancer metastasis patient specimens. RNA was extracted from the following cancer metastasis tissues, colon metastasis to lung, lung metastasis to lymph node, lung metastasis to skin, and C breast metastasis to lymph-node, as well as from normal bladder normal lung normal breast (NBr), and normal 00 0 ovary(NO). Northern blots with 10 plg of total RNA/lane were probed with 193P1E1B sequence. Size standards in Skilobases (kb) are indicated on the side. The results show expression of 193P1E1B in all four different cancer metastasis samples but not in normal tissues.

Figure 20. Expression of 193P1E1B in pancreas, ovary and testis cancer patient specimens. RNA was extracted 'from pancreatic cancer ovarian cancer (P2, P3), and testicular cancer (P4, P5) isolated from cancer patients, as well as from normal pancreas (NPa). Northern blots with 10 pg of total RNAlane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in pancreatic, ovarian and testicular cancer specimens but not in normal pancreas.

Figure 21. Expression of 193P1E1B in Normal versus Patient Cancer Specimens. First strand cDNA was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate, bladder, kidney, colon, lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, 'metastasis cancer pool, LAPC prostate xenograft pool and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in Figure 14, was performed was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1B expression in normal 'testis amongst all normal tissues tested. 193P1E1 B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.

Figure 22. Expression of 193P1E1B in Normal versus Patient Cancer Specimens. First strand cDNA was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate, bladder, kidney, colon lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPC prostate xenograft pool and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in Figure 14, was performed was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1B expression In normal testis amongst all normal tissues tested. 193P1E1B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.

00 0 DETAILED DESCRIPTION OF THE INVENTION 0 Outline of Sections Definitions SII.) 193P1E1B Polynucleotides II.A.) Uses of 193PIE1B Polynucleotides C II.A.1.) Monitoring of Genetic Abnormalities II.A.2.) Antisense Embodiments II.A.3.) Primers and Primer Pairs II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid Molecules Recombinant Nucleic Acid Molecules and Host-Vector Systems CIII.) 193P1E1B-related Proteins 00 SIII.A.) Motif-bearing Protein Embodiments 11.B.) Expression of 193P1E1B-related Proteins III.C.) Modifications of 193P1E1B-related Proteins III.D.) Uses of 193P1E1B-related Proteins IV.) 193P1E1B Antibodies 193P1E1B Cellular Immune Responses VI.) 193P1E1B Transgenic Animals VII.) Methods forthe Detection of 193P1E1B VIII.) Methods for Monitoring the Status of 193P1E1B-related Genes and Their Products IX.) Identification of Molecules That Interact With 193P1E1B Therapeutic Methods and Compositions Anti-Cancer Vaccines 193P1E1B as a Target for Antibody-Based Therapy 193P1E1B 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 andlor HTL Peptides Adoptive Immunotherapy Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 193P1E1B.

XII.). Inhibition of 193P1E1B Protein Function XIIA.) Inhibition of 193P1E1B With Intracellular Antibodies XII.B.) Inhibition of 193P1EIB with Recombinant Proteins XII.C.) Inhibition of 193P1E1B Transcription or Translation XII.D.) General Considerations for Therapeutic Strategies XIII.) Identification, Characterization and Use of Modulators of 193P1E1 B XIV.) KITSIArticles of Manufacture Definitions: 00 O Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are Sintended 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 commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms "advanced prostate cancer', "locally advanced prostate cancer", "advanced disease" and "locally 0 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 compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the 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 193P1E1B (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 193P1E1B. 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 wilh another molecule a 193P1ElB-related protein). For example, an analog of a 193PIE1B protein can be specifically bound by an antibody or T cell that specifically binds to 193P1E1B.

The term "antibody" is used in the broadest sense. Therefore, an "antibody" can be naturally occuring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-193P1E1B 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-193PIE1B antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-193P1E1B 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 andlor 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 00 S combinatorial chemical library, such as a polypeptide mutein) library, is formed by combining a set of chemical building N blocks called amino acids in every possible way for a given compound length the number of amino acids in a Spolypeplide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical j building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).

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

Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded Speptides (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 at., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal CK peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), 00 Sanalogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),.

0 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 Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, Liang 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; thiazolidinones and metathiazanones, U.S.

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

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

Devices for the preparation of combinatorial libraries are commercially available (see, 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, 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 apparalus 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 andlor 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 andlor variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine; dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retslrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaia officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At 2 1131, 1125, ys 0 Re 186 Rela 8 Sm 153 Bi212 213, P32 and radioactive isotopes of Lu including Lu 1 n. Antibodies may also be conjugated to an anticancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

00 0 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 0 Table a "cancer mRNA", "mRNA of a cancer listed in Table etc. In one embodiment, the cancer protein is encoded by a nucleic acid of Figure 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of Figure 2. In one embodiment a cancer amino acid sequence is used to determine LC sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure 2. In another embodiment, the sequences are sequence variants as further described herein.

7 "High throughput screening" assays for the presence, absence, quantification, or other properties of particular nlucleic 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 00 proteins; U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding arrays); Swhile U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.

C

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

The term "homolog" refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues Ilal 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, 8" 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 pg/ml ssDNA,"in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1% SDS are above 55 degrees C.

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 silu environment. For example, a polynudeoide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 193P1E1B genes or that encode polypeplides other than 193P1E1B gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 193P1E1B polynudeotide. A protein is said to be "solated," for example, wh3n physical, mechanical or chemical methods are employed to remove the 193P1E1B proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 193P1E1 B protein. Alternatively, an isolated protein can be prepared by chemical means.

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

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

Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, 0\ rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, N skeletal radiography, andlor bone lesion biopsy.

00 0 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 capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, a nucleic acid or protein sequences, or effects of cancer sequences signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By "neutralize" is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, at zero concentration or below the level of detection.

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

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

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

I

00 0 Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.

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

A motif, as in biological motif of a 193P1E1B-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function prolein-protein interaction, protein-DNA C interaction, etc) or modification that is phosphorylated, glycosylated or amidated), or localization secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with 00 .a certain function or property. In the context of HLA motifs, "motif' refers to the pattern of residues in a peptide.of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino r 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 residues. A "pharmaceutical excipient" comprises a material such'as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

"Pharmaceutically acceptable' refers to a non-toxic, inert, andlor composition that is physiologically compatible with Shumans 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 adds 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. Altematively, 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 peplides 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): 00 O Examples of Medical Isotopes: SIsotope N Description of use 0) .Actinium-225 C(AC-225) See Thorium-229 (Th-229) Actinium-227 (AC-227) Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton resulting from cancer breast and prostate cancers), and cancer radioimmunotherapy Bismuth-212 (Bi-212) SSee Thorium-228 (Th-228) 00 SBismuth-213 0(Bi-213) r See Thorium-229 (Th-229) Cadmium-109 (Cd-109) Cancer detection Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical supplies Copper-64 (Cu-64) A positron emitter used for cancer therapy and SPECT imaging Copper-67 (Cu-67) Betalgamma emitter used in cancer radioimmunotherapy and diagnostic studies breast and colon cancers, and lymphoma) Dysprosium-166 (Dy-166) Cancer radioimmunotherapy Erbium-169 (Er-169) Rheumatoid arthritis trealment, particularly for the small joints associated with fingers and toes Europium-152 (Eu-152) Radiation source for food irradiation and for sterilization of medical supplies Europium-154 (Eu-154) Radiation source for food irradiation and for sterilization of medical supplies Gadolinium-153 (Gd-153) Osteoporosis detection and nuclear medical quality assurance devices Gold-198 (Au-198) Implant and Intracavity therapy of ovarian, prostate, and brain cancers Holmium-166 00 0 (Ho-166) SMultiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone marrow ablation, Sand rheumatoid arthritis treatment Iodine-125 S(1-125) Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, radiolabeling, tumor imaging, mapping of receptors in the brain, interstilial radiation therapy, brachytherapy for treatment of i

N

prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs 0Iodine-131 Cr (1-131) Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as other nonmalignant thyroid diseases Graves disease, goiters, and hyperthyroidism), treatment of leukemia, 0 lymphoma, and other forms of cancer breast cancer) using radioimmunotherapy 00 I ridium-192 (Ir-192) Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries arteriosclerosis and restenosis), and implants for breast and prostate tumors Lutetium-177 (Lu-177) Cancer radioimmunotherapy and treatment of blocked arteries arteriosclerosis and restenosis) Molybdenum-99 (Mo-99) Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver, lungs, heart, and other organs.

Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging of various cancers and diseases involving the brain; heart, liver, lungs; also used in detection of deep vein thrombosis of the legs Osmium-194 (Os-194) Cancer radioimmunolherapy Palladium-103 (Pd-103) Prostate cancer treatment Platinum-195m (Pt-195m) Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug Phosphorus-32 (P-32) Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer 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 (P-33) Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of blocked arteries arteriosclerosis and restenosis) Radium-223 (Ra-223) See Actinium-227 (Ac-227) Rhenium-186 (Re-186) Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma and bone, breast, colon, and iver cancers using radioimmunotherapy 00 Rhenium-188 (Re-i 88) Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, treatment of rheumatoid arthritis, and treatment of prostate cancer 1- Rhodium-lOS C1 (Rh-105) Cancer radjoinimunotherapy Samariuni-145 (Sm-145) Ocular cancer treatment Samarium-I 53 C1 (Sm-i 53) 00 Cancer radioimmunotherapy and bone cancer pain relief Scandium-47 *-(Sc-47) Cancer radiolmmunotherapy and bone cancer pain relief Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool Bone cancer detection and brain scans Strontium-89 (Sr-89) Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy Technetium-99m (Tc-99m) See Molybdenum-99 (Mo-99) Thorium-M2 (Th-228) Parent of Bismuth-21 2 (Bi-21 2) which is an alpha emitter used in cancer radioimmunotherapy Thorium-229 (Th-229) Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-21 3 (Bi-21 3) which are alpha emitters used in cancer radioinimunolherapy Thulium-i (Tm-170) Gamma source for blood irradiators, energy source for implanted medical devices Tin-I 17m (Sn-I 17m) Cancer immunotherapy and bone cancer pain relief Tungsten-I 88 (-188) Parent for Rhenium-i 88 (Re-i 88) which is used for cancer diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment and treatment of blocked arteries arteriosclerosis and restenosis) Xenon-i 27 00 (Xe-127) Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, and cerebral blood tC~ flow studies Ytterbium-175 (Yb-175) Cancer radioimmunolherapy Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment I Yttrium-91 (Y-91) A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy lymphoma, Sbreast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers) 00 (O 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 (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, a library is "fully randomized," with no sequence preferences or constants at any position. In 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 nucleolides or amino acid residues are randomized within a defined class, of hydrophobic amino adds, 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.

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

Non-limiting examples of small molecules include compounds that bind or interact with 193P1E1B, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 193P1E1B 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, 193P1E1B protein; are not found in naturally occurring metabolic pathways; andlor are more soluble in aqueous than non-aqueous solutions "Stringency" of hybridization reactions is readily 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 00 O details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, CK Wiley Interscience Publishers, (1995).

C "Stringent conditions" or high stringency conditions", as defined herein, are identified by, but not limited to, those Sthat employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citratel0.1% sodium dodecyl sulfate at 50oC; employ during hybridization a denaturing agent, such as formamide, for example, 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 oC; or employ 50% formamide, 5 x SSSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x 01 Denhardfs solution, sonicated salmon sperm DNA (50 pg/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 oC, followed by a high-stringency wash consisting of 0.1 x SSC conlaining EDTA at 55 oC. "Moderately stringent conditions" are described by, but not limited to, 00 0 those in Sambrook ef 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 Denhardts solution, 10% dextran sulfate, and 20 mglmL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50oC. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

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

Overall phenotypic frequencies of 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 A3: A3, All, A31, A*3301, A*6801, A*0301, A*1 101, A3101 7: B7, 8*3501-03, 6*51, B*5301, B'5401, B*5501, B'5502, B*5601, 8*6701, B"7801, B*0702, B*5101, 8B5602 B44: B*3701, B*4402, B*4403, 8*60 (B*4001), B61 (B*4006) Al: A*0102, A'2604, A*3601, A*4301, A*8001 A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003 B27: B*1401-02, B*1503, B*1509, B*1510, 8*1518, B*3801-02, 8*3901, B83902, 8*3903-04, B*4801-02, B*7301, 8*2701-08 B58: B'1516, B*1517, B*5701, 8*5702, 858 B62: B*4601, B52, B*1501 (B62), 8*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 "lransgenic 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" 1 00 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, C" 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.

SThe 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.

SThe term varianr 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 193P1E16 protein shown in Figure 2 or Figure 3. An analog is an example of a variant protein. Splice isoforms and single nucleolides polymorphisms (SNPs) are further examples of variants.

00 The "193P1 E1 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 Sexperimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 193P1E1B proteins or fragments thereof, as well as fusion proteins of a 193P1E1B protein and a heterologous polypeptide are also included. Such 193P1E1B proteins are collectively referred to as the 193P1E1B-related proteins, he proteins of the invention, or 193P1E1B. The term "193P1E1B-related protein" refers toa polypeplide fragment or a 193P1E1B 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 25 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, 600, 625, 650, or 664 or more amino acids.

II.) 193P1E1B Polynucleotides One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 193P1E1B gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 193P1E1B-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonudeotides complementary to a 193P1E1B gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleolides that hybridize to a 193P1 E B gene, mRNA or to a 193P1E1B encoding polynucleotide (collectively, "193P1E1B polynucleotides"). In all instances when referred to in this section, T can also be U in Figure 2.

Embodiments of a 193P1E1B polynucleotide include: a 193P1E1B polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 193P1 E1B as shown in Figure 2 wherein T is U; at least 10 contiguous nucleolides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 193P1E1B nucleotides comprise, without limitation: a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in Figure 2, wherein T can also be U; (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2A, from nucleotide residue number 805 through nucleotide residue number 2043, including the stop codon, wherein T can also be U; r 00 (I1I) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure r 2B, from nucleotide residue number 805 through nucleotide residue number 2043, including the stop codon, Swherein T can also be U; (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure r1 2C, from nucleotide residue number 805 through nucleotide residue number 2043, including the a stop codon, wherein T can also be U; C a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D, from nucleotide residue number 805 through nucleolide residue number 2043, including the stop codon, Swherein T can also be U; 00 0(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure i2E, from nucleotide residue number 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 2G, from nucleotide residue number 805 through nucleotide residue number 2043, including the stop codon, wherein T can also be U; (IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2H,;from nucleotide residue number 805 through nucleotide residue number 2043, 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 989 through nucleotide residue number 1981, including the stop codon, wherein T can also be U; (XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2J, from nucleotide residue number 805 through nucleotide residue number 1971, including the stop codon, wherein T can also be U; (XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2K, from nucleotide residue numnber 989 through nucleotide residue number 1909, including the slop 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 805 through nudeotide residue number 1026, including the stop codon, wherein T can also be U; 00 O (XIV) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure S2M, from nucleotide residue number 952 through nucleotide residue number 2070, including the stop codon, Swherein T can also be U; (XV) a polynudeolide that encodes a 193P1E1B-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-M; (XVI) a polynucleotide that encodes a 193P1E1B-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, S98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-M; (XVII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX; 00 (XVIII) .a polynucleotide that encodes a peptide region of at least 5, 6,7,8,9,10, 11, 12,13,14,15,16,17,18, 019, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A-C in any Swhole number increment up to 412 that includes at least 1, 2,3,4, 5,6, 7, 8,9,10,11, 12, 13, 14,15,16,17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than in the Hydrophilicity profile of Figure (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 peplide of Figure 3A-C in any whole number increment up to 412 that indudes 1, 2, 3, 41 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; (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-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; (XXI) a polynucleolide 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-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16,17,18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (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-C in any whole number increment up to 412 that indudes 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 Beta-turn profile of Figure 9; (XXIII) 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 acds of a peptide of Figure 30 in any whole number increment up to 330 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 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 CL" Hydrophilicity profile of Figure (XXIV) a polynucleotide that encodes a peptide region of atleast 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 Snumber increment up to 330 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; (XXV) a polynucleotide thatencodes a peptide region of at least 5, 6, 7,8,9,10, 11, 12, 13,14,15,16,17, 18, O 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 (-i 00 number increment up to 330 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 (Ni Percent Accessible Residues profile of Figure 7; (XXVI) a polynucteotide 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 330 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; (XXVII) a polynucleotide thatencodes 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 330 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 (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 3E in any whole number increment up to 388 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 (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 3E in any whole number increment up to 388 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; (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, 19, 20, 21, 22, 23, 24, 25,26, 27,28, 29, 30, 31,32, 33,34,35 amino acids ofa peptide of Figure 3E in any whole number increment up to 388 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; 00 (XXXI) a polynudeotide that encodes a peptide region of at least 5, 6,7,8,9,10,11,12,13,14,15,16,17,18, C 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a peptide of Figure 3E in any whole number increment up to 388 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; (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 3E in any whole number increment up to 388 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 Betaturn profile of Figure 9 00 (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 3F in any whole number increment up to 308 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 (XXXIV) apolynucleotide thatencodesa peptide region of atleast 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 3F in any whole number increment up to 308 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 valueless than 0.5 in the Hydropalhicity profile of Figure 6; (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 3F in any whole number increment up to 308 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; (XXXVI) 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 adds of a peptide of Figure 3F in any whole number increment up to 308 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; (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 3F in any whole number increment up to 308 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 (XXXVIII) a polynuceotide that encodes a peptide region of at least 5,6,7,8,9,10, 11, 12,13,14,15,16,17,18, 19, 20, 21, 22, 23, 24,25, 26,27,28,29,30, 31.32,33, 34, 35 amino acids of a peptide of Figure 3G in any whole 00 number increment up to 73 that indudes 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 Hydrophilicity profile of Figure (XXXIX) apolynudeotidethatencodes a peptideregionofatleast 5,6,7,8, 9,10,11,12,13,14,15,16,17,18, Cl 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3G in any whole number increment up to 73 that includes 1, 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 Hydropathicity profile of Figure 6; (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, 00 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3G in any whole Snumber increment up to 73 that includes 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 Percent Accessible Residues profile of Figure 7; (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 3G in any whole number increment up to 73 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; (XLII) 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 adds of a peptide of Figure 3G in any whole number increment up to 73 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 (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 3H in any whole' number increment up to 372 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 (XLIV) a polynucleotide that encodes a peptideregion 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 372 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; (XLV) 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 372 that indudes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 00

O

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 r C Percent Accessible Residues profile of Figure 7; r (XLVI) 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 372 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 posilion(s) having a value greater than 0.5 in the O Average Flexibility profile of Figure 8; (XLVII) 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 00 number increment up to 372 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 Betaturn profile of Figure 9 (XLVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XLVII).

(XLIX) a peptide that is encoded by any of to (XLVIII); and a composition comprising a polynudeotide of any of (I)-(XLVIII) or peptide of (XLIX) together with a pharmaceutical excipienl and/or in a human unit dose form.

(LI) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of in a method to modulate a cell expressing 193P1E1B, (LII) a method of using a polynucleolide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B (LIll) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1 B, said cell from a cancer of a tissue listed in Table I; (LIV) a method of using a polynudeotice of any (I)-(XLVIll) or peptide of (XLIX) or a composition of in a melhod to diagnose, prophylax, prognose, or treat a a cancer; (LV) a method of using a polynucleotide of any (l)-(XLVIII) or peptide of (XLIX) or a composition of in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and, (LVI) a method of using a polynuceolide of any (I)-(XLVIII) or peplide of (XLIX) or a composition of in a method to identify or characterize a modulator of a cell expressing 193P1 E1 B.

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

Typical embodiments of the invention disclosed herein include 193P1 E1B polynucleotides that encode specific portions of 193P1E1B mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

I

00 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, 65,70, s 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, 225, 250, 275, 300, 325,350, 375, 400, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; the maximal Slengths relevant for other variants are: variant 5, 412 amino acids; variant 6, 412 amino acids, variant 9, 330 amino acids, variant 10, 388 amino acids, variant 11,308 amino acids, variant 12, 73 amino adds, and variant 13, 372 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 193P1E1B protein shown in Figure S2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 193P1E1B protein shown in O\ Figure 2 or Figure 3, polynudeotides encoding about amino acid 20 to about amino acid 30 of the 193P1E1B protein shown 0in Figure 2 or Figure 3, polynudeotides encoding about amino acid 30 to about amino acid 40 of the 193P1E1B protein Sshown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 40 to about amino acid 50 of the 193P1E18 00 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino add 50 to about amino acid 60 of the S193P1EIB protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 193P1E1B protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid of the 193P1E1B protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid of the 193P1E1B protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino add 100 of the 193P1E1B 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 add sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 193P1E1B protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynudeotides encoding relatively long portions of a 193P1E1B protein are also within the scope of the invention.

For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or or 50 etc.) of the 193P1E1B protein "or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 193P1E1B sequence as shown in Figure 2.

Additional illustrative embodiments of the invention disclosed herein include 193P1E1B polynucleotide fragments encoding one or more of the biological motifs contained within a 193P1E1B protein "or variant" sequence, induding one or more of the motif-bearing subsequences of a 193P1E1B protein 'or variant" set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 193P1E1B protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 193P1E1B protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylalion 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 Peplides listed in Table LVII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative.to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position one must add the value "X minus 1" to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 1, 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

0 0 II.A.) Uses of 193P1E1B Polynucleotides SII.A.I.) Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have a numberof different specific uses. The human 193P1E18 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 193P1E1B." For C' example, because the 193P1E1B gene maps to this chromosome, polynucleolides thai encode different regions of the 193P1E18 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et Mutat. Res. 382(3-4): 81-83 (1998); Johansson et Blood 86(10): 3905-3914 (1995) and Finger et al, 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 193P1E1B proteins provide new Stools that can-be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the 00 chromosomal region that encodes 193P1E1 B that may contribute to the malignant phenolype. In this context, these Spolynucleotides 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 Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 193P1E1B was shown to be highly expressed in bladder and other cancers, 193P1E1B polynucleotides are used in methods assessing the status of 193P1E1B gene products in normal versus cancerous tissues.

Typically, polynucleotides that encode specific regions of the 193P1E1B proteins are used to assess the presence.of ,perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 193P1E1B gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-slrand conformation polymorphism (SSCP) analysis (see, Marrogi et al., J. Cutan. Pathol.

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

II.A.2.) Antisense Embodiments Other specifically contemplated nucleic acid relaled embodiments of the invention disclosed herein are genomic DNA, cDNAs, rbozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 193P1E1B. For example, antisense molecules can be RNAs or other molecules, including peplide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 193P1E1B polynucleotides and polynudeolide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleolides:lhat bind to a target polynucleoUde located within the cells. The term *antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, 193P1E1B. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 193P1E1B antisense oligonudeotides 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 phosphorothioales) are isoelectronic analogs of an oligonucleotide (O-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,l-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 193P1E1B antisense oligonudeolides of 00 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 193P1E1B antisense oligonucleotides of the present invention typically can be RNA or DNA that is |Ll complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 193P1 E1 B genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 193PIE1B mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 193P1E1B antisense oligonudeotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 193P1E1B mRNA. Optionally, 193P1E1B antisense oligonucleotide is a 30-mer oligonuceotide that is 0 complementary to a region in the first 10 5' codons or last 10 3' codons of 193P1E1B. Alternatively, the antisense molecules S are modified to employ ribozymes in the inhibition of 193P1E1 B expression, see, L. A. Couture D. T. Stinchcomb; 00 Trends Genet 12: 510-515 (1996).

SII.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 193P1E1B polynudeotide in a sample and as a means for detecting a cell expressing a 193P1E1B protein.

Examples of such probes include polypeptides comprising all or part of the human 193P1E1B cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 193P1E1B mRNAs are also described in the Examples.

As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 193P1E1B mRNA.

The 193P1E1B 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 and/or detection of the 193P1E1 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 193P1E1B polypeptides; as tools for modulating or inhibiting the expression of the 193P1E1B gene(s) and/or translation of the 193P1 E1 B transcript(s): and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 193P1E1B or 193P1E1B 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.

II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid Molecules The 193P1E1B cDNA sequences described herein enable the isolation of other polynucleolides encoding 193P1E1B gene product(s), as well as the isolation of polynucleotides encoding 193P1E1B gene product homologs, altematively spliced isofonns, allelic variants, and mutant forms of a 193P1E1B gene product as well as polynucleotides that encode analogs of 193P1E1B-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 193P1E1B gene are well known (see, for example, Sambrook, J. et Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et at., 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 dones containing 193P1E1B gene cDNAs can be identified by probing with a labeled 193P1E1B cDNA or a fragment thereof. For example, in one embodiment, a 193P1E1B 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 193P1 E1 B 00 gene. A 193P1E18 gene itself can be isolated by screening genomic DNA libraries, bacterial artficial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 193P1E1B DNA probes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems The invention also provides recombinant DNA or RNA molecules containing a 193P1E1B 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).

SThe invention further provides a host-vector system comprising a recombinant DNA molecule containing a 0193P1 El B polynudeotide, 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 00 0various 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 193P1E1B or a fragment, analog or homolog thereof can be used to generate 193P1E1B proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 193P1E1B proteins or fragments thereof are .available, see for example, Sambrook et at., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors Sfor mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the relroviral vector pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 193P1 E1 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 systems of the invention are useful for the production of a 193P1E1B protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 193P1E1B and 193P1E1B mutations or analogs.

Recombinant human 193P1E1B protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 193P1E1B-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 193P1E1B or fragment, analog or homolog thereof, a 193P1E1B-related protein is expressed in the 293T cells, and the recombinant 193P1E1B protein is isolated using standard purification methods affinity purification using anti-193P1E1B antibodies).. In another embodiment, a 193P1E1B coding sequence is'subdoned into the retroviral vector pSRaMSVIkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 193P1E1B 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 193P1E1B coding sequence can be used for the generation of a secreted form of recombinant 193P1E1B protein.

As discussed herein, redundancy in the genelic code permits variation in 193P1E1B gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/~nakamuralcodon.html.

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

00 O Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications C include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in SKozak, 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)).

II.t1 193P1EiB-related Proteins O Another aspect of the present invention provides 193P1E1B-related proteins. Specific embodiments of 193P1E1B :proteins comprise a polypeptide having all or part of the amino acid sequence of human 193P1E1B as shown in Figure 2 or Figure 3. Alternatively, embodiments of 193P1E1B proteins comprise variant, homolog or analog polypeptides that have C: alterations in the amino acid sequence of 193P1E1B shown in Figure 2 or Figure 3.

00 Embodiments of a 193P1E1B polypeptide include: a 193P1E1B polypeptide having a sequence shown in Figure 2, Sa peptide sequence of a 193P1E1B 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 193P1E1B peptides comprise, without limitation: .a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in Figure 2A-M or Figure 3A-H; (II) a 193P1E1B-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-M; (111) a 193P1E1B-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-M or 3A-H; (IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of Figure 2; a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of Figure 2; (VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of Figure 2; (VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2; (VIII) a protein that comprisesat 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 adds of a protein of Figure 3A, 38, 30, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively that includes at least 1, 2, 00 0 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, amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure a polypeplide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 30, 3E, 3F, 3G, or 3H in Sany whole number increment up to 412,412, 412, 330, 388, 308, 73, or 372 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, amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6; 0\ (XI) a polypeptide comprising atleast5, 6, 7, 8, 9, 10,11,12,13,14, 15,16,17,18,19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in 0 any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that includes at least 1, O 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, t^ 35 amino acid posilion(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; (XII) a polypeptide comprising atleast 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, 38, 3C, 30, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 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, amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XIII) a polypeptide comprising at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412. 330, 388, 308, 73, or 372 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, amino acid position(s) having a value greater than 0.5 in the Bela-turn profile of Figure 9; (XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively; (XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively; (XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively; (XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively; (XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XIX) a peptide that occurs at least once in Tables Vll-XXI, and at least twice in tables XXII to XLIX; (XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX; (XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX; (XXII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide: i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that 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 00 ii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment C 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; Siii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment Sup to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5,0.6, 0.7,0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; Siv) a region of at least 5 amino adds of a particular peptide of Figure 3, in any whole number increment 0\ up to the full lenglh 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, 0 v) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment 0up to the full length of that protein In Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9; (XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.

(XXIV) a method of using a peptide of or an antibody or binding region thereof or a composition of (XXIII) in a method to modulate a cell expressing 193P1E1B, (XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B (XXVI) 'a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B, said cell from a cancer of a tissue listed in Table I; (XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer; (XXVIII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and, (XXIX) a method of using a a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to identify or characterize a modulator of a cell expressing 193P1E18.

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

Typical embodiments of the invention disclosed herein include 193P1 E1 B polynucleotides that encode specific portions of 193P1E1B 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, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; the maximal lengths relevant for other variants are: variant 5, 412 amino acids; variant 6, 412 amino acids, variant 9, 330, variant 10, 388 amino acids, variant 11, 308 amino acids, variant 12, 73, and variant 13, 372 amino acids..

00 O In general, naturally occurring allelic variants of human 193P1E1B share a high degree of structural identity and homology 90% or more homology). Typically, allelic variants of a 193P1E1B protein contain conservative amino acid substitutions within the 193P1E1B sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 193P1E1B. One class of 193P1E1B allelic variants are proteins that share a high degree of homology with at least a small region of a particular 193P1E1B amino acid sequence, but further contain a radical departure from the r- sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology Sand 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 CKl in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 00 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine 0 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" 2n d 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 193P1E1B proteins such as polypeptides having amino acid insertions, deletions and substitutions. 193P1E1B variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Sitedirected mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10.6487 (1987)), cassette mutagenesis (Wells etal., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells of al., Philos. Trans. R.

Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 193P1E1B 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 adds are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine.

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

As defined herein, 193P1E1B variants, analogs orhomologs, have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 193P1E1B 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 193P1E1B variant also specifically binds to a 193P1E18 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 00 specifically binds to the starting 193P1 E1 B protein. Those skilled in the art understand that antibodies that recognize Cproteins bind to epitopes of varying size, and a grouping of the order of about four or five amino adds, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See; Nair et al., J. Immunol 2000 165(12): 6949- [J 6955; Hebbes etal., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 193P1E1 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 193P1E1B protein variants or analogs comprises one or more of the 193P1E1B biological motifs described herein or presently known in the art. Thus, Sencompassed by the present invention are analogs of 193P1E1B fragments (nucleic or amino acid) that have altered Sfunctional 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.

SAs discussed herein, embodiments of the claimed invention include polypeptides containing less than the full 00 Samino acid sequence of a 193P1 E1 B protein shown in Figure 2 or Figure 3. For example, representative embodiments of Sthe 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 193P1E1B 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 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 193P1E1B protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 193PIE1B protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 193P1E1 B aminoacid 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 193P1E1B protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positionsin this paragraph refer to the specified position as well as that position plus or minus 5 residues.

193P1E1 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 193P1E1B-related protein. In one embodiment, nucleic add molecules provide a means to generate defined fragments of a 193P1E1B protein (or variants, homologs or analogs thereof).

III.A.) Motif-bearlng Protein Embodiments Additional illustrative embodiments of the invention disclosed herein include 193P1E1B polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 193P1E1B polypeptide sequence set forth in Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Intemet sites (see, URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edulseq-search/struc-predict.html; psortims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uklinterprolscan.html; expasy.chftools/scnpsitl .html; Epimatrix T M and Epimer T M Brown University, brown.edulResearch/TB-HIV_Lablepimabix/epimatrix.html and BIMAS, bimas.dcrtnih.gov/.).

00 0 Motif bearing subsequences of all 193P1 E1 B variant proteins are set forth and identified in Tables VllI-XXI and

XXII-XLIX.

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

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

Polypeptides comprising one or more of the 193P1E1B motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 193P1E B motifs discussed above are associated with growth dysregulation and because 193P1 E1B is overexpressed in certain cancers (See, Table I).

SCasein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be c associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest, 78(2): 165-174 (1998);

O

0 Gaiddon ef al, Endocrinology 136(10): 4331-4338 (1995); Hall et Nucleic Acids Research 24(6): 1119-1126 (1996); 0 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)). Amidalion is another protein modification also associated with cancer and cancer progression (see: e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (.13):.169-175 (1992)).

S 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 193P1E1B protein that are capable of optimally binding to specified HLA alleles Table IV: Epimatrix T and Epimer T M Brown University, URL brown.edu/Research/TB- HIV_Lab/epimalrix/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 epilope that bears a CTL or HTL motif (see, the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epilope 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 et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et at., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.

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

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

00 SRelated embodiments of the invention include polypeptides comprising combinations of the different motifs set forth CN 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 Spredicted HTL epitopes of Tables XLVI-XLIX, andlor, one or more of the T cell binding motifs known in the art. Preferred Sembodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of Sthe polypeptides. In addition, embodiments which include a number of either N-terminal andlor C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on Seither side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

0 193P1E1B-related proteins are embodied in many forms, preferably in isolated form. A purified 193P1E1B protein molecule will be substantially free of other proteins or molecules that impair the binding of 193P1E1B to antibody, T cell or ri other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 00 193P1E1B-related proteins include purified 193P1E1B-related proteins and functional, soluble 193P1E1B-related proteins.

O In one embodiment, a functional, soluble 193P1E1B protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

SThe invention also provides 193P1E1B proteins comprising biologically active fragments of a 193P1E1B amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 193P1E1B protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 193P1E1B 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.

193P1 E1B-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-193P1E1B antibodies orT cells or in identifying cellular factors that bind to 193P1E1B. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, 1981; Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles.

can be generated, and immunogenic peptide fragments.identified, using the method of Kyte, J. and Dooliltle, 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. PepL Protein Res. 32242-255. Beta-turn profiles can be generated, andimmunogenic 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 193P1E1B 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 and Epimer

TM

Brown University, URL (brown.edu/ResearchfTB- HIV_Lab/epimatrixlepimatrix.html); and BIMAS, URL bimas.dcrtnih.gov/). Illustrating this, peptide epitopes from 193P1E1B that are presented in the context of human MHC Class I molecules, HLA-A1, A2, A3, All, A24, B7.and 835 were predicted (see, Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 193P1E1B protein and relevant portions of other variants, for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeilhi.bmiheidelberg.com/.

00 The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide CN sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et J. Immunol. 149:3580-7 (1992); Parker et J. Immunol. 152:163-75 S(1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8- 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine or methionine at position 2 and a valine or leucine at the C-terminus (see, Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 193P1E18 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and SXXII-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, C mers,.for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an S* 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 ofpeptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigenprocessing defective cell line T2 (see, Xue et at, Prostate 30:73-8 (1997) and Peshwa et 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, Epimer

T

M and Epimatrix

T

M sites, or specified bythe HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be 'applied" to a 193P1E1B protein in accordance with the invention. As used in this context "applied" means that a 193P1E1B protein is evaluated, eg., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art.

Every subsequence of a 193P1E1B protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention; Ill.B.) Expression of 193P1EiB-related Proteins In an embodimentdescribed in the examples that follow, 193P1E1B 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 193P1E1B 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 tofacilitate the production of a secreted 193P1E1B protein in transfected cells. The secreted HIS-tagged 193P1E1B in the culture media can be purified, using a nickel column:using standard techniques.

III.C.) 'Modifications of 193P1E18-related Proteins Modifications of 193P1E1B-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 193P1E1B polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 193P1E1B protein. Another type of covalent modification of a 193P1E1B polypeptide included within the scope of this invention comprises altering the native glycosylation pattem of a protein of the invention. Another type of covalent modification of 193P1 E1B comprises linking a 193P1E1B polypeptide to one of a variety of nonproteinaceous polymers, e.g., 00 polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; C 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

SThe 193P1E1B-related proteins of the present invention can also be modified to form a chimeric molecule Scomprising 193P1E1B fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinanlly. A chimeric molecule can have a protein of the invention fused to another tumorassociated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 193P1E1 B sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can 0comprise multiples of the same subsequence of 193P1 E1B. A chimeric molecule can comprise a fusion of a 193P1E1Brelated protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or-with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a 193P1E1B protein. In an alternative embodiment, the chimeric molecule can comprise a fusion ofa 193P1E1 B-related protein with an Simmunoglobulin ora 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 193P1E1B 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 immunoglobutin fusions see, U.S. Patent No. 5,428,130 issued June 27, 1995.

III.D.) Uses of.193P1E1B-related Proteins The proteins of the invention have a number of different specific uses. As 193P1E1B is highly expressed in prostate and other cancers, 193P1E1 B-related proteins are used in methods that assess the status of 193P1E1B gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeplides from specific regions.of a 193P1E1B 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 193P1E1B-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 193P1 El 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, 193P1E1B-relaled proteins that contain the amino acid residues of one or more of the biological motifs in a 193P1 E1B protein are used to screen for factors that interact with that region of 193P1E1B.

193P1E1B protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies antibodies recognizing an extracellular or intracellular epitope of a 193P1E1B protein), for identifying agents or cellular factors that bind to 193P1E1B 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 193P-1E1 B genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 193P1E1B gene product Antibodies raised against a 193P1E1B protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 193P1E1B protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 193P1E1B-related nucleic acids or proteins are also used in generating HTL or CTL responses.

00 Various immunological assays useful for the detection of 193P1E1B proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays S(ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents Scapable of detecting 193P1E1B-expressing cells in radioscintigraphic imaging methods). 193P1E1B proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 193PiE1B Antibodies S. Another aspect of the invention provides antibodies thatbind to 193P1E1B-related proteins. Preferred antibodies O specifically bind.to a 193P1E1B-related protein and do not bind (or bind weakly) to peptides or proteins that are not 193P1E1Brelated proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate CNl buffered saline; 2) Tris-buffered-saline containing 25mM Tris and 150 mM NaCI; or saline NaCI); 4) animal serum such as 00 0 human serum; or 5) a combination of any of 1) through these reactions preferably taking place at pH 7.5, altematively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4 C to 37'C. For.example, antibodies that bind 193P1E1B can bind 193P1E1B-related proteins such as the homologs or analogs thereof.

193PIE1B antibodies of the invention are particularly useful in cancer (see, Table I)diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodiiare useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 193P1 E1B 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 193P1E1B is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detectionand quantification of 193P1E1B and mutant 193P1E18-related proteins. Such assays can comprise one or more 193P1E1B antibodies capable of recognizing and binding a 193P1E1B-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), 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 193P1E1B are also provided.by the invention, including but not limited to radioscintigraphic imaging methods using labeled 193P1E1B antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 193P1E1B expressing cancers such as prostate cancer.

193P1E1B antibodies are also used in methods for purifying a 193P1E1B-related protein and for isolating 193P1E1B homologues and related molecules. For example, a method of purifying a 193P1E1 B-related protein comprises incubating a 193P1E1B antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 193P1E1B-related protein under conditions that permit the 193P1E1B antibody to bind to the 193P1E1B-related protein; washing the solid marix to eliminate impurities; and eluting the 193P1E1B-related protein from the coupled antibody. Other uses of 193P1E1B antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 193P1E1B protein.

Various methods for the preparation of antibodies are.well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 193P1E1B-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY(1989)). In addition, fusion proteins of 193P1E1B can also be used, such as a 193P1E18 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino add sequence of Figure 2 or Figure r 00 S 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 193P1E1B-related Sprotein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 193P1E1 B-related S protein or 193P1E1B expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et at., 1997, Ann. Rev. Immunol. 15: 617-648).

CKl The amino acid sequence of a 193P1E1B protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regiocs of the 193P1E1B protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 0 193P1E1B amino acid sequence are used to identify hydrophilic regions in the 193P1E1B structure. Regions of a 193P1E1B 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-Schultzor Jameson-Wolf C analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, 1981, Proc. Natl. Acad.

00 Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, 1982, J.

O 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 P.K., 1988, Int. J. Pept Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, Roux B., -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 193P1E1B 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 caierprotein. 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 193P1E1B 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, liters of antibodies can be taken to determine adequacy of antibody formation.

193P1E18 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 193P1E1B-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 193P1E1B protein can also be produced in the context of chimeric or complementaritydetermining region (CDR) grafted antibodies of multiple species origin. Humanized or human 193P1E1 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 for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et 1993, Proc. NaU. Acad. Sci. USA 89:4285 and Sims et aL, 1993, J. Immunol. 151: 2296.

Methods for producing fully human monodonal antibodies include phage display and transgenic methods (for-review, see Vaughan et 1998, Nature Biotechnology 16: 535-539). Fully human 193P1E1B monoconal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. Nottingham Academic, pp 45-64 (1993); Burton and Barbas.

Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 193P1E1B monoclonal antibodies can also be 00 O produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application SW098/24893, Kuchedapali and Jakobovits et al, published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.

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

C1 Reactivity of 193P1E1B antibodies with a 193P1E1B-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA. and FACS analyses using, as appropriate, 193P1E.1B-related proteins, 193P1E1 B-expressing cells or extracts thereof. A 193P1E1B 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 Sradioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more 193P1E1B epitopes are generated using methods generally 00 known in the art Homodimeric antibodies can also be generated by cross-linking techniques known in the art Wolff et 0al., Cancer Res. 53: 2560-2565).

193P1E1B Cellular Immune Responses The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions:of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-.

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

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Biuis, S. et a/,Cell47:1071, 1986; Babbitt, B. P. et 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, e.g; Southwood, et J. Immunol. 160:3363, 1998; Rammensee, et Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (1 34 2 9 6.221/scripts.hlaserver.dllhome.htm); Sette, A.

and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. Curr. Opin. Immunot. 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 J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et 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 bome 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 al, Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stem et al., Structure 2:245, 1994; Jones, E.Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C.

et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, .1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, 0. H. et al., Science 257:919, 1992; Saper, M. Bjorkman, P. J. and Wiley, D. J. Mol. Biol. 219:277,1991.) Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of 00 Sassociation of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.

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

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 S(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 S51Cr-release assay involving peptide sensitized target cells.

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

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

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

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et at., Immunity 7:97, 1997; Bertoni, R. et al, J. Clin. Invest. 100:503,1997; Threlkeld, S. C. et al., 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 piesenting 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 51 Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 193PIE1B Transgenic Animals Nucleic acids that encode a 193P1E1 B-related protein can also be used to generate either Iransgenic 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 193P1El E1B can be used to clone genomic DNA that encodes 193P1E1B. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 193P1E 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 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 193P1E1B transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 193P1E1B can be used to examine the effect of increased expression of DNA that encodes 193P1E1B. Such animals can be used as tester animals for reagents.thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance.with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 193P1E1B can be used to construct a 193P1E1B "knock out" animal that has a defective or altered gene encoding 193P1E1B as a result of homologous recombination between the endogenous 00 S gene encoding 193P1E1B and altered genomic DNA encoding 193P1E1B introduced into an embryonic cell of the animal.

0 For example, cDNA that encodes 193P1E1B can be used to clone genomic DNA encoding 193P1E18 in accordance with established techniques. A portion of the genomic DNA encoding 193P1E1B can be deleted or replaced with another gene, S such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, Thomas and Capecchi, Cell, 51:503 C (1987) for a descriplion 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 at., 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 00 harboring the homologously recombined ONA 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 193P1E1B polypeptide.

VII.) Methods for the Detection of 193P1E1B Another aspect of the present invention relates to methods for detecting 193P.1E1B polynuceotides and 193P1E1Brelated proteins, as well as methods for identifying a cell that expresses 193P1E1B. The expression profile of 193P1E1B makes it a diagnostic marker for metastasized disease. Accordingly, the status of 193P1E1B 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 193P1E1B 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 193P1E1B polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 193P1E1B polynucleotides include, for example, a 193P1E1B gene or fragment thereof, 193PiE1B mRNA, alternative splice variant 193P1E1B mRNAs, and recombinant DNA or RNA molecules that contain a 193P1E1B polynucleotide. A number of methods for amplifying and/or detecting the presence of 193P1E1B 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 193P1E1B 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 193P1E1B polynucleotides as sense and antisense primers to amplify 193P1E1B cDNAs therein; and detecting the presence of the amplified 193P1E1B cDNA. Optionally, the sequence of the amplified 193P1E1B cDNA can be determined.

In another embodiment, a method of detecting a 193P1 E1B gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 193P1E1B polynudeotides as sense and antisense primers; and detecting the presence of the amplified 193P1E1B gene. Any number of appropriate sense and antisense probe combinations can be designed from a 193P1E1B nucleotide sequence (see, Figure 2) and used for this purpose.

The invention also provides assays for detecting the presence of a 193P1E1B protein in a tissue or other biological sample such as seum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 193P1E1B-related 00 O protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Westem blot analysis, C molecular binding assays, ELISA, ELIFA and the like. For example, a melhod of detecting the presence of a 193P1E1 B-related .0 protein in a biological sample comprises first contacting the sample with a 193P1E1B antibody, a 193P1E1B-reactive CT fragment thereof, or a recombinant protein containing an antigen-binding region of a 193P1E1B antibody; and then detecting the binding of 193P1E1B-related protein in the sample.

Methods for identifying a cell that expresses 193P1E1B are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 193P1E18 gene comprises detecting the presence of 193P1E1B mRNA in the cell.

SMethods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using s complementary DNA probes (such as in situ hybridization using labeled 193PIE1B riboprobes, Northem blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 193P1E1B, C and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an 00 assay for identifying a cell that expresses a 193P1E1 B gene comprises detecting the presence of 193P1E1B-related protein in the cell or secreted by the cell. Various methods for the detection of proteinsare well known in the art and are employed for the detection of 193P?1E1 B-related proteins and cells:that express 193P1E1B-related proteins.

193P1E1B expression analysis is also useful as a tool for identifying and evaluating agents that modulate 193P1E1B gene expression. For example, 193P1E1 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 193P1E1 B expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identifiedby using a screen that quantifies 193P1E1B expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.). Methods for Monitoring the Status of 193P1E1B-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 e al., Cancer Surv. 23:19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 193P1E1B 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 193P1E1B in a biological sample of interest can be compared, for example, to the status of 193P1E1B 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 193P1E1B in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, Grever et al., J. Comp. Neurol. 1996 Dec9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 193P.1 E1B status in a sample.

The term"status" in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skiled 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 193P1E1B expressing cells) as well as the level, and biological activity of expressed gene products (such as 193P1E1B mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 193P1EiB comprises a change in the location of 193P1E1B and/or 193P1E1B expressing cells and/or an increase in 193P1E1B mRNA and/or protein expression.

193P1E1B status in a sample can be analyzed by a number of means well known in the art, including without limitation, Immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 193P1 E1 B gene and gene products are found, 00 O for example in Ausubel et a. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern C\ Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 193P1E1B 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 f perturbations in a 193P1E1B gene), Northern analysis and/or PCR analysis of 193P1E1B mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 193P1E18 mRNAs), and, Western and/or r immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localizalion within a sample, alterations in expression levels of 193P1 E1B proteins and/or associations of 193P1E1 B proteins with polypeptide binding partners). Detectable 193P1E1B polynudeotides include, for example, a 193P1E1B gene or fragment thereof, 193P1E1B mRNA, alternative splice variants, 193P1E1B mRNAs, and recombinant DNA or RNA molecules containing a 193P1E1B polynucleotide.

C The expression profile of 193P1E1B makes it a diagnostic marker for local and/or metastasized disease, and 00 provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 193P1E1B provides 0information useful for predicting susceptiblity to particular disease stages, progression, andfor tumor aggressiveness. The invention provides methods and assays for determining 193P1E1B status and diagnosing cancers that express 193P1 E B, such.

as cancers of the tissues listed in Table I. For example, because 193P1E1B mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 193P1E1B mRNA transcripts or proteins in a ,biological sample can be used to diagnose a disease associated with 193P1E1B dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 193P1E1B 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 193P1E1B: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 193P1E1B in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 193P1E1B 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 193P1E1B expressing cells those that express 193P1E1B.mRNAs or proteins). This examination can provide evidence of dysregulated cellular growlh;.for example, when.193P1E1B-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 193P1E1B in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells .from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, Murphy et al., Prostate 42(4): 315-317 (2000);Su et Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman etal., J Urol 1995 Aug 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 193P1E1B gene products by determining the status of 193P1E1B 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 193P1E1B gene products in a corresponding normal sample. The presence of aberrant 193P1E1B gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

00 O In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 193P1E1B mRNA or protein expression in a test cell or tissue sample relative 3) to expression levels in the corresponding normal cell or tissue. The presence of 193P1E1B mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 193P1E1B expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding CK normal tissues do not express 193P1E B mRNA or express it at lower levels.

In a related embodiment, 193P1E1B status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 193P1E1B protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 193P1E1B expressed in a corresponding normal sample. In one embodiment, the presence of 193P1E1B protein is evaluated, for example, using immunohistochemical methods. 193P1E1B antibodies or N binding partners capable of detecting 193P1E1B protein expression are used in a variety of assay formats well known in the art for 00 this purpose.

O In a further embodiment, one can evaluate the status of 193P1E1B nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions andthe like. Such evaluations are useful because perturbations in the nucleotide and amino add sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, Marrogi et al., 1999, J.

Cutan. Pathol. 26(8):369-378). For example, a inutation in the sequence of 193P1E1B may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 193P1E1B indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino add sequences are well known in the art.

For example, the size and structure of nucleic acid or amino acid sequences of 193P1E1B gene products are observed by the Northern, Southern;Westem, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino add 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 193P1E1B gene in a biological sample. Aberrant demethytation 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 at., 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 spedfic gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylaled DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et a. eds., 1995.

00 SGene amplification is an additional method for assessing the status of 193P1E1B. Gene amplification is measured in a sample directly, for example, by conventional Southem blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein C- duplexes. The antibodies in turn are labeled and the assay carded 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, l Northern, dot blot orRT-PCR analysis to detect 193P1E1B expression. The presence of RT-PCR amplifiable 193P1E1B mRNA 0provides 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 00 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 1995, J. Clin. Oncol. 13:1195-2000; Heston etal., 1995, Clin. Chem. 41:1687c-l 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 193P1E1B mRNA or 193P1E1B.protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 193P1E18 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 193P1E18 in prostate or other tissue is examined, with the: presence of 193P1E1B 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 193P1E1B nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 193P1E1B 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 193P1E1B mRNA or 193P1E1B protein expressed by.tumor cells, comparing the level so determined to the levelof 193P1E1B mRNA or 193P1E1B protein expressed in a corresponding normal tissue.taken from the same individual or a normal tissue reference sample, wherein the degree of 193P1E1B mRNA or 193P1E1B 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 193P1E1B is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrily of 193P1E1B nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

S Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over.time comprise determining the level of 193P1E1B mRNA or 193P1E1B protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 193P1E1B mRNA or 193P1E1B protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 193P1E1B mRNA or 193P1E1B protein expression.in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 193P1E1B expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 193P1E1B nucleotide and amino acid sequences in a biological 00 O 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.

SThe above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic Sprotocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence S between the expression of 193P1E1B gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B C 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 0with malignancy PSA. PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, Bocking etal.,.1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995; Hum. Palhol. 26(2):223-9; Thorson et al., 1998, Mod.

O Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between Sthe expression of 193P1E1B8 gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B gene 00 products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific Sfactors 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 193P1E1B gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B gene products) and another factor associated with.malignancy •ehtals detecting the overexpression of 193PIE1B mRNA or protein in a tissue sample, detecting the overexpression of PSA -mRNA or proteinin a tissue sample (or PSCA or PSM expression), and observing a coincidence of 193P1E1B mRNA or protein and PSA mRNA-or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 193P1E1 B and PSA mRNA in prostate tissue is examined, where the coincidence of 193P1E1B 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 193P1E1B mRNA or protein are described herein, and standard nudeicacid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 193P1 El B mRNA include in situ hybridization using labeled 193P1 E1B riboprobes, Northern blot :;and related techniques using 193P1E1B polynucleotide probes, RT-PCR analysis using primers specific for 193P1E1B, 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 193P1E1B mRNA expression. Any number of primers capable of amplifying 193P1E1B 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 193P1E1B protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 193P1E1B The 193PIE1B protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 193P1E1B, as well as pathways activated by 193P1E1B 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 proteinprotein 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).

Alternatively one can screen peptide libraries to identify molecules that interact with 193P1E1B protein sequences.

In such methods, peptides that bind to 193P1E1B are identified by screening libraries that encode a random or controlled 00 GO collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, Sthe bacteriophage particles are then screened against the 193P1E1B protein(s).

0 Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are S 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 193P1E1 B protein sequences are Cdisclosed 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 193P1E1B are used to identify protein-protein interactions mediated by 193P1E1B. Such interactions can be examined using immunoprecipitation techniques (see, Hamilton etal.

r Biochem. Biophys. Res. Commun. 1999, 261:646-51). 193P1E1B protein can be immunoprecipitated from 193P1E1Bexpressing cell lines using anti-193P1E1B antibodies. Alternatively, antibodies against His-tag can be used in a cell line Sengineered to:express fusions of 193P1E1B and a His-tag (vectors mentioned above). The immunoprecipitated complex can 00 be examined for protein association by procedures such as-Western blotting, 3 5 S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

ri Small molecules and ligands that interact with 193P1E1B can be identified through related embodiments of such screening assays. For example, small molecules can be identified (hat interfere with protein function, including molecules that interfere with 193P1E1B's ability to mediate phosphorylation and de-phosphorylation, interaction with DNAor RNA molecules as'an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 193P1 E1 B-related ion channel, protein pump, or cell communication functions are identified and used.to treat patients that have a cancer that expresses 193PiE1B (see, Hille, Ionic Channels of Excitable Membranes 2n.Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate 193P1E1B function can be identified based on their ability to bind 193P1E1B 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 193P1E1B and a DNA-binding:protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriplional ectivator 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 193P1E1B.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a 193P1E1B amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with'a 193P1E1B amino acid sequence, allowing the population of molecules and the 193P1E1B amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 193P1E1B amino acid sequence, and then separating molecules that do not interact with the 193P1E1B 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 193P1E1B amino acid sequence. The identified moleculecan be used to modulate a function performed by 193P1E1B. In a preferred embodiment, the 193P1E1B amino acid sequence is contacted with a library of peptides: Therapeutic Methods and Compositions The identification of 193P1E1B as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers.

00 O As contemplated herein. 193P1E1B functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

SAccordingly, therapeutic approaches that inhibit the activity of a 193P1E1B protein are useful for patients suffering Sfrom a cancer that expresses 193P1E1B. These therapeutic approaches generally fall into two classes. One class I' comprises various methods for inhibiting the binding or association of a 193P1 E1B protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 193P1E1B gene or translation of 193P1E1B mRNA.

Anti-Cancer Vaccines The invention provides cancer vaccines comprising a 193P1E1B-related protein or 193P1E1B-related nucleic acid. In C view of the expression of 193P1E1B, cancer vaccines prevent and/or treat 193P1E1B-expressing cancers with minimal or no 0 effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral andlor cell-mediated imrmune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int J. Cancer 63:231-237; Fong et 1997; J. Immunol. 159:3113-31,17):.

Such.methods can be readily practiced by employing a 193P1E1B-related protein, or a 193P1E1B-encoding nucleic add molecule and recombinant vectors capable of expressing and presenting the 193P1E1B immunogen(which typically comprises.a number of antibody or T cell epitopes). 'Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, Heryln et Ann Med 1999 Feb 31(1):66- 78; Maruyama et Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response humoral andlor cell-mediated) in a mammal, comprise the steps of: exposing the mammals immune system to an immunoreactive epitope an epitope present in a 193P1E1B protein shown in Figure 3 or-analog or homolog thereof) so that the mammal generates an immune response that is specific for that epilope generates antibodies that specifically recognize that epitope). In a preferred method, a 193P1E1B immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 193P1E1 B indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.

The entirel193P1E18 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeplides (e.g.,Vitiello, A. et J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) microspheres (see, Eldridge, ef 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 e.g.c Tam, J. P., Proc. Nail. 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 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. et al., 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. H. et al:, Sem. Hematol. 30:16, 1993; Falo, L. Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. Vogel, F. and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. eta., 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., 00 0 Sam. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as S Ithose of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

In patients with 193P1E1B-associated cancer, the vaccine compositions of the invention can also be used in S conjunction with other treatments used for cancer, surgery, chemotherapy, drug therapies, radiation therapies, etc.

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

Ci Cellular Vaccines: CTL epitopes can be determined using specific algorithms to identify peptides within 193P1E1B protein that bind corresponding HLA alleles (see Table IV; Epimer T M and Epimatrix

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Brown University (URL brown.edu/Research/TB- HIV_Lab/epimatrixtepimatrix.htmi); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/).

SIn a preferred embodiment, a 193P1E1B 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 00 acids specified by an HLA Class I motif/supermotif Table IV Table IV or Table IV(E)) and/or a peptide of at 0 least 9 amino acids that comprises an HLA Class II motif/supermotif Table IV or Table IV As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10,-or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class IImolecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino add 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 IIepitopes 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 adds.

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 tie mammal's immune system to an immunogenic epitope on a protein a 193P1E1B protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 193P1E18 in a host, by contacting the host with a sufficient amount of at least one 193P1E1B B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 193P1E1 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 193P1E1B-related protein or a man-made multiepitopic peptide comprising: administering 193P1E1B immunogen a 193P1E1B protein or a peptide fragment thereof, a 193P1E1B fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, sudi 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. mmunol. 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 altemative method comprises generating an immune response in an individual against a 193P1E1B immunogen by: administering in vivo to muscle or skin of the individuals body a DNA molecule that comprises a DNA sequence that encodes a 193P1E1B 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 sultoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 193P1E1B, in order to generate a response to the target antigen.

00 SNucleic Acid Vaccines: Cl Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode Sprotein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 193P1E1B.

Constructs comprising DNA encoding a 193P1E1B-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 193P1E1B proteinlimmunogen. Alternatively, a vaccine comprises a 193P1E1B-related protein.

SExpression of the 193P1E1B-related protein immunogen results in the generation of prophylactic or therapeutic humoral and 0\ cellular immunity against cells that bear a 193P1E1B protein. Various prophylactic and therapeutic genetic immunization Stechniques known in the art.can be used (for review, see information and references published at Internet address C genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as 00 :0U.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 DNA- S based delivery technologies include 'naked DNA', facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and.partide-mediated ('gene gun") or pressure-mediated delivery (see, U.S. Patent No. 5,922,687). n For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to,:vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Nail. Cancer Inst. 87:982-990 (1995)). Non-viral delivery.systems can also be employed by introducing naked DNA encoding a 193P1E1 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, 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 1herapeutic 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 193P1E1B-related nucleic acid molecule. In one embodiment, the full-length human 193P1E1B cDNA is employed. In another embodiment, 193P1E1B nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

.Ex Vivo Vaccines Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 193P1E1B 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 a., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 193P1E1B peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 193P1E1B peptides capable of binding to MHC class I andlor class II molecules. In another embodiment dendritic cells are pulsed with the complete 193P1E1B protein. Yet another embodiment involves engineering the overexpression of a 193P1E1B 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), lentivirus, adeno- 00 0 associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection

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N (Ashley et 1997, J. Exp. Med. 186:1177-1182). Cells that express 193P1E1B can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

SX.B.) 193P1E1B as a Target for Antibody-based Therapy 193P1E1B 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 193P1E1B is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 193P1E1 -munoreactive compositions are prepared that exhibit O excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive CN composition to non-target organs and tissues. Antibodies specifically reactive with domains of 193P1E1B are useful to treat 00 193P1E1B-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

S193P1E1B antibodies can be introduced into a patient such that the antibody binds to 193P1E1B and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 193P1E1B, 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 193P1E1B 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 Sto antibodies specific for a molecule expressed by that cell 193P1E1B), 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- 193P1E1B antibody) that binds to a marker 193P1E1B) 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 193P1E1B, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 193P1E1B 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-193P1E1B 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 a., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (7hong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of 00 naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or 1131 to anti-CD20 antibodies Zevalin T m

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r Pharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceplin T M (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 193P1E1B antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin 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, platform, ImmunoGen, Cambridge, MA, also see US Patent 5,416,054).

SAlthough 193P1 E1B antibody therapyis useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or melastalic 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 00 chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, Santibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan etal. (Cancer Res. 53:4637-4642,1993), Prewett et al.

(InternationalJ. ofOnco. 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 193P1EIB 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 lte 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 193P1 E1B expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 193P1 E1 B imaging, or other techniques that reliably indicate the presence and degree of 193P1E1B 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-193P.1 E1B 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-193P1E1B 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 interactior with effector cell Fc receptor sites on complement proteins. In addition, anti-193P1E1B mAbs that exert a direct biological effect on tumor growth are useful to treatcancers thatexpress 193P1E1B. 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-193P1E1B 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 (hat bind specifically to the target 193P1E1B 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-193P1E1B mAbs as well as S combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that Srely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti- 193P1E1B mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators IL-2, GM-CSF), surgery or radiation. The anti- 193P1E1B mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

SAnti-193P1E1B 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, S. intradermal, and:lhe like. Treatment generally involves repeated administration of the anti-193P1E1B antibody preparation, 00 0 via an acceptable route of administration such as intravenous injection typically at a dose in the range of about 0.1, .2, 2, 3, 4, 5, 6,7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

SBased on clinical experience with the Herceptin T M mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 193P1E1B mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as.

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 193P1E1B expression in the patient, the extent of circulating shed 193P1E1B antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used.in combination with the treatment method of the invention, as well as the health status of a particular patient.

SOptionally, patients should be evaluated for the levels of 193P1E1B in a given sample the levels of circulating 193P1E1B antigen and/or 193P1E1B 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' mmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti193P1E1 B antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 193P1E1B-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-193P1E1B antibodies that mimic an epitope on a 193P1E1B-related protein (see, for example, Wagner et 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.

193P1E1B 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. Alteratively, 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 00

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

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

Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS). Moreover, an adjuvant such-as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10-to.100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000)) Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host.becomes atleast partially immune to later development of cells that express or overexpress 193P1 E1B 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 ofsuch a composition comprises class I and class II epitopes in accordance with the invention. An:allernative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a, cross reactive H-TL epitope such as PADRE T M (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 peplides 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.

S 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.

59 00 O Sufficient supermotif bearing-peptides, or a sufficient array of allele-spedfic motif-bearing peplides, are N selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, Fpopulation coverage.

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

Of particular relevance are epitopes referred to as "nested epitopes.' Nested epitopes occur where at 0 least two epitopes overlap in a given peplide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective Is to provide the greatest number of epitopes per Ssequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal c epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, 00 Ssuch as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it Sdoes not have pathological or other deleterious biological properties.

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

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

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 J. Immunol. 157:822 1996; Whitton, J. L. et at., 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 193P1E1B, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 193P1E1B (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 CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells Iransfecled with the DNA plasmid. Thus, these 00 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, Sthe amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated Sand included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes,-a 0\ ubiquitination signal sequence, andlor an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and l HTL epitopes maybe improved by including synthetic poly-alanlne) or naturally-occurring flanking sequences adjacent C to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

00 0 The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus.

0 strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified Sand 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. coliorigin of replication; and an E.

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

T

M, Epimmune, San Diego, CA), Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II 00 0 pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune 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 Spurification. 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 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.

SPurified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is O reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA," is Scurrently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of CN 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 also be used in the formulation (see, as described by WO 93124640; Mannino Gould-Fogerite, BioTechniques 6(7): S682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, etal., Proc. Nat'lAcad. Sci. USA 84:7413 (1987). In Saddition, 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 epilopes. 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 melhod used will be dependent on the final 'formulation. Electroporation can be used for naked" DNA, whereas cationic lipidsallow 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 (s5Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by s 5 Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epilopes. 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 Simmunization, 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 cylolysis of peptide-loaded, s 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 epilopes, 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 altemative 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 epilopes of the invention can be incorporated into a viral vector such as vaccinia.

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

00 0 For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence N which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, Ala, Gly, or other neutral spacers of nonpolar amino adds or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or.carboxy S.terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be C. acylated.

00 SIn certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a S. genetically diverse population. This can be accomplished by selecting peplides 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: 48), Plasmodium falciparum drcumsporozoite (CS) protein at positons 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 49), and Streptococcus 18kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 50); 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 95107707). 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: AKXVAAWTLKAAA (SEQ ID NO: 51), where is either cyclohexylalanine, phenylatanine, or tyrosine, and a is.either D-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 alternative of a pan-DR binding epitope comprises all natural amino adds and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance toproteases and thus extend their serum half life, or they can be Sconjugated: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 S 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 peptide (see, Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, 00 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.

SX.C.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor HTL Peptides .An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a i 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 Progenipoietin T M (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4.

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

Cr The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 193P1E1B.

00 Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be Sincluded to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 193P1E1B.

X.D: Adoptive Immunotherapy Antigenic 193P1E1B-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 S'not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the 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 dendrilic cells may also be used as antigen presenting cells.

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 193P1E1 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 ihe 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 193P1E1 B. The peptides or DNAencoding.

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 193P1E1B-associated cancer.

This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary 00 according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses Cr 193P1E1B, a vaccine comprising 193P1E1B-spedfic CTL may be more efficacious in killing tumor cells in patient with Sadvanced disease than alternative embodiments.

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

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is 0 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 0\ human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient Boosting dosages of between about 0 pg to about 50,000 pg of peptide pursuant to a boosting regimen over weeks to months may be administered depending 0 upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from 00 0 the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the C-i 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 ofextraneous 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 pg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, intravenously, subcutaneously, intradermally, or intramuscularly: Thus, the invention provides.

compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended ini an acceptable carrier, preferably an aqueous carrier.

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

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.

00 O A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a K human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, Remington's SPharmaceutical Sciences, 17b Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 jig, generally 100-5,000 pg, for a 70 kg patient.

For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 pg) Scan also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then 0 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-193P1E1 B antibody preparation, S via an acceptable route of administration such as intravenous injection typically at a dose in the range of about 0.1 to 00 Sabout 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- 193P1E1B 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 Scomposition; the binding affinity of an Ab, the immunogenicity of a substance, the degree of 193P1E1B expression in the patient, the extent of circulating shed 193P1E1B antigen, the desired steady-state concentration level, frequency of Streatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, *500pg 1mg, Img 50mg; 50mg 100mg, 100mg 200mg, 200mg 300mg, 400mg 500mg, 500mg 600mg,-600mg 700mg, 700mg 800mg, 800mg 900mg, 900mg 1g, or 1mg 700mg. In certain embodiments, the dose is in a range of 2mg/kg body weight, wilh follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/kg body weight followed, in two, three or four weeks by weekly doses; 0.5 10mg/kg body weight, followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375,400mg m 2 of body area weekly; 1-600mg m 2 of body.area weekly; 225-400mg m 2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

i In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors,-including the sequence of the polynucleolide, molecular weight of the polynudeoide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynudeotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5,1, 2, 5,10, 20, 30, 40, 50, 60, 70, 80,90,100, 200, 300, 400 or 500 mg/kg up toan 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 mglkg. 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 Sto 100 mg/kg, 100 to 200 mglkg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mgfkg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of 00 parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 S cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5 x 1010 cells.

A dose may also about 106 cells/m 2 to about 10 10 cells/m 2 or about 106 cellslm 2 to about 108 cells/m 2 Proteins(s) of the invention, andlor nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the Speptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or.immunogenic compositions. Thus, liposomes either filed or decorated with a desired peptide of the invention.

S can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use 00 0 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, el Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S.

SPatent 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., Santibodies 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 Sesters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, Jauric, palmitic, stearic, lino!eic, 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 193P1E1B.

As disclosed herein, 193P1E1B polynudeotides, polypeptides, reactive cytotoxicT 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 the Example entitled "Expression analysis of 193P1E1B in normal tissues, and patient specimens").

193P1E1B 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. 163(2): 00 503-5120 (2000); Polascik et J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635- S1640(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 Prey 2000;24(1):1-12). Therefore, C' this disclosure of 193P1 E1B polynucleotides and polypeplides (as well as 193P1E1B polynucleotide probes and anti- 193P1E1B 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 193P1E1B polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, PSA 0 polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes 0 (for example-in.Northern analysis, see, Sharief et 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 193P1 E1 B polynucleotides described herein can be utilized in the same way to detect 193P1E1B overexpression or the metastasis of Sprostate and other cancers expressing this gene. Altematively, 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 al., Pathol. Res. PracL 192(3):233-7 (1996)), the 193P1E1B polypeptides described herein can be utilized to generate antibodies for use in detecting 193P1E1B 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 193P1E1B polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example,.when a biological sample from lissue that does not normally contain 193P1E1B-expressing cells (lymph node) is found to contain 193P1E1B-expressing cells such as the 193P1E1B expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Allermaively 193P1E1B polynucleotides and/or polypeptides can be used to provide evidence of cancer, for S.example, when cells in a biological sample that do not normally express 193P1E1B or express 193P1E1B at a different level are found to express 193P1E1B or have an increased expression of 193P1E1B (see, the 193P1E1B 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 193P1E1B) such as PSA, PSCA etc. (see, Alanen et Pathol. Res. Pract.

192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 193P1E1B 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 polynucleolide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, Caetano-Anolles, G.

Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled "Expression analysis of 193P1E1B in normal 00 tissues, and patient specimens," where a 193P1E1B polynudeotide fragment is used as a probe to show the expression of C 193P1El 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 C 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).

Polynudeotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence a 193P1E1 B polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that C specifically binds to that epitope are used in methods of monitoring PSA. 193P1E1B polypeptide fragments and polypeptide Sanalogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide Svariants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems 00 such as fusion proteins being used by practitioners (see, Current Protocols In Molecular Biology, Volume 2, Unit 16, SFrederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an :antibody or T cell is reactive: Typically, skilled artisans create a variety of different polypeptide fragments that can be used in S-order to generate immune responses specific for different portions of a polypeptide of interest (see, U.S. Patent No.

5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the S193P1EiB biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one:of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence a 193P1E1B polypeptide shown in Figure 3).

As shown herein, the 193P1E1B polynucleotides and polypeptides (as well as the 193P1E1B polynucleotide probes and anti-193P1E1 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 1. Diagnostic assays that measure the presence of 193P1E1 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 Ssuccessfully 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, Smaterials such.as 193P1E1B polynucleotides and polypeptides (as well as the 193P1E1B polynucleotide probes and anti- 193P1E1B antibodies used to identify the presence of these molecules) need to be employed to confirm a melastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 193P1E1B 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 193P1E1B gene maps (see the Example entitled "Chromosomal Mapping of 193P1E1B" .below). Moreover, in addition to their use in diagnostic assays, the 193P1E1B-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci nt 1996 Jun 28;80(1-2): 63-9).

S Additionally, 193P1E1B-related proteins or polynucleotides of the invention can be used to treat a pathologic Scondition characterized by the over-expression of 193P1E1B. 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 193P1E1B antigen.

Antibodies or other molecules that react with 193P1E1B can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

00 0 XII.) Inhibition of 193P1E1B Protein Function The invention includes various methods and compositions for inhibiting the binding of 193P1E1B to its binding 3) partner or its association with other protein(s) as well as methods for inhibiting 193P1E1 B function.

XII.A.) Inhibition of 193P1E1B With Intracellular Antibodies C In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 193P1E1B are introduced into 193P1E1B expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti- 193P1E1B antibody is expressed intracellularly, bindsto 193P1E1B 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 inhibitory Sactivity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and 0 Marasco, 1995, TIBTECH vol. 13). Intrabodies havebeen shown to virtually eliminate the expression of otherwise abundant 0 cell surface receptors (see, Richardson et al., 1995, Proc. Nati. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J.

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

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

Intrabodies intended to exert activity in the nucleus are engineered to include a nudear 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 193P1E1B in the nucleus, thereby preventing its activity within the nucleus. :Nudear targeting signals are engineered into such 193P1E1B intrabodies in order to achieve the desired targeting. Such 193P1E1B intrabodies are designed to bindspecifically to a particular 193P1E1B domain. In another embodiment, cytosolic intrabodies that specifically bind to a 193P1E1 B protein are used to prevent 193P1E1B from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus preventing 193P1E1B from forming transcription complexes with other factors).

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

XII.B.) Inhibition of 193P1E1B with Recombinant Proteins In another approach, recombinant molecules bind to 193P1E1B and thereby inhibit 193P1E1B function. For example, these recombinant molecules prevent or inhibit 193P1 E1 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 193P1E1 8 specific antibody molecule. In a particular embodiment, the 193P1E18 binding domain of a 193P1E1B binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 193P1E1B 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 Gi3 domains and 00

O

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 193P1E1B, whereby the dimeric fusion protein specifically binds to 193P1E1B and blocks 193P1E1B interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins [i using known antibody linking technologies.

XII.C.) Inhibition of 193P1E1B Transcription or Translation The present invention also comprises various methods and compositions for inhibiting the transcription of the i 193P1E1B gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 193P1E1B.

mRNA into protein.

0In one approach, a method of inhibiting the transcription of the 193P1E1B gene comprises contacting the 0 193P1E1B gene with a 193P1 E1B antisense polynucleotide. In another approach, a method of inhibiting 193P1E1B mRNA 0 translation comprises contacting a 193P1E1B mRNA with an antisense polynucleotide. In another approach, a 193P1E1B c specific ribozyme is used to cleave a 193P1E1B message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory.regions of the 193P1E1B gene, such as 193P1E1B promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 193P1E1B gene transcription factor are used to inhibit 193P1E1BmRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of193P1E1B by interfering with 193P1E1B transcriptional activation are also useful to treat cancers expressing 193P1E1B. Similarly, factors that interfere with 193P1E1B processing are useful to treat cancers that express 193P1E1B. 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 193P1E1B antisense, ribozyme, polynucleotides encoding intrabodies and other 193P1E1 inhibitory molecules). A number of gene therapy approaches are known in theart. Recombinant vectors encoding 193P1 El B antisense polynudeotides, ribozymes, factors capable of interfering with 193P1E1B 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 193P1E1B to a binding partner, etc.

In vivo, the effect of a 193P1E1B 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 00 O osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

SIn vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in C 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 O 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 0 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,.

00 Remington's Pharmaceutical Sciences 16 th Edition, A. Osal., Ed., 1980).

O 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, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacterioslatic 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 193PIE1B 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 pattem, 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.

00 O Modulator-related Identification and Screening Assays: CKl Gene Expression-related Assays Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated CT 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 throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent 0 Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986- 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 00 compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is Sdone 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.

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

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

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

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

In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries' are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired 00 characteristic activity. The compounds thus identified can serve as conventional 'lead compounds," as compounds for r screening, or as therapeutics.

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

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

SIf required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the 00 cells, using known lysis buffers, electroporalion, etc., with purification and/or amplification such as PCR performed as Sappropriate. 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 signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be.an enzyme; such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a Sproduct that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as; an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, *thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.

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

A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency Sconditions 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 00 are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states,

C

r forming a gene expression profile.

SBiological Activily-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 cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention.

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

In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, 00 pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells cell-cell contacts), In 0 another example, the determinations are made at different stages of the cell cyde process. In this way, compounds that Smodulate 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.

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

In one embodiment, a method for modulating the status of a cell lhat 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 transduclion, 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 Agar Growth and Colony Formation to Identify and Characterize Modulators Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit 00 0 abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow C- 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 FAnimal 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 SNormal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, Iransformed 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, C similarly an MTT or Alamar.blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect 00 Ssame. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a Snormal phenotype and become contact inhibited and would grow to a lower density.

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

limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with 3 H)-thymidine is determined by.

incorporated cpm.

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

Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators Transformed cells have lower serum dependence than their normal counterparts (see, Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle 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) isreleased 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 (1.974); 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 Matriqel to Identify and Characterize Modulators The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between 00 malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic C cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used.

SBriefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, Freshney (1984), supra.

SEvaluation of Tumor Growth In Vivo to Identify and Characterize Modulators 0 Effects 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., r- mammals such.as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out 00 0 transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in Sthe 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 transgenic chimeric animals, mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is reimplanted into.a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of Which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan el 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).

Altematively, 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. trealed 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. Northem hybridization, RNAse protection, dot blotting, are preferred. The level of 00 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.

SAlternatively, 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 C 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).

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

C

particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.

In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the 00 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 used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.

Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments ulilize 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

TM

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.

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

0 Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays d 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 C1 the like.

A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound 0 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.

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

Alternatively, more than one.component is labeled with different labels, 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 "competitor." The competitor is a binding moiety that binds to the target molecule a cancer protein of the invention).

Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor.displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient .to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and Incubation periods are typically optimized, to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

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

In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicales 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 Ihe 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.

00 0 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 Sdrug 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.

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

SA variety of other reagents can be included in the screening assays. These include reagents like salts, neutral 00 proteins, e.g.albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific 0 or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nudease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.

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

Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), a nucleic add complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, a cancer protein of the invention, mRNA, ora subsequence thereof. Binding of the antisense polynucleolide to the mRNA reduces the translation and/or stability of the mRNA.

In the context of this invention, antisense polynuceotides 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 nucleolides of the invention. See, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynuceolides 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 alkytated derivatives is also well known to those of skill in the art 00 O Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonudeotides can, CK 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 S(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 San antisense or a sense oligonuceotide, based upon a cDNA sequence encoding a given protein is described in, Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

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

The.general features of hairpin ribozymes are described, in Hampel et al., Nud. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, WO 94126877; Ojwang et al., Proc. Nat. Acad. Sci. USA 90:6340.-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699- 703 (1995); Leavitt et al., Human Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205:121-126 (1994)).

Use of Modulators in Phenotypic Screening In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By "administration" or "contacting" herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, PCT US97/0.1019. 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 genelprotein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invenlion 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

I

00 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 genefic markers by S Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second Smessengers such as cGNIP.

Methods of Identifyinq Characterizing Cancer-associated Sequences Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or C variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, determining the presence of, all or part, the sequence of at least one endogenous cancer gene in Sa cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying 0 the cancer genotype of an individual, determining all or part of the sequence of at least one gene of the invention in the Sindividual. This is generally done in at least one tissue of the individual, a tissue set forth in Table I, and may include Cthe evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the.

sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of.the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.

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

Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as Iranslocations, 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 delectably 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 andlor 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 0 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 kil.

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

)J In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid.

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

rli 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 polynudeolide for use in examining the mRNA expression profile of a cell,. together with reagents used for this purpose.

00 The container can alternatively hold a composition which is effective for treating, diagnosis, prognosing or Sprophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or "K a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 193P1E1B and modulating the function of 193P1E1B.

The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a 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 indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasiaof 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 193P1E1B 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 xenograft tissues. LAPC-9AD xenograft was obtained from Dr.

Charles Sawyers (UCLA) and was generated as described (Klein et at., 1997, Nature Med. 3:402-408; Craft et al., 1999, Cancer Res. 59:5030-5036). LAPC-9AD 2 was generated from LAPC-9AD xenograft by growing LAPC-9AD xenograft tissues within a piece of human bone implanted in SCID mice. Tumors were then harvested and subsequently passaged subcutaneously into other SCID animals to generate LAPC-9AD 2 The 193P1 E1 B SSH cDNA sequence was derived from a subtraction consisting of a prostate cancer xenograft LAPC-9AD 2 minus prostate cancer xenograft LAPC-9AD. By RT-PCR, the 193P1E1B cDNA was identified as highly expressed in the prostate cancer xenograft pool (LAPC4-AD, LAPC4-AI, LAPC9-AD, LAPC9-AI), bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, with low expression observed in the prostate cancer pool, and no expression observed in vital pool 1 (kidney, liver, lung), and in vital pool 2 (stomach, colon, pancreas) (Figure 14).

00 The 193P1E1 B SSH cONA of 227 bp is listed in Figure 1. The full length 193P31E1B cDNAs and ORFs are descibed in Figure 2 with [he protein sequences listed in Figure 3. 193PlE1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11, v.12 and v.13 are novel proteins and have not been previously described. 193PIE18 v.9 shows 99% identity to a hypothetical protein, MGC4832. 193PIE18 v.10 shows 100% identity to a novel unnamed protein BAC03484.1.

CK1 Materials and Methods RNA Isolation: C) Tumor tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 mlI g tissue or 10 mVI 108 cells to isolate total RNA. Poly A RNA was purified fromtotal RNA using Qliagen's Oligotex mnRNA Mini and Midi kits.

Total and mnRNA were quantified by spectrophotometric analysis 2601280 nm) and analyzed by gel electrophoresis.

Oligonucleotides: 00 The following HPLC puriied oligonucleotides were used.

r~l DPNCDN (cDNA synthesis primer): 5'TTTrGATCAAGCTTo3' (SEQ ID NO: 52) Adaptor 1: 5'GTMATACGACTCAOTATAGGGCTCGAGCGGCCGCOCGGGCAG3' (SEQ ID NO: 53) (SEQ ID NO: 54) Adaptor 2: 5'GTAATACGACTCACTATAGGGCAGGGTGGTCGGGGCCG.AG3 (SEQ ID NO: (SEQ ID NO: 56) PCR primer 1: 5'CTAATAGGACTCACTATAGGGC3' (SEQ ID NO: 57) Nested primer (NP)1: 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 58) Nested primer (NP)2: 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 59) Suppression Subtractive Hybridization: Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from prostate cancer xenograft LAPC-9AD2.

The gene 193PIE1B6 was derived from a prostate cancer xenograft LAPC-9AD 2 minus prostate cancer xenograft LAPO-9AD tissues. The S-SH DNA sequence (Figure 1) was identified.

The cDNA derived from prostate cancer xenog raft LAPC-9AD tissue was used as the source of the 'driver" cONA, while the cDNA from prostate cancer xenograft LAPC-9AD 2 was used as the source of the "tester' cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 pg of poly(A)* RNA isolated from the relevant tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as 00 O primer. First- and second-strand synthesis were carried out as described in the Kits user manual protocol (CLONTECH C Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37oC. Digested cDNA was extracted with phenol/chloroform and ethanol precipitated.

STester cDNA was generated by diluting 1 pl of Dpn II digested cDNA from the relevant tissue source (see above) O (400 ng) in 5 pl of water. The diluted cDNA (2 pl, 160 ng) was then ligated to 2 pl of Adaptor 1 and Adaptor 2 (10 pM), in separate ligation reactions, in a total volume of 10 pl at 16oC overnight, using 400 u of T4 DNA ligase (CLONTECH).

Ligation was terminated with 1 pi of 0.2 M EDTA and healing at 72°C for 5 min.

SThe first hybridization was performed by adding 1.5 pll (600 ng) of driver cDNA to each of two tubes containing pl (20 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 00 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 680C. The second hybridization was then diluted in 200 pi of 20 mM Hepes, p1H 8.3, 50 mM C NaCI, 0.2 mM EDTA, heated at 70C 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 pl of PCR primer 1 (10 pM), 0.5 p1 dNTP mix pM), 2.5 pl 10 x reaction buffer (CLONTECH) and 0.5 pi 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 pl. PCR 1 was conducted using the following conditions: 75°C for 5 min., 94oC for 25 sec., then 27 cycles of 94oC for 10 sec, 660C for 30 sec, 720C 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 pM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94oC for 10 sec, 6BoC for 30 sec, and 720C 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 ml of bacterial culture using Ihe conditions of PCRI and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

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

RT-PCR Expression Analysis: First strand cDNAs can be generated from 1 pg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's 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: 60) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 61) to amplify p-actin.

First strand cDNA (5 pl) were amplified in a total volume of 50 pl containing 0.4 pM primers, 0.2 pM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM 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 00 sec, followed by a 18, 20, and 22 cycles of 94CC for 15, 6500°C for 2 min, 72oC for 5 sec. A final extension at 720C was 0 carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 bp B-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal -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.

CNl To determine expression levels of the 193P1E1 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.

A typical RT-PCR expression analysis is shown in Figure 14. RT-PCR expression analysis was performed on first 0strand cDNA generated using pools of tissues from multiple samples. The cDNA samples were shown to be normalized using beta-actin PCR. Strong expression of 193P1E1B was observed in prostate cancer xenograft pool, bladder cancer 00 pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, and metastasis pool.

Low expression was observed in prostate cancer pool, but no expression was detected in VP1 and VP2.

Example 2: Isolation of Full Length 193PIE1B Encoding cDNA To isolate genes that are involved in prostate cancer, an experiment was conducted using the prostate cancer xenograft LAPC-9AD 2 The gene 193P1E1B was derived from a subtraction consisting of a prostate cancer xenograft LAPC- 9AD 2 minus prostate cancer xenograft LAPC-9AD. The SSH DNA sequence (Figure 1) was designated 193P1E1B.

Thirteen variants of 193P1E1B were identified (Figures 2 and cDNA clone 193P1E1B v.1 and 193P1E1B v.5 were cloned from bladder cancer pool cDNA. 193P1E1B v.9 was cloned from LAPC-4AD cDNA library. All other variants were identified by bioinformatic analysis.

193P1E1B v.1 through v.8 differ from each other by one nucleic acid substitution. 193P1E1B v.1, v.2, v.4 v.7 and v.8 code for the same protein, whereas 193P1E1B v.5 and v.6 contain one amino acid substitution as shown in Figure 12.

Absence of a 62-nucleotide sequence was identified in 193P1E1B v.9, nucleic acid positions 907-969 of 193P1E1B v.1. This resulted in an 82-amino acid truncation at the amino terminus of 193P1E1B v.9. Other splice variants were identified and referred to as 193P1E1B v.10, v.11, v.12 and v.13.

193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11, v.12 and v.13 are novel proteins and have not been previously described.

193P1E1B v.9 shows 99% identity to a hypothetical protein, MGC4832. 193P1E1B v.10 shows 100% identity to a novel unnamed protein BAC03484.1.

Example 3: Chromosomal Mapping of 193P1E1B Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), humanlhamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).

193P1E1B maps to chromosome 13q11, using 193P1E1B sequence and the NCBI BLAST tool: (http://www.ncbi.nlm.nih.gov/genomelseqlpage.cgi?F=HsBlast.html&&ORG=Hs). This 13q11 region has been previously implicated in bladder cancer (Wada T, Louhelainen J, Hemminki K, Adolfsson J, Wijkstrom H, Norming U, Borgstrom E, Hansson J, Sandstedt B, Steineck G. Bladder cancer: allelic deletions at and around the retinoblastoma tumor suppressor gene in relation to stage and grade. Clin Cancer Res. 2000 Feb;6(2):610-5.).

00 SExample 4: Expression Analysis of 193P1E1B Expression of 193P1E1B was analyzed using 2 sets of primers as illustrated in Figure 14A. First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool, kidney Cr cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from 2 different patients. Normalization was performed by 0 PCR using primers to actin and GAPDH. Semi-quantitative PCR, using Primer Set A or Primer Set B to 193P1E1B, was performed at 30 cycles of amplification. A schematic diagram depicting the location of the 2 primer sets A and B is 0 shown in Figure 14A. Primer Set A detected a PCR product of 190 bp which is identical in all variants of 193P1E1B (Figure 14B). .Expression of 193P1E18 was observed in prostate cancer xenograft pool, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, Spancreas cancer pool, as well as the 2 prostate metastasis to lymph node, but not in VP1 and VP2 (Figure 14B). In order to test abundance of expression of 193P1E1B v.1 through v.8 compared to 193P1E1B v.9, an experiment was conducted in which RT-PCR was performed using Primer Set B (Figure 14C). Primer Set B detected a PCR product of 239 bp from 193P1E1B v.1 through v.8, and of 177 bp from 193P1E1B v.9 (Figure 14C). Figure 14C shows that the transcipt encoding encoding 193P1E1B v.1 through v.8, is expre-sed ale higher levels that the transcript encoding 193P1E1B v.9. But both transcripts are expressed at similar proportion in all tissues tested.

Extensive northern blot analysis of 193P1 E1 B in 16 human normal tissues confirms the expression observed by RT-PCR (Figure 15). Two transcripts of approximately 3.5 kb and 2 kb are only detected in testis and thymus, but not in any other normal tissue tested.

Figure 16 shows expression of 193P1E1B in prostate cancer xenografts. RNA was extracted from normal prostate, and from prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 ug of total RNA/lane was probed with 193P1E1B SSH sequence. Northern blot analysis shows expression of 193P1E1B in all 4 tissues, LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, with the lowest expression detected in the LAPC-9AD tissue, but not in normal prostate.

To test expression of 193P1E1B in patient cancer specimens, RNA was extracted from a pool of three patients for each of the following, bladder cancer, colon cancer, ovary cancer and metastasis cancer, as well as from normal prostate normal bladder normal kidney normal colon Northern blots with 10 ug of total RNAlane were probed with 193P1E1B SSH sequence (Figure 17). Results show expression of 193P1E1B in bladder cancer pool, colon cancer pool, ovary cancer pool and metastasis cancer pool, but not in any of the normal tissues tested.

Analysis of individual bladder cancer tissues by northern blot shows expression of 193P1 E1B in the 2 bladder cancer cell lines and in the 3 bladder cancer patient specimens tested, but not in normal bladder tissues (Figure 18).

Figure 19 shows expression of 193P1E1B in cancer metastasis patient specimens. RNA was extracted from the following cancer metastasis tissues, colon metastasis to lung, lung metastasis to lymph node, lung metastasis to skin, and breast metastasis to lymph node, as well as from normal bladder normal lung normal breast (NBr), and normal ovary Northern blots with 10 ug of total RNAlane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in all four different cancer metastasis samples but not in the normal tissues tested.

Figure 20 shows expression of 193P1E1lB in pancreatic, ovarian and testicular cancer patient specimens. RNA was extracted from pancreatic cancer ovarian cancer (P2, P3), and testicular cancer (P4, P5) isolated from cancer patients, as well as from normal pancreas (NPa). Northern blots with 10 ug of total RNA/lane were probed with 193P1E18 00 0 sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in Spancreatic, ovarian and testicular cancer specimens but not in normal pancreas.

SFigure 21 shows expression of 193P1E1B in normal compared to patient cancer specimens. First strand cDNA L was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate, bladder, Skidney, colon, lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, C kidney cancer pool, colon cancer pool lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPC prostate xenograft pool and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in Figure 14, was performed was performed at 26 and 30 cycles of amplification. Samples were 0.run on an agarose gel, and PCR products were quanlitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1 B expression in normal 00 testis amongst all normal tissues tested. 193P1E1B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.

193P1E1B was also shown to be expressed in uterus, melanoma and bone cancer patient specimens. First strand cDNA was prepared from a panel of uterus patient cancer specimens melanoma and bone cancer specimens Semiquantitative PCR, using primers to 193P1E1B, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products-were quantitated using the Alphalmager software. Expression was recorded as absent, low, or strong. Results show expression of 193P1 E1B in the majority of uterus patient cancer specimens tested, as well as in the 2 melanoma specimens and in the bone tumor tested.

193P1E1B expression is reminiscent of a cancer-testis gene. Its restricted normal tissue expression to normal testis, and the upregulation detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovary cancer, breast cancer and pancreatic cancer suggest that 193P1E1B is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5: Transcript Variants of 193P1E B Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for .translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding andlor non-coding or 3' end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different limes. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length 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 compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice 00 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 Sfor antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

SMoreover, computer programs are available in the art that identify transcript variants based on genomic D) 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.oml.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 Sexpressed sequence tags, Proc. Nal. 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 Sfull-length cloning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see Proteomic Validation: 00 Brennan, et 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 al., 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 altemative transcripts or splice variants of the gene are modulated as well.

Disclosed herein is that 193P1E1B has a particular expression profile related to cancer. Alternative transcripts and splice variants of 193P1E1B 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, three transcript variants were identified, designated as 193P1E1B v.7, v.8 and v.9. Compared with 193P1E1B v.1, transcript variant 193P1E1B v.7 has spliced out exons 10 and 11 from variant 193P1E1B v.1, as shown in Figure 12. Variant 193P1E1B v.8 inserted 36 bp in between 1931 and 1932 of variant 193P1E1B v.1 and variant 193P1E1B v.9 replaced with 36 bp the segment 1136-1163 of variant 193P1E1B v.1.

Theoretically, each different combination of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant.

Tables LI through LXX are set forth on a variant-by-variant bases. Tables LI, LV, LIX, LXIII, and LXVII show the nucleotide sequence of the transcript variant. Tables LII, LVI, LX, LXIV, and LXVIII show the alignment of the transcript variant with nucleic acid sequence of 193P1E1B v.1. Tables LIII, LVII, LXI, LXV, and LXIX show the amino add translation of the transcript variant for the identified reading frame orientation. Tables LIV, LVIII, LXII, LXVI, and LXX display alignments of the amino add sequence encoded by the splice variant with that of 193P1 E1B v.1.

Example 6: Single Nucleotide Polymorphisms of 193P1E1B 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: AT, CIG, GIC and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the 00 O protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to S environmental factors including diet and drugs among individuals. Therefore, SNPs andlor combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, Sidentification of genes responsible for diseases, and analysis of the genetic relationship between individuals Nowolny, J.

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

i 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 0disease 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).

S SNPs are identified by a variety of art-accepted methods Bean, "The promising voyage of SNP target C- discovery," Am: Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res. 1998 Jul; S8(7):691-697; M. M. She, "Enabling large-scale pharmacogenelic studies by high-throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation'of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs Gu, L Hillier and P. Y. Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998; 12(4):221-225}~.SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct 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 SNPs were identified in the original transcript, 193P1E1B v.1, at positions 57 792 804 1253 (GIA), 1564 2268 (CfT) and 2387 The transcripts or proteins with alternative alleles were designated as variants 193P1E1B v.2, v.3, v.4, v.5 and v.6, respectively. Figure 10 shows the schematic alignment of the SNP variants. Figure 11 shows the schematic alignment of protein variants, corresponding to nudeotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in Figure 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes) and in any. one of the transcript variants (such as 193P1E1B v.9) that contains the sequence context of the SNPs.

Example 7: Production of Recombinant 193P1E1B In Prokaryotic Systems To express recombinant 193P1E1B in prokaryotic cells, the full or partial length 193P1E1B 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 193P1E1B are expressed in these constructs, amino adds 1 to 412 of variant 5 or variant 2; or amino acids 1 to 388 of variant 10, 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 adds from 193P1E1B, variants, or analogs thereof. In certain embodiments a region of 193P1E1B is expressed that encodes an amino acid not shared amongst at least variants.

A. In vitro transcription and translation constructs: oCRIl: To generate 193P1E1B sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of a 193P1E1B cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 193P1E18 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 193P1E18 at the RNA level.

00 S Transcribed 193P1E1B RNA representing the cDNA amino acid coding region of the 193P1E1B gene is used in in vitro S translation systems such as the TnTT u Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize ,Q 193P1E1B protein.

B. Bacterial Constructs: pGEX Constructs: To generate recombinant 193P1E1B proteins in bacteria that are fused to the Glutathione S- 1 transferase (GST) protein, all or parts of a 193P1E1 B cDNA protein coding sequence are fused to the GST gene by cloning into pGEX-6P-1 or any other GST- fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 193P1E1B protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. The GST and 6X His tags permit purification of the recombinant Sfusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti- CK GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, 00 Se.g., 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 193P1E1B-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 193P1E1B proteins that are fused to maltose-binding protein (MBP), all or parts of a 193P1E1B 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 193P1E1B protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxylterminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate 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 Stag from 193P1E1B. 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 193P1E1B in bacterial cells, all or parts of a 193P1E1BcDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 193P1E1B protein in bacteria with and without fusion to proteins that enhance solubility, such as SNusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag M that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of a 193P1E1B protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs: pESC Constructs: To express 193P1E1B in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of a 193P1E18 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 FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 193P ElB. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 193P1E1B in the yeast species Saccharomyces pombe, all or parts of a 193P1E1B cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 193P1E1B protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag

T

M epitope tag allows detection of the recombinant protein with anti- Flag

T

M

antibody.

00

O

O Example 8: Production of Recombinant 193P1EIB in Higher Eukaryotic Systems A. Mammalian Constructs: 0) To express recombinant 193P1E1B in eukaryotic cells, the full or partial length 193P1E1B 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 193P1E1B are expressed in these constructs, amino acids 1 to 412; 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 193P1E1B, variants, or analogs thereof.

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

r Transfected 293T cell lysales can be probed with the anti-193P1E1B polyconal serum, described herein.

0 pcDNA4HisMax Constructs: To express 193P1E1B in mammalian cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven 00 from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressM and six histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth C hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plismid in E. coli.

pcDNA3.1/MvcHis Constructs: To express 193P1E1B in mammalian cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B with a consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for 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.

pcDNA3.1/CT-GFP-TOPO Construct: To express 193P1E1B in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the 193P1E1B ORF, or portions thereof, of 193P1E1B with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylalion signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E coli Additional constructs with an amino-terminal GFP fusion are made in pcONA3.1/NT-GFP-TOPO spanning the entire length of the 193P1E1B proteins.

PAPtaq: The 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pAPtag-5 (GenHunter Corp.

Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of the 193P1E1B proteins while fusing the IgGic 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 193P1E1B proteihs. The resulting recombinant 193P1E1 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 the 193P1E1B proteins. Protein expression is driven from 00 O 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 Scells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

The 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 193P1E1B protein with an amino-terminal C" IgGr signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 193P1E1B 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 193P1E1B proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for 0selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E.

CN2 coli.

O

00 PsecFc: The 193P1E1 B ORF, or portions thereof, of 193P1E1B are also cloned into psecFc. The psecFc vector 0 was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgGI Fc fusion at the carboxyl-terminus of the 193P1E1B proteins, while fusing the IgGK signal sequence to N-terminus. 193P1E1B fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 193P1E1 B proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with the 193P1E1B protein. Protein expression is driven from the CMV promoter. The hygromydn resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRao Constructs: To generate mammalian cell lines that express 193P1E1B constitutively, 193P1E1B ORF, or portions thereof, of 193P1E1 B are cloned into pSRa constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSRo constructs into the 293T-10A1 packaging line or co-transfection of pSRao 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, 193P1E1B, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRa constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 193P1E1B sequences to allow detection using anti-Flag antibodies. For example, the FLAGT sequence 5' gat tac aag gat gac gac gat aag 3 (SEQ ID NO: 62) 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 193P1E1B proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 193P1E1B. High virus titer leading to high level expression of 193P1E1B is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The 193P1E1B coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombinalion and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 193P1E1B 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.

00 Regulated Expression Systems: To control expression of 193P1E18 in mammalian cells, coding sequences of 193P1E1B, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System S(Invitrogen), the GeneSwitch System (Invitrogen) and the lighlly-regulated Ecdysone System (Sratagene). These systems D) allow the study of the temporal and concentration dependent effects of recombinant 193P1E18. These vectors are thereafter used to control expression of 193P1E1B in various cell lines such as PC3, NIH 3T3, 293 or ral-1 cells.

SB. Baculovirus Expression Systems To generate recombinant 193P1E1B proteins in a baculovirus expression system, 193P1E1B ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus.

c Specifically, pBlueBac-193P1E1 B is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera Sfrugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is Sthen collected from cell supernatant and purified by plaque assay.

00 Recombinant 193P1E1B protein is then generated by infection of HighFive insect cells (Invitrogen) with purified Sbaculovirus. Recombinant 193P1E1B protein can be detected using anti-193P1E1B oranti-His-tag antibody. 193P1E1B C protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monodonal antibodies specific for 193PIE1B.

Example 9: Antigenlcity Profiles and Secondary Structure Figure 5. Figure 6, Figure 7, Figure 8, and Figure 9 depict graphically five amino acid profiles of the 193P1E1B amino acid sequence (variant each assessment is available by accessing the ProtScale website (URL www.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.

These profiles: Figure 5, Hydrophilicity, (Hopp Woods 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 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.

Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of 193P1E1B protein. Each of the above amino acid profiles of 193P1E1B 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 add 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 Hydrophilicily and Percentage Accessible Residues profiles, 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-tum (Figure 9) profiles determine stretches of amino acids values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.

Antigenic sequences of the full length 193P1E1B protein (variant 1) indicated, by the profiles set forth in Figure 5, Figure 6, Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-193P1E1B antibodies. The immunogen can be any 5,6,7,8,9, 11, 12,13,14, 15,16,17,18,19, 20, 21, 22, 23, 24, 25, 3 0 35 40 4 5 50 or more than 50 contiguous amino acids, or thecorresponding nucleic acids that encode them, from 193P1E1B protein. In particular, peptide immunogens of the 00 invention can comprise, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 412 that includes an amino acid position having a value S less than 0.5 in the Hydropathicity profile of Figure 6; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible CKl Residues profile of Figure 7; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 8; and, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9. Peptide immunogens of the invention can also 0comprise nucleic acids that encode any of the forgoing.

C) All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised 00 by a composition that includes a pharmaceutical excipient compatible with human physiology.

SThe secondary structure of 193P1E1B, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence of 193P1E1B variant 1 using the HNN Hierarchical Neural Network method (Guermeur, 1997, httpJ/pbil.ibcp.fr/cgi-bin/npsa_.automaLp?page=npsa_nn.html), accessed from the ExPasy molecular biology server (http:llwww.expasy.ch/toolsl). The analysis indicates that 193P1E1B is composed 29.13% alpha helix, 9.95% extended strand, and 60.92% random coil (Figure 13).

Analysis of 193P1E1B using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server(http://www.expasy.ch/tools/) did not predict.the presence of such domains, confirming that 193P1E1B is a soluble protein.

Example 10: Generation of 193PIE1B Polyclonal Antibodies Polydonal 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 the full length 193P1E1B protein, 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 Example 9 entitled "Antigenicity Profiles and Secondary Structure"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9 for amino acid profiles that indicate such regions of 193P1E1B).

For example, 193P1E1B recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 193P1E1B are used as antigens to generate polydonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 20-43, amino acids 100-164, amino acids 241-261, or amino acids •310-331. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 241-261 of 193P1E1B is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of a 193P1E1B protein, analogs or fusion proteins thereof. For example, a 193P1E1B 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. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

00 0 In one embodiment, a GST-fusion protein containing an entire 193P1E1B coding sequence is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that can be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 193P1E1B in S 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).

C In addition to bacterial-derived fusion proteins, mammalian-expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see Example 8 entitled "Production of Recombinant 193P1E1B in Eukaryotic Systems"), and retain post-translational modifications such as -glycosylations found in native protein. In one embodiment, an entire 193P1E1B coding sequence is cloned into the 0mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supematants of 293T cells stably expressing the recombinant vector. The purified Tag5193P1E1B protein is then used as 00 immunogen.

0During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response-cf 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 193P1E1B protein or KLH-coupled peptide encoding amino acids 241-261, the full-length 193P1EiB cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled "Production of Recombinant 193P1E1B in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-193P1E1B serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 193P1E1B protein using the Western blot technique. Immunoprecipitation and flow cytometric analyses of 293T and other recombinant 193P1E1B-expressing cells determine recognition of native protein by the antiserum. In addition, Westem blot, immunoprecipitation, fluorescent microscopy, and flow cylometric techniques using cells that endogenously express 193P1E1B are carried out to test specificity.

The anti-serum from the Tag5 193P1E1B immunized rabbit is affinity purified by passage over a column composed of the Tag5 antigen covalently coupled to Affigel matrix (BioRad, Hercules, Calif.). The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Serum from rabbits immunized with 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. Sera from other His-tagged antigens and peplide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11: Generation of 193P1E1B Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 193P1E1B comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 193P1E1B, for example those that would disrupt its interaction with ligands, proteins, or substrates that mediate its biological activity. Immunogens for generation of such mAbs include those designed to encode or contain an entire 193P1E1B protein or its variants or regions of a193P1E1B protein predicted to be antigenic from computer analysis of the amino acid sequence (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, 00 and Example 9 entitled "Antigenicity Profiles and Secondary Structure"). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, 'cells expressing high levels of 193P1E1B, such as 293T-193P1E1B or 300.19-193P1E1B murine Pre-B cells, are used to S immunize mice.

To generate mAbs to 193P1E1B, mice are first immunized intraperitoneally (IP) with, typically, 10-50 pg of protein CKl immunogen or 107 193P1E1B-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 Sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding S193P1E1 B sequence is used to immunize mice by direct injection of the plasmid DNA. For example, an entire coding Csequence of 193P1E1B, amino acids 1-412 of 193P1E1B variant 1, is cloned into the Tag5 mamnialian secretion vector 00 Sand the recombinant vector is used as immunogen. In another example the amino acids are cloned into an Fc-fusion 0 secretion vector in which a 193P1E1B 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 193P1E1B.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor liter and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Westem 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, monoclonal antibodies are derived that distinguish between, the various 193P1E1B variants, the amino terminal truncated splice variant 3, encoding amino acids 83-412 and the full length protein encoding amino acids 1-412. In one method, two different Fc-fusion proteins are derived, one encoding amino acids 1-82, and the other encoding amino acids 83-412. These are expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 pg of the Tag5-193P1E1B protein mixed in complete Freund's adjuvant.

Mice are subsequently immunized every two weeks with 25 pg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to the full length 193P1E1B protein and to amino terminal truncated variant 3 is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding each of the respective 193P1E1B cDNAs (see the Example entitled "Production of Recombinant 193P1E1B in Eukaryotic Systems"). Other recombinant 193P1E1B-expressing cells or cells endogenously expressing 193P1E1B 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 using standard procedures (see, Harlow and Lane, 1988). Supematants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 193P1E1 B specific antibodyproducing clones.

The binding affinity of a 193P1E1B monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 193P1E1B monoclonal antibodies preferred, for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BlAcore 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 00 biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

SExample 12: HLA Class I and Class II Binding Assays HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed Cprotocols 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 al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 1 25 1-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction 0 of peptide bound.is determined. Typically, in preliminary experiments, each MHC preparation is (itered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the 00 total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

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

Binding assays as outlined above may be used to analyze HLA supermotif and(or HLA motif-bearing peptides (see Table IV) Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epilopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer searches and alqorithms for identification of supermotif and/or motif-bearing epitopes The searches. performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles" and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 193P1E1B set forth in Figures 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 193P1E1B protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: "AG" ai x aix x amn 00 where a- 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 peplide 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 D position i in the peptide, it is assumed to contribute a constant amount j to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

S 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 J. Immunol. 160:3363- S3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding CK (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate ofji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an ileralive procedure.

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

Selection of HLA-A2 supertype cross-reactive peptides Protein sequences from 193P1E1B are scanned utilizing molif identification software, to identify 9- 10- and 11mer sequencescontaining the HLA-A2-supermolif 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 193P1E1B protein sequence(s) scanned above is also examined for the presence of peptides with the HLA- A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A'0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3supertype alleles. The peptides that bind at least one of the two alleles. with binding affinities of 500 nM, often 200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles A'3101, A*3301, and A'6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-87 supermotif bearing epitopes The 193P1E1B protein(s) scanned above is also analyzed for the presence of 9- 10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peplides are synthesized and tested for binding to HLA-B'0702, the molecule encoded by the most common B7-supertype allele the prototype 87 supertype allele). Peptides binding 8*0702 with ICso of <500 nM are identified using standard methods. These peptides are then tested for binding to other common B7- Ssupertype molecules B*3501, 8*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five

B

7 -supertype alleles tested are thereby identified.

Selection of Al and A24 motif-bearinq epilopes 00 To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine Scompositions. An analysis of the 193P1E1B 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 Immunocenicity Cross-reactive candidate CTL A2-supermolif-bearing peptides that are identified as described herein are selected r to confirm in vitro immunogenicity. Confirmation is performed using the following methodology: 0Target 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 B- 00 lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and C 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 i g/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, Lglutamine and penicillinstreptomycin). The monocytes are purified by plating 10 x 10 6 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 24x10 CD8* T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30pglml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20x10scells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (1 4 0pl beads/20x10 6 cells) and incubated for 1 hour at4°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 PBSIAB serum containing 100plml detacha-bead® reagent and 30 pglml 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 6 /ml in the presence of 3pg/ml 92- microglobulin for 4 hours at 20 0 C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1x10 5 cells/mi) are co-cultured with 0.25ml of CD8+ T-cells (at 2x10 6 cellml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IUlml.

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 00 O 2x106 in 0.5 ml complete medium per well and incubated for 2 hours at 37 0 C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10pg/ml of peptide in the S presence of 3 pg/ml .2 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37°C. Peptide solution from each well is S aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent C 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 Critical Reviews in Immunology 018(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 5 sCr release assay. In some experiments the cultures are assayed for peptide-spedfic recognition in the in situ IFNy ELISA at the time of the second 0restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays C for a side-by-side comparison.

0 Measurement of CTL lytic activity by s'Cr release.

SSeven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 5 1 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 37 0

C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200pCi of s 5 Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37°C. 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 37°C. At that time, 100 pl of supematant are collected from each well and percent lysis is determined according to the formula: (cpm of the lest sample- cpm of the spontaneous 5'Cr release sample)/(cpm of the maximal 5 1 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 higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In situ Measurement of Human IFNy Production as an Indicator of Peptide-specific and Endogenous Recognition Immulon.2 plates are coated with mouse anti-human IFNI monoclonal antibody (4 pg/ml 0.1M NaHC03, 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 piwell) and targets (100 ld/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 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 microlilerlwell and the plate incubated for two hours at 37°C. The plates are washed and 100 pI of biotinylated mouse anti-human IFNgamma monoclonal antibody (2 microgram/ml in PBS/3%FCS10.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6x with wash buffer, 100 microliterlwell developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliterlwell 1M H3P04 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.

00 O CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets andlor 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: 1x10 6 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x105 irradiated (8,000 rad) EBV- transformed cells per Sml, and OKT3 (anti-CD3) at 30ng per ml in RPMI-1640 containing 10% human AB serum, non-essential amino acids, r sodium pyruvate, 25pM 2-mercaptoethanol, L-glutamine and penicillinlstreptomycin. Recombinant human IL2 Is added 24 hours later at a final concentration of 2001U/ml and every three days thereafter with fresh media at 501UIml. The cells are Ssplit if the cell concentration exceeds 1xl0 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'Cr release assay or at 1x10 6 /ml in the in situ IFNy assay using the same targets as before the expansion.

SCultures are expanded in the absence of anti-CD3' as follows. Those cultures that demonstrate specific lytic C activity against peptide and endogenous targets are selected and 5x104 CD8+ cells are added to a T25 flask containing the 00 following: 1x10 6 autologous PBMC per ml which have been peptide-pulsed with 10 pg/ml peptide for two hours at 37"C and O 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 coss-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 193P1E1B. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 immunogenicity HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

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

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

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

Analoging at Primary Anchor Residues Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

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

SAlternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate S 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 S capacity of the parent wild type (WT) peptide to bind at least weakly, bind at an ICso of 5000nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient -quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross- C reactivity by T cells specific for the parent epitope (see, Parkhurst et al, J. Immunol. 157:2539, 1996; and Pogue et al., 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 00 to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

C

i Analoging of HLA-A3 and B7-supermotif-bearinq 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. 87 supermotif-bearing peptides are, for example, engineered to possess a preferred residue I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. Immunol. 157:3480-3490, 1996).

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

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

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

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 193P1E1Bexpressing tumors.

Other analoging strategies 1 00 O Another form of peplide analoging, unrelated to anchor positions, involves the substitution of a cysteine with a- Samino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the )Q .peptide structurally so as to reduce binding capacity. Substitution of a-amino butyric acid for cysteine not only alleviates this Sproblem, but has been shown to improve binding and crossbinding capabilities in some instances (see, the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and 1. Chen, John Wiley Sons, England, 1999).

S

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

Example 16: Identification and confirmation of 193PE1 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 C methodology similar to that described for HLA Class I peptides.

00 0 Selection of HLA-DR-supermotif-bearinq epitopes.

To identify 193P1E1B-derived, HLA class II HTL epitopes, a 193P1E1B 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 193P1E1B-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7.

Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 p1, DR2w2 p2, 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 SDR8w2 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. 193P1E1B-derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 motif peptides Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epilopes. 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 193P1 E1B antigens are-analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1pM or better, less than 1 pM. Peptides are found Ihat meet this binding criterion and qualify as HLA class II high affinity binders.

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

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

CD

Example 17: Immunogenicity of 193P1E1B-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 0 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; Cl patients who have 193P1E18-expressing tumors.

00 SExample 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 andlor motifs. In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1-(SQRT(1af)) (see, Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1-(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, the B7-like supertype-confirmed alleles are: B7, B'3501-03, B51, B'5301, B*5401, B*5501-2, B*5601, 8*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

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

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

00 O 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 simulaton 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 C. 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 0 described herein-recognize endogenously synthesized, native antigens.

SEffector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 00 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are 0assayed 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 5'Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 5 sCr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 193P1E1B expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize Sendogenously synthesized 193P1E1B antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb.transgenic mice, several other 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 epitopes.

Example 20: Activity Of CTL-HTL Coniugated Epitopes In Transgenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 193P1E1B-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 193P1E1B-expressing tumor. The peptide composition can comprise multiple CTL andlor HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicily can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

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

Immunol. 159:4753-4761, 1997). For example, A2/K b 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 subculaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTUHTL conjugate, in OMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS- .activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxidty assays are Jurkat cells transfected with the HLA-A2.IKb chimeric gene Vitiello et at., J. Exp. Med. 173:1007,1991) 0 In vitro CTL activation: One week after priming, spleen cells (30x10 6 cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x10 6 cells/flask) in 10 ml of culture medium/f25 flask.

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

Assay for cytotoxic aclivily: Target cells (1.0 to 1.5x10 6 are incubated at 37"C in the presence of 200 pl of After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a Cl concentration of 1 pg/ml. For the assay, 104 5t Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 pl) in U-bottom 96-well plates. After a six hour incubation period at 37"C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity _s determined in a Micromedic automatic gamma counter. The percent specific S lysis is determined by the formula: percent specific release 100 x (experimenlal release spontaneous release)/(maximum.

release spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, release data is expressed as lytic units/10 6 cells.. One lytic unit is arbitrarily defined as the number of effector cells 00 required to achieve 30% lysis of 10,000 target cells in a six hour s'Cr release assay. To obtain specific lytic units/106, the lytic units/1 0 obtained in the absence of peptide is subtracted from the lytic units/1 06 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 Stargets) in the absence of peptide and 5:1 5x104 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 CTLHTL 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 concomitanlly that an HTL response is induced upon administration of such compositions.

Example 21: Selection of CTL and HTL epitopes for inclusion in a 193P1E1B-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 193P1E1B clearance. The number of epitopes used depends on observations of patients who spontaneously clear 193P1E1B. For example, if it has been observed that patients who spontaneously clear 193P1E1B-expressing cells generate an immune response to at least three epitopes from 193P1E1B antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

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

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

00 GO When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peplide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine D) 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 adminislralion 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 Sthis native sequence, whereby one or more of the epilopes 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 Sprophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system 00 processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylaclic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif- Cl bearing 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 193P1E1B, thus avoiding.' the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acidvaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in theart, 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 dears cells that bear or overexpress 193P1E1B.

Example 22: Construction of "Minigene" Multi-Epitope 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 epilopes and/or DR3 epitopes. HLA class. I supermotif or molif-bearing peptide epitopes derived 193P1E1B, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 193P1E18 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

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

For example, the li protein may be fused to one or more HTL epilopes as described in the art, wherein the CLIP sequence of the 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.

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

For example, a minigene is prepared as follows. For a first PCR reaction, 5 pg of each of two oligonucleotides are Sannealed and extended: In an example using eight oligonucleotides, four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 pl reactions containing Pfu polymerase buffer (lx= 10 mM KCL, 10 mM (NH4)2S0 4 mM Tris-chloride; pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 pg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu r polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and 00 the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 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 et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, Kageyama et al, J. Immunol. 154:567-576, 1995).

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

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

Splenocytes from Immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a s 5 Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the iri vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

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

00 STo confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for Sthose epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized intramuscularly with 100 pg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a Sgroup of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant CD4+ T cells, ie. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective 1 compositions (peptides encoded in the minigene). The HTL response is measured using a 3 -thymidine incorporation proliferation assay, (see, Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL Sresponse, 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 Scombination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein Nc- Bamett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for 0 example, expressing a minigene or DNA encoding the complete protein of interest (see, Hanke et al., Vaccine-16:439- S445, 1998; Sedegah etal., Proc. Natl. Acad. SciUSA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177- 181, .1999; and Robinson et al., 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 pg of a DNA minigene encoding the immunogenic peplides 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 pg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the.vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-spedfic activity in an ELISPOT assay.

Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vacdnia, 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 analysiscan 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 193P1E1B expression in persons who.are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, Is administered to individuals at risk for a 193P1 E B-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes.

The vaccne is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 pg, generally 100-5,000 pg, for a kg patient The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 193P1E1B-associated disease.

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

00

O

Example 25: Polyepitopic Vaccine Compositions Derived from Native 193PiEiB Sequences 0 A native 193P1E1B polyprotein sequence is analyzed, preferably using computer algorithms defined for each class S I andlor 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 Ccontains multiple distinct or overlapping, "nested' epitopes can be used to generate a minigene construct The construct is engineered to express the peptide, which corresponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is Sselected because it has maximal number of epitopes contained within the sequence, it has a high concentration of Sepitopes. As noted herein, epitope motifs may be nested or overlapping frame shifted relative to one another). For 00 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.

SThe vaccine composition will include, for example, multiple CTL epitopes from 193P1E1B 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 193P1E1B, 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 193P1E1B peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 193P1E1B and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 193P1E1B as well as tumor-associated antigens that are often expressed with a target cancer associated with 193P1E1B expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Altematively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27: Use of peptides to evaluate an immune response Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTLor HTL directed to 193P1E1B. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

00 In this example highly sensitive human leukocyte antigen tetrameric complexes ("tetramers') are used for a cross- 0 sectional analysis of, for example, 193P1 E1 B HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 193P1E1B 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' Iriphosphate 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 lelramer-phycoerythrin.

00 For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes 0and resuspended in 50 il 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 193P1E1 B epitope, and thus the status of exposure to 193P1E1B, 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 193P1E1B-associated disease or who have been vaccinated with a 193P1E18 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 193P1E1 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) supplemented with L-glutarnine (2mM), penicillin (50U/ml), streptomycin (50 pg/ml), and Hepes (10mM) containing heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 pg/ml to each well and HBV core 128-140 epitope is added at 1 pg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4 x 105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 pilwell 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, rlL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51 Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med.

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

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

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

SCytolylic activity is determined in a standard 4-h, split well s'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 Scytotoxidty is determined from the formula: 100 x ((experimental release-spontaneous release)/maximum release- S spontaneous release)]. Maximum release is determined by lysis of targets by detergent Triton X-100; Sigma Chemical 00 SCo., St. Louis, MO). Spontaneous release is <25% of maximum release for all experiments.

0 The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 193P1E18 or a 193P1E1B vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5x10 5 cells/well and are stimulated with 10 pg/ml synthetic peptide of the invention, whole 193P1E1B 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-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3 H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3Hthymidine 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 pig peptide composition; Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 pg of peptide composition.

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

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

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

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

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

The vaccine is found to be both safe and efficacious.

00 O Example 30: Phase II Trials In Patients Expressing 193PIE1B C Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 193P1E1B. The main objectives of the trial are to determine an effective dose and regimen for Sinducing CTLs in cancer palients that express 193P1E1B, 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, r1 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 Sand reversibility) are recorded.

C<K There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and 00 0 the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each S group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 193P1E1B.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 193P1El Bassociated 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 g) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x10 9 pfu. An alternative recombinant virus, such.as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine.

Peripheral blood 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 193P1E1B is generated.

Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC) Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professional" APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL 00 and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 193P1E1B protein from which the L"K 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- 1- 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 Sreinfused 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.

Cl In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For 00 0 example, PBMC generated after treatment with an agent such as Progenipoietin T M are injected into patients without 'purification of the DC. The total number of PBMC that are administered often ranges from 108 to 101. 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 Progenipoietin T mobilizes 2% DC in 'the peripheral blood of a given patient, and that patient Is to receive 5 x 10 6 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 Progenipoietin TM is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex vivo activation of CTUHTL responses Alternatively, ex vivo CTL or HTL responses to 193P1E1B 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 motif4bearing 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 onlya single type of HLA molecule.

These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 193P1E1B. Peptides produced by endogenous antigen processingof peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, by mass spectral analysis Kubo et al., J.

Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an altemative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfecled with an expression construct encoding a single HLA allele. These cells can then be used as described, they can then be transfected with nucleic acids that encode 193P1E1B to isolate peptides corresponding to 193P1E1B that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

00 As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele Sand subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize Sthat means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

CExample 34: Complementary Polynucleotides Sequences complementary to the 193P1E1 B-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 193P1E1B. Although use of oligonucleotides comprising from about base pairsis described, essentially the same procedure is used with smaller or with larger sequence fragments.

SAppropriate oligonucleotides are designed using, OLIGO 4.06 software (National Biosciences) and the coding sequence of 193P1E18. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and 00 used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 193P1E1B-encoding transcript.

Example 35: Purification of Naturally-occurring or Recombinant 193P1E1B Using 193P1E1B-Specific Antibodies Naturally occurring or recombinant 193P1E1B is substantially purified by immunoaffinity chromatography using antibodies specific for 193P1E1B. An immunoaffinity column is constructed by covalently coupling anti-193P1E1B antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 193P1E1B are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 193P1E1B high ionic strength buffers in the presence of detergent).

The column is eluted under conditions thai disrupt antibody/193P1E1B 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 193P1E1B 193P1E1B, or biologically active fragments thereof, are labeled with 121 1 Bollon-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 193P1E1B, washed, and any wells with labeled 193P1E1B complex are assayed. Data obtained using different concentrations of 193P1E1B are used to calculate values for the number, affinity, and association of 193P1E1B with the candidate molecules.

Example 37: In Vivo Assay for 193P1 E1B Tumor Growth Promotion The effect of a 193P1E1B protein on tumor cell growth can be confirmed in vivo by gene overexpression in a variety of cancer cells such as those in Table For example, SCID mice can be injected SQ on each flank with 1 x 106 prostate, kidney, colon or bladder cancer cells (such as PC3, LNCaP, SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839 cells) containing IkNeo empty vector or 193P1E1B.

At least two strategies can be used: Constitutive 193P1E1B expression under regulation of a promoter such as a constitutive promoter obtained from the genonmes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems.

00 Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used Sprovided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors or by following serum markers such as PSA. Tumor development is followed over time to validate that S 193P1E1B-expressing cells grow at a faster rate and/or that tumors produced by 193P1E1B-expressing cells demonstrate characteristics of altered aggressiveness enhanced metastasis, vascularization, reduced responsiveness to C' chemotherapeutic drugs). Tumor volume is evaluated by caliper measurements. Additionally, mice can be implanted with the same cells orthotopically in the prostate, bladder, colon or kidney to determine if 193P1 E B has an effect on local growth, in the prostate, bladder, colon or kidney or on the ability of the cells to metastasize, specifically to lungs or S lymph nodes (Saffran et at., Proc Nati Acad Sci U S A. 2001,98: 2658; Fu, et al., Int J. Cancer, 1991. 49: 938-939; Chang, eta/., Anticancer Res., 1997, 17: 3239-3242; Peralta, E. et al., J. Urol., 1999. 162: 1806-1811). For instance, the orthotopic growth of PC3 and PC3-193P1E1 B can be compared in the prostate of SCID mice. Such experiments reveal 00 the effect of 193P1E1 B on orthotopic tumor growth, metastasis and/or angiogenic potential.

Furthermore, this assay is useful to confirm the inhibitory effect of candidate therapeutic compositions, such as rK 193P1E1B antibodies or intrabodies, and 193P1E1B antisense molecules or ribozymes, or 193P1E1B directed small molecules, on cells that express a 193P1E1B protein.

Example 38: 193P1E1B Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo.

The significant expression of 193P1E1B, in cancer tissues, together with its restricted expression in normal tissues makes 193P1E1B an excellent target for antibody therapy. Similarly, 193P1E1B is a target for T cell-based immunotherapy.

Thus, the therapeutic efficacy of anti-193P1E1B mAbs is evaluated, in human prostate cancer xenograft mouse models using androgen-independent LAPC4 and LAPC-9 xenografts (Craft, et al. Cancer Res, 1999. 59(19): p. 5030-5036), kidney cancer xenografts (AGS-K3, AGS-K6), kidney cancer metastases to lymph node (AGS-K6 met) xenografts, and kidney cancer cell lines transfecled with 193P1E1B, such as 769P-193P1E1 B, A498-193P1E1B.

Antibody efficacy on tumor growth and metastasis formation is studied, in mouse orthotopic prostate cancer xenograft models and mouse kidney 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-193P1E1B mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-193P1E1B tumor xenografts. Anti-193P1E1B mAbs.also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-193P1E1B mAbs in the treatment of local and advanced stages of, prostate cancer. (See, Saffran, D., et al., PNAS 10:1073-1078 or located on the World Wide Web at (.pnas.org/cgi/doi/10.1073/pnas.051624698). Similarly, anti-193PlE1B mAbs inhibit formation of AGS-K3 and AGS-K6 tumors in SCID mice, and prevent or retard the growth A498- 193P1E18 tumor xenografts. These results indicate the use of anti-193P1E1B mAbs in the treatment of prostate and/or kidney cancer.

Administration of the anti-193P1E1B mAbs leads to retardation of established orthotopic 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 193P1E1B is an attractive target for immunotherapy and demonstrate the therapeutic use of anti- 193P1E1B mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 193P1E1B monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice.

Tumor inhibition using multiple unconjugated 193P1E1B mAbs 00 Materials and Methods 193P1E1B Monoclonal Antibodies: Monoclonal antibodies are obtained against 193P1E1B, as described in Example 11 entitled: Generation of n 193P1E1B Monoclonal Antibodies (mAbs), or may be obtained commercially. The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 193P1E1B. Epitope mapping data for the anti- S 193P1E1B mAbs, as determined by ELISA and Western analysis, recognize epitopes on a 193P1E1B protein.

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

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supematants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are.

performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a Cr mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or 00 orthotopic injections of, LAPC-9 prostate tumor xenografls.

SCancer Xenograffs and Cell Lines 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 subcutaneous trocar implant (Craft, et al, 1999, Cancer Res. 59:5030-5036). The AGS-K3 and AGS-K6 kidney xenografts are also passaged by subcutaneous implants in 6- to 8- week old SCID mice. Single-cell suspensions of tumor cells are prepared as described in Craft, et al. The prostatecarcinoma cell line PC3 (American Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS, and the kidney carcinoma line A498 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% FBS.

PC3-193P1 El B and A498-193P1E1B cell populations are generated by relroviral gene transfer as described in Hubert, et al., STEAP: A Prostate-specific Cell-surface Antigen Highly Expressed in Human Prostate Tumors, Proc Nat. Acad. Sci. U S A, 1999. 96(25): p. 14523-14528. Anti-193P1E1l staining is detected by using, an FITCconjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL f low cytometer.

Xenoqraft Mouse Models.

Subcutaneous tumors are generated by injection of 1 x 10 6 LAPC-9, AGS-K3, AGS-K6. PC3, PC3- 193P1E1B, A498 or A498-193P1E1B cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumorcell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-193P1E1B mAbs are determined by a capture ELISA kil (Bethyl Laboratories, Montgomery, TX). (See, (Saffran, et al., PNAS 10:1073- 1078 or www.pnas.orglcgil doi/10.1073/pnas.051624698) Orthotopic prostate injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5 x 10 5 mixed with Matrigel are injected into each dorsal lobe in a 10 pl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. For kidney orthotopic models, an incision is made through the abdominal muscles to expose the kidney. AGS-K3 or 00 O AGS-K6 cells mixed with Matrigel are injected under the kidney capsule. The mice are segregated into groups for C\ appropriate treatments, will anti-193P1E1B or control mAbs being injected i.p.

SAnti-193P1 E1B mAbs Inhibit Growth of 193P1 E1B-Expressing Xenoqraft-Cancer Tumors T The effect of anti-193P1E1B mAbs on tumor formation is tested by using, LAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, et al, PNAS supra; Fu, et Int J Cancer, 1992.

S52(6): p. 987-90; Kubota, J Cell Biochem, 1994. 56(1): p. The features make the orthotopic model more Srepresentative of human disease progression and allow for tracking of the therapeutic effect of mAbs on clinically relevant end points.

S, Accordingly, tumor cells are injected into the mouse prostate or kidney, and the mice are segregated into two 00 0 groups and treated with either: a) 200-500pg, of anti-193P1E1B Ab, or b) PBS for two to five weeks.

SAs noted, a major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, et al., Proc Nat. Acad. Sc. U S A, 1999. 96(25): p. 14523-14528) or anti-G250 antibody for kidney cancer models. G250 is a clinically relevantmarker for renal clear cell carcinoma, which is selectively expressed on tumor but not normal kidney cells (Grabmaier K et al, Int J Cancer. 2000, 85: 865).

Mice.bearing established orthotopic LAPC-9 tumors are administered 500-1000 pg injections of either anti- 193P1E1B mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate/kidney and lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anli-193P1E1B antibodies on initiation and/or progression of prostate and kidney cancer in xenograft mouse models. Anti-193P1 E1B antibodies inhibit tumor formation of both androgendependent and androgen-independent prostate tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-193P1E1B 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. Similar therapeutic effects are seen in the kidney cancer model. Thus, anti-193P1E1B mAbs.are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39: Therapeutic and Diagnostic use of Anti-193P1E1B Antibodies in Humans.

Anti-193P1 E1B 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-193P1E1B mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 193P1E1B in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-193P1E1B 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-193P1 E B mAb specifically binds to carcinoma cells. Thus, anti-193P1E1B 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 193P1E1B. Shedding or release of an extracellular domain of 193P1E1B into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. Hepatology 27:563-568 00 O (1998)), allows diagnostic detection of 193P1E1B by anti-193P1E1B antibodies in serum and/or urine samples from suspect patients.

Anti-193P1E1B antibodies that specifically bind 193P1E18 are used in therapeutic applications for the treatment of Scancers that express 193P1E1B. Anti-193P1E1B 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 CKl prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-193P1E1 B antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, kidney cancer 0 models AGS-K3 and AGS-K6, (see, the Example entitled "193P1E1B Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo"). Either conjugated and unconjugated anti-193P1E1B antibodies are used as a Stherapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

00 O Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-193P1E1B Antibodies In vivo Antibodies are used in accordance with the present invention which recognize an epitope on 193P1E1B, and are usedin the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 193P1E1B 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-193P1E1B antibodies in Scombination with a chemotherapeutic or antineoplastic agent andlor radiation therapy. Primary cancer targets, such as those lisled in Table I, are treated under standard protocols by the addition anti-193P1E1B 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-193P1E1B antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamydn (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-193P1E1B 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 to anti-193P1E1B antibodies, the radiolabeled antibodies are utilized as a diagnostic andlor imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 193P1E1B. In connection with the use.of the anti-193P1E1 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 1 n)-193P1E1B antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 193P1E1B (by analogy see, Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)).

Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified Dose and Route of Administration 00 0 As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison 0 with the analogous products that are in the clinic. Thus, anti-193P1E1B antibodies can be administered with doses in the o range of 5 to 400 mg/m 2, with the lower doses used, in connection with safety studies. The affinity of anti-193P1E1 B Santibodies 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-193P1E1B antibodies that are fully human antibodies, as compared to ri the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-193P1E1 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 Sopposed to the conventional measurement of dose in mglkg, 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.

SThree distinct delivery approaches are useful for delivery of anti-193P1E1B antibodies. Conventional intravenous CK delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, 0 0 such as tumors of the ovaries, biliary duct, other ducts, and the like, intrapeitoneal administration may prove favorable for 0obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of.antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP) Overview: The CDP follows and develops treatments of anti-193P1E1B 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-193P1E1B antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 193P1E1B 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 193P1E1B. Standard tests and follow-up are utilized to monitor each of these safety concerns.

Anti-193P1E1B antibodies are found to be safe upon human administration.

Example 41: Human Clinical Trial Adiunctive Therapy with Human Anti-193P1E1B Antibody and Chemotherapeutic Agent A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-193P1E1B 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-193P1 E1 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-193P1E1 B antibody with dosage of antibody escalating from approximately about 25 mg/m 2to about 275 mglm 2 over the course of the treatment in accordance with the following schedule: 00 O Day0 Day 7 Day14 Day21 Day28 mAb Dose 25 75 125 175 225 275 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 Chemotherapy CK (standard dose) Patients are closely followed for one-week following eachadministration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: cytokine release syndrome, Le., hypotension, O fever, shaking, chills; (ii) the development of an immunogenic response to the material development of human N antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that 00 express 193P1E1B. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also 0 assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-193P1E1B antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42: Human Clinical Trial: Monotherapy with Human Antl-193PIE1B Antibody Anti-193P1E1B 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 safely 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-193P1E1B antibodies.

Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-193P1E1B 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-193P1E1B 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 el al. J. Nat. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44: Hoinology Comparison of 193P1E1B to Known Sequences The 193P1E1B protein has several forms, including 3 SNPs and 5 splice variants (Figure 4G). Three variants, namely 193P1E1B v.1, v.5 and v.6, consist of 412 amino acids each, with calculated molecular weight of 46.25 kDa, and pl of 5.18, and differ from each other by one amino acid. 193P1E1B v.10, v.9 and v.12 are progressively smaller proteins, with 388, 330, and 73 amino acids respectively. These variants differ with regards to their molecular weights and isoelectic points, as shown in Table L. All variants of 193P1E18 are predicted to be nuclear proteins, with possible localization to the mitochondria (193P1E3B v.1, v.5, v.6, v.9, v.10, v.11 and v.13) or cytoplasm (193P1E3B v.12). Motif analysis revealed no known motifs.

All protein variants of 193P1E1B show best homology to a human un-named protein (gi 21748775) of unknown function, with 193P1E1B v.5 showing 100% identity with gi 21748775 over the entire length of the protein, and 193P1E1B sharing 99% identity with the same protein. Similarly, the other variants show highest homology to the human un-named protein (gi 21748775). The variant with the lowest homology to gi 21748775 is 193P1 E1B v.12, with 89% identity and 89% homology over the first 39 amino acids of the protein (Figure 4A-D).

The 193P1E1 B protein shows homology to a protein of known function, namely the arginlne repressor (gi14349114) of E coli, also known as carbamate kinase. Variant 193P1E1B v.1 shows 30% identity and 57% homology with 00 O that protein (Figure 4E). This homology indicates that 193P1E1B may regulate ATP synthesis and metabolism (Marina A et al, Eur J Biochem 1998, 253:280; Alcantara C et al, FEBS Lett. 2000, 484:261), a key factor in cell growth and biological Sfunction.

rr In addition, 193P1E1B also exhibit some homology to human double-stranded RNA-specific adenosine deaminase (ADAR-c isoform) (gi 7669475). 193P1E1Bv.1 shares 26% identity and 40% homology with ADAR-c (Figure 4F). Similar Cl results were obtained with 193P1E1Bv.5, v.6, v.9, v.10 and v.13. This suggests that 193P1E1B has the ability to bind specifically to double stranded RNA or DNA (Schwartz et al, Nature Struc. Biol. 2001, 8:761). Adenosine deaminases Sacting on RNA have been shown to be involved in RNA editing (Raitskin, et al., Proc. Nail. Acad. Sci 2001, 98:6571).

Recent studies have associated adenosine deaminase with cancer and cellular proliferation (Eroglu A, et al., Med Oncol.

0 2000,17:319-24; Barry and, Lind, Cancer Res. 2000, 60:1887-94). In addition, adenosine deaminase is highly expressed in tumor tissue relative to normal tissues in such cancers as colon, leukemia and other lymphoid cancers (Blatt, J.; O 0 et al., N Engl J Med. 1980; 303:918; Eroglu, et al., Med Oncol. 2000, 17:319). Adenosine deaminase has been 0 considered a potential marker for lymphoid malignancies (Blatt J et al., N Engl J Med. 1980;303; 918). In addition, inhibition -of adenosine'deaminase was found to result in cell death of epithelial cells (Barry, and, Lind, Cancer Res. 2000, 60:1887).

This information indicates that 193P1E1B plays a role in the Iransformation of mammalian cells, supports cell survival and proliferation, and regulates geine transcription by regulating events in the nucleus.

Accordingly, when 193P1E1B functions as a regulator of cell Iransformation, tumor formation, or as a modulator of transcription involved in activating genes associated with inflammation, tumorigenesis or proliferation, 193P1E1B is used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Example 45: 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). In particular, adenosine deaminase has been found to associate with G-proteins, thereby regulating several signaling pathways (Ciruela F et al, FEBS Lett. 1996, 380:219). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 193P1E1B and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 193P1E1B, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as Smitogenic/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).

To confirm that 193P1E1B directly or indirectly activates known signal transduclion pathways in cells, ludferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of.these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

1. NFkB-luc; NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress 2. SRE-luc, SRFITCFIELK1; MAPK/SAPK; growth/differentiation 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress 4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis p53-luc, p53; SAPK; growth/differenatiaon/apoptosis 6. CRE-luc, CREBIATF2; PKA/p38; growthlapoptosis/stress 00 SGene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be Sintroduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.

C Signaling pathways activated by 193P1E1B are mapped and used for the identification and validation of therapeutic targets. When 193P1E1B is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

O Example 46: Regulation of Transcription C The nuclear localization of 193P1E1B and its ability to regulate adenosine deaminase indicate that it is effectively Sused as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., 0 by studying gene expression in cells expressing or lacking 193P1E1B. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 193P1E1B-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000.83:246). Resting cellsas well as cells treated with FBS, pheromones, or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen Ketal. Thyroid. 2001. 11:41.).

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

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, 193P1E1B plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 47: Involvement in Tumor Progression The .193P1E1 B gene can contribute to the growth of cancer cells. The role of 193P1E1B in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, colon, bladder and kidney cell lines, as well as NIH 3T3 cells engineered to stably express 193P1E1B. Parental cells lacking 193P1E1B and cells expressing 193P1E1B are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, et al., Prostate 2000;44:61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7:288). The effect of 193P1E1B can also be observed on cell cycle progression. Control and 193P1E1B-expressing cells are grown in low serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide incorporation by FACS analysis.

To confirm the role of 193P1E1B in the transformation process, its effect in colony forming assays is investigated.

Parental NIH-3T3 cells lacking 193P1E1B are compared to NIH-3T3 cells expressing 193P1E1B, 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 193P1E1B in invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Control cells, induding prostate, and bladder cell lines lacking 193P1E1B are compared to cells expressing 193P1E1B. Cells are 00 O loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane c analog the Transwell insert) and used in the assay. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.

r 193P1E1B can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 193P1E1B are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol.

S 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 193P1E1B, including normal and tumor prostate, and kidney cells. Engineered and parental cells are treated with various Schemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells C are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 193P1E1 B 00 Scan play a critical role in regulating tumor progression and tumor load.

0 When 193P1E1B plays a role in cell growth, ransformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 48: Involvement in Angiogenesis Angiogenesis or new capillary blood.vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell.

1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of phsophodieseterase inhibitors on endothelial cells, 193P1E1B plays a role in angiogenesis (DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 193P1E1B in angiogenesis, enhancement or inhibition, is confirmed. For example, endothelial cells engineered to express 193P1E1B are evaluated using.tube formation and proliferation assays. The effect of 193P1E1B is also confirmed in animal models in vivo. For example, cells either expressing or lacking 193P1E1B are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques.

193P1E1B affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative andfor therapeutic purposes.

Example 49: Involvement in Cell Adhesion Cell adhesion plays a critical role in tissue colonization and metastasis. 193P1E1B can participate in cellular organization, and as a consequence cell adhesion and motility. To confirm that 193P1E1B regulates cell adhesion, control cells lacking 193P1E1B are compared to cells expressing 193P1E1B, using techniques previously described (see, Haler et al, Br. J. Cancer. 1999, 80.1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. In another embodiment, cells lacking or expressing 193P1E1B are analyzed for their ability to mediate cell-cell adhesion using similar experimental techniques as described above. Both of these experimental systems are used to identify proteins, antibodies and/or small molecules that modulate cell adhesion to extracellular matrix and cell-cell interaction. Cell adhesion plays a critical role in tumor growth, progression, and, colonization, and 193P1E1B is involved in these processes. Thus, it serves as a diagnostic, prognostic, preventative and/or therapeutic modality.

Example 50: Protein-Protein Association 00 L i Several adenosine deaminases have been shown to interact with other proteins, therby regulating gene transcription, protein function, as well as cell growth (Railskin Set al above; Morimoto and Schlossman S. Immunol Rev. 1998, 161:55). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 193P1E1B. Immunoprecipitates from cells expressing 193P1E1B and cells lacking 193P1E1B are compared for specific protein-protein associations.

SStudies are performed to confirm the extent of association of 193P1ElB with ci effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates, etc. Studies comparing 193P1E1B positive and 193P1E1B negative cells as well as C 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 193P1E1B-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 193P1E1B 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 193P1E1B. Thus it is found that 193P1E1B associates with proteins and small molecules. Accordingly, 193P1E1B and these proteins and small molecules are used for diagnostic, prognostic, preventative 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).

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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, 00 125A

O

1 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 Sdescribed herein, will become apparent to those skilled n the art from the foregoing Sdescription and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practised without departing from the true scope and spirit of the invention.

Throughout this specification the word "comprise", or variations such as S"comprises" or "comprising", will be understood to imply the inclusion of a stated N element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The following deposits have been made: ATCC No. Description Deposit Date Depositor PTA-3815 E. Coli w/plasmid p193P1E1B-V.1 25 Oct. 2001 Pia Challita-Eid PTA-3816 E. Coli w/plasmid p193PIE1B-V.2 25 Oct. 2001 Pia Challita-Eid PTA-3817 E. Coli w/plasmid p193P1E1B-V.3 25 Oct. 2001 Pia Challita-Eid

TABLES:

TABLE 1: Tissues that Express 193PI El B: a. Malignant Tissues Prostate Bladder Kidney Colon Lung Ovary Breast Pancreas Testis Uterus Skin Bone TABLE 11: 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. hislidine.

Q GIn glulainine R Arg arginine Itie isoleucine M Met meihionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp asparlic acid E Glu glutamic acid G Gly glycine 00TABLE III: Amino Acid substitution MatrixC Adapted from the GOG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL ikp.urnibe.chmanualiblosum6 2 .html) A C D E F GR1I K L M N P Q R S T V WY.

4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -I -1I 1 0 0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1I 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 (I8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H 00 4 -3 2 1-3-3 -3 -3-2 -1 3-3 -11 5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L C1S -2 -2 0 -1 -1 -1 1 -1 -1I M 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 I0 1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 0 -2 -2 T 4 -3 -1 V 11 2 W 7 Y TABLE IV: HLA Class till AlotifsiSupermotifs TA13LE IV HLA Class I Supermotits/Motifs SUPERMOTIF POSITION POSITION

POSITION

2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Al I L -VMSAnchor) Al TILVMST FWY A2 LIMATQIVMATL A3

RK

A24 YF WIVLMT 137 P

_________VILFMWYA

B27 RHK B44 ED

FWYLIMVA

B58 ATS B62 QLIVMP

FWYMIVLA

MOTIFS

Al TSM

Y

Al DEAS

Y

A2.1 LMVOIAT

VLIMAT

A3 LMVISATFCGD

KYRHFA

All 1_ VTMLISAGNCDF_

KRYH

A24 YFWM

FLIW

A*3101 MVTALIS

RK

A*3301 MVALFIST

RK

A*6801 -AVTMSLI

RK

63*0702 P B*3501 P

LMFWVYIVA

651 P ______LIVFW4YAM 6*5301 p IMFWYAL V 63*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 HIA Class 11 Supermotif TAB3LE IV HLA Class if Motifs 00 MOTIFS I' anchorl1 2 3 4 5 1' anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPAUfM MH MH deleterious W R WOE D~iprfered MFIVW PMQVMATSPLJC M AVM deleterious C OH FD W GDE D DR7 preferred MFLIVWY' M W A IVMSACTPL M IV deleterious C G GRO N G DR3 MOTIFS I* anchor 1 2 3 1l*anchor 4 5 1' anchor 6 Motif a preferred LIVMFY D Motif b preferred -LIVMFAY DNQEST KRH DR Supermotif MFLIVWY

VMSTACPUI

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

SUPER-

MOTIFS

Al 10 Anchor 1 0 Anchor TIL VMS

FWY

A2 10 Anchor 1* Anchor LIVMVATO

LIVMAT

A3 Preferred 1* Anchor YFW YFW YFW P 10 Anchior VSMATL/

RK

deleterious DE

DE

P A24 1' Anchor 10* Anchor YFWIVLMT FlY WLM 137 Preferred FWY 1' Anchor FWY FWY l 0 Anchor LIVM P VILFMWiYA deleterious DE DE G ON DE B27 1* Anchor l 0 Artchor RHK FYLWA4IVA 844 I* Anchor 10 Anchor ED

FWYLIMVA

858 1 0 Anchor l 0 Anchor ATS

FWYIJVMA

862 10* Anchor 1' Anchor QIJVMP

FW'(MIVLA

Italicized residues indicate less preferred or "tolerated" residues TABLE IV HLA Class I Motifs

I

3 4 5 6 7 8 9 00 terminus or C-terminus Al preferred GFYW l 0 Anchor DEA YFW P DEQN YFW 1l*Anchor 9-nier SIM

Y

deleterious DE RHKLIVMP A G A Al preferred GRHK ASTCLIVM 1 0 Anchor GSTO ASTO LIVMV DE 1 Anctior 9-mer DEAS

Y

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

Y

mer deleterious GP RHKGLIVM DE RHK ONA RHKYFW RHK A Al preferred YFW STCLIVMV 1 *Anchor A YFW PG G YFW 1 'Anchor DEAS

Y

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

VUIMAT

deleterious *DEP DERKH RKH DERKH POSITION: 1 2 3 4 5 6 7 8 9 C- Terminus A2.1 preferred AYFW 1'Anchor LVIM G G FYWL I'Anchor LMIVQAT vim VLIMAT mer deleterious DEP DE RKHA P RKH DERK RKH

H

A3 preferred RHK 1*Anchor YFW PRHKYF A YFW P 1'Anchor LMVISATFGGD W

KYRHFA

deleterious DEP DE All preferred A l'Anchor YFW YFW A YFW YFW P 1'Anchor VTLMISAGNCD

KRYH

F

deleterious DEP A G A24 preferred YFWRHK l'Anchor' STC YFW YFW i'Anchor 9-mer YFWM

FLIW

deleterious DEG DE G QNP DERH G AQN

K

A24 Preferred 1 'Anchor P YFWP P 1 *Anchor 110- YFWM

FLIW

mer Deleterious GDE QN. RHK DE A QN DEA A310 Preferred RHK l'Anchor YFW P YFW YFW AP 1'Anchor 1 M'JITALIS

RK

Deleterious DEP DE ADE DE DE DE A330 Preferred 1'Anchor YFW AYFW 1'Andior I MVALF/ST

-RK

Deleterious GP DE A680 Preferred YFWSTC I Anchor YFWLIV YFW P 1 'Anchor 1AVTMSLI M

RK

deleterious GP DEG RHK A B070 Preferred RHKFWY 1 'Anchor RI-K RHK RHK RHK PA 1 *Anchor 2 P

LMFWYAI

V

deleterious DEQNP DEP DE DIE GDE ON OF POSITION 1 3 4 5 6 7 8 9 Cor C-terminus Al preferred OFYW 1 *Anchor DEA YFW P DEON YFW 1VAndior 9-mer STM y deleterious DE RHKLIVMVP A G A Al preferred GRHK ASTCLIVM l 0 Anchor GSTC ASTC LIVM DE l 0 Anchor 9-met DEAS y deleterious A RHKDEPYFW DE PON RHK PG GP B350 Preferred FWYLIVM 1 *Anchor FWY FVVY l'Aridior 1P

LMFWY/V

A

deletefious AGP G G B51 Preferred LIVMFWY l 0 Anchor FWY STC FWVY G FVWY l 0 Anchor P

IVFWVYA

deleterious AGPOER DE G DEON ODE

HKSTC

8530 preferred UIVMFWY 1 0 Anchor FWY STC FVVY LIVMFW FVVY 1 0 Anchor: 1P Y IMFWYAL

'I

deleterious AGPQN G RHKQN DE B540 preferred FWY I *Anchior FWYLIVM LIVM ALIVMA FWYA I*Anctor 1 P P All VLMF deleterious GPQNDE GDESTC RHKDE DE QNDGE DE TABLE IV [Sum marv of HLA-sucertvoes Overall Dhenotypic frequencies of HLA-supertypes in different ethnic pulations 00 ____Specificity _____Phenoty ic frequency___ Suetp osition 2 C-TerminusCaucasianIN.A. Black Japanes Chinese H-ispani verag B7 ______ILMVFW 43.2 5.1 57.1 3.0 R9.3 49.5 A3 IdLMVST RK 7.5 K2.1 45.8 52.7 43.1 4.2 A2 LMVT AILMVT 5.8 39.0 42.4 5.9 43.0 2.2 4 F (WIVLMT) -1 (YWLM) 3.9 38.9 58.6 0.1 08.3 0.0 :WYLIMV 3.0 21.2 429 39.1 39.0 1 1 (LVMS) WYPx 7.1 16.1 21.8 1 4.7 26.3 5.2 27 HK( :YL (WMI) 28.4 26.1 13.3 13.9 35.3 3.4] 62 L (IVMP) -WY (MIV) 12.6 .8 3.5 5.4A 1. 1 18.1 58 Is FWY f I \A 10.0 51 .6 0 .9 10.3 TABLE IV lCalculated oulation coverage afforded by different HLA-supertype combinations HIA-supertypes Phenotypic frequency Caucasian N.A Blacks Paans 'hinese isanc verage 83.0 6G.1 P7.5 38.4 36.3 36.2 A2, A3 and 87 99.5 38.1 100.0 )9.5 )9.4 )9.3 A2, A3, B7, A24, 844 9.9 39.6 100.0 )9.8 39.9 39.8 and Al A2, A3, B7, A24, 1344, Al, B27,8B62, and B 58 Motifs indicate the residues defining supertype specificites. The motf incorpor ate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

Table V: Frequently Occurring Motifs Name 'vr, Description Potential Function Nucleic acid-binding protein functions as transcription factor, nudlear location zf-C2H2 34% Rno finger, C21-2 type robable Cytochrome b(N- nembrane bound oxidase, generate cytochrome .b N 68% terminalyb6/petB 3uperoxide morains are one hundred amino acids ong 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 repeal protein interaction may function in targeting signaling POZ 23% PDZ domain molecules to sub-membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involvedin protein-protein interactions conserved catalytic core common to both serinelthreonine and tyrosine Pkinaseprotein kinases containing an ATP 00

O

0)

C)

0

,_N

qj fc o o

(N

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_q1 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 Rv 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

F

n3 20% Fibronectin type III domain bonds seven hydrophobic transmembrane regions, with the N-terminus located 7 transmembrane receptor extracellulary while the C-terminus is 7tm_1 19% (rhodopsin family) cytoplasmic. Signal through G proteins Table VI: Motifs and Post-translational Modifications of 193P1E1B N-glycosylation site Number of matches: 3 1 246-249NKSE (SEQID NO: 63) 2 316-319 NSSS (SEQ ID NO: 64) 3 340-343 NLTD (SEQ ID NO: cAMP- and cGMP-dependent protein kinase phosphorylation site 107-110 KKNS (SEQ ID NO: 66) Protein kinase C phosphorylaion site Number of matches: 1 22-24 TAR 2 53-55 TLK 3 103-105 SPR 4 152-154 SPR 149-151 SEK 6 103-105 SPR 7 152-154 SPR 8 203-205 TPK 9 217-219 TPK 203-205 TPK Casein kinase II phosphorylation site Number of matches: 12 1 16-19 STLD (SEQ ID NO: 67) 002 34-37 SDFE (SEQ ID NO: 68) 3 53-56 TLKD (SEQ ID NO: 69) ~~I4 110-113SVHE (SEQ ID NO: 119-122 SDPE (SEQ IDNO: 71) 6 124-127 SNCE (SEQ ID NO: 72) 7 276-279 SDAE (SEQ ID NO: 73) 8 318-321 SSND (SEQ ID NO: 74) 9 336-339 TCFE (SEQ ID NO: 350-353 SSYE (SEQ ID NO:- 76) 11 360-363 TPPE (SEQ ID NO: 77) 12 408-411 SNKE (SEQ ID NO: 78) ri N-myristoylation site 239-244 GLKNAR (SEQ ID NO: 79) 00 Table VII: 00 Search Peptides variant 1: 9-mers, lO-mers and 1 5-mers (SEQ ID NO:.

MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI

PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN

LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD

NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGSEYTMCLNEDYTMGL

KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK

NSIALVSTNYPLSKTNSSSNOLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT

PPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN

variant 9-mers PVASSCISEKSPRSPQL (SEQ ID NO:- 81) PPVASSCISEKSPRSPQLS (SEQ ID NO: 82) DDLSDPPVASSCISEKSPRSPQLSDFGLE (SEQ ID NO: 83) Variant 6: 9-mers NKSEEAIDAESRLND NV (SEQ ID NO: 84) NNKSEEAIDAESRLND NVF (SEQ ID NO: LKNARNNKSEEAIDAESRLND NVFATPSP (SEQ ID NO: 86) Variant 9-mers KIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 87) 1 0-mers TKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 88) 1 TPPEVTKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 89) Variant 12: 9-mers RALDGEESLLSKYNSN (SEQ ID NO: 1 0-mars QRALDGEESLLSKYNSNL (SEQ ID NO: 91) 00 ETARLQRALDGEESLLSKYNSNLATPIA (SEQ ID NO: 92) 00 Tables VIII XXI: Table VIII-VI-HLA-AI-9mersi93P1EIB_ Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position pu eiht.

S ubear quec Score F19 CETARLOR [5.o] F9 LSFGLERY [37T500 F-341 FLTDPSSPTI 125.000 132 GIDFIKAtK 20.000 1 78 11 LSDPPVASS 15.000 Fi2l NQEGIDFIK 113.500I 272 QLEKSOAEY 9.000 28 ALDGEESDF 5.000 253 DTESRLNDN 2.250 31 GEESDFEDY 2.250 ]33] ESOFEDYPM 1500 F306 VSTNYPLSK 1.500 LSNCENFQK] 1.500 ISETMCLN 1.350 F37 EDYPMRILY I F228 YTMCLNEDY 1.250 19l SNDLEVEDR 1.250 [7111 KTDVI(DDLS 1.250 F237 LNEDYTMGL 1125 3581 TPTPPEVTK [.o 21 CVTPKEHF 1.000 389 KAVPPSKRF 1.000 I TLDCETARL F1.001 277 DAETNSPL 0.900 I321 DLEVEDRTS 0.900 123 EVEDRTSLV 0.900 344 PSSPTISSY 0.750 349 ISSYENLLR I 0.750 1 333 11 NSDTCFENL 1 0.750 7[ KSDAEYTNS 0.750 SHSEVQTLKD r 0.675 F233 NEDYTMGLK If I F382 LATPIAIKA 0.500 I 8Fj TNSPLVPTF_ 0.500 251 AIDTESRNII 0.500 E_ DILLLSKY 0.500 1 263 FATPSPIIQ 0.500 1302 SIALVSTNY 0.500 I 97 ]jQLSDFGLER 0.500 12191 KLEHFGISE 4550 Table VIII-VI-HLAAl-9mers- 193PiEIB 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 Subsequence 381 NLATPIAIK 0.400 -36 MGLKNAR][ (1250 116-1[ STLDCETAR ][0.250] F91 VPPSKRFLK 0.250 1 [67-1 SPIIQQLEK 209 KMDDFECVT 0.250 FI-21 I LLDKARLEN 0.250 189 VTPPTKQSL 0.250 1 7I IPELSNCEN 0.225 367 IPEOILQLL 0.225 I171 ~jVHEQEAINS ]0.2251 35 DFEDYPMRI] 0.225 142 LMEKNSMDI 0.225 182 YKEEPVIVT 0.25 175 EAINSDNYK 201f LKTPKCALK -2-0 1 110 1 QVLPNPPQA 0.200 1 -83 VASSCISGK I I200 1102 GLERYIVSQ 0.180 146 NSMDIMKIR 0.150 173 EQEAINSDN 0.135 i KSEEAIDTE 10.135] 2 CPOLKIPS I 0.125 147 SMDIMKIRE 0.125 264 ATPSPlIQQ 0.125 30 OGEESOFED 113] F86 SCISGKSPR 0.100 Ii301 LVLNSDTCF IL1 881 IVTPPTKQS .1 0.100 ji1h8 AVNLLDKAR ~10.100 F20 KCALKMDDF 0.100 r160_ YGYSPRVKK I 0.100 37 KATKVLMEK 0.100 390 I AVPPSKRFL 0.100 126 FRLENGEGID 0.090 1183 ]f KEEPVIVTP If f 651 NCENFQKTD 0.090 212 DECVTPKL F 0.090 12 RSLASTLDC 0.075 NSDNYKEEP _0.075 Table VIII-VI -HLA-AI -9mers- 193PIEIB 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 IStart Subsequence IIScore [IKI IRSFCGKLRS1 0.075 31671 NSSSNDLEV 0.0757 F350 ]SSYENLLRT 0.0757 YW[ QSLVVLKT 10.075 KQSLVKVLK 0.060 F2-87 PTFCTPGLK If o V7 if DVNIPELSN T112T[ LPNPPQAVN 0.050 1280 YTNSPLVPT 0.050 I108 YIVSQVLPN 110.050] 224 GISEYTMCL 0.050 11541 REYFQKYGY I -2571 RLNDNVFAT 15 I 369 EDILQLLS( 0.050 55 KDDVNIPELI 0.050 FI-52[ KIREYFQIY II 0.050I 1368 I IPEDILQL 0.050 F67 ENFQKTDVK ]IW51 IZi5 IKDDLSDPPV 121411 ECVTPKLEH Table VIII-V5.HLA-AI-9mers- 1931EIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start osition lus ei h Start fl Subseuence l Score VASSCISEK 0.200 F-5 SCISEKSPR [1 6 JfCISEKSPRS II0.020 7f3 ASSCISEKS 7 ][ISEKSPRSP j0.014 [797 EKSPRSPQL 0.010 Table SSCISEKSP I 0.002 1 j j PVASSCISE 0.00 13 I8 1 SEKSPRSPQ 0.0001 Table VIII.V6-HLA-A-9mers- 00 00 193PIEIB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is (he sart position plus eight.

Start[ Subseune] cr 8j[ -C DAESRLNDN 10.900 .6 AIDAESRLN Ij00 121] KSEEAIDAE ]j 0.135- F3 ]1 SEEAIDAES _j0.9 EAIDAESRL jooo -4 1 EEAIDAESR I os iT][ NKSEEAIDA J100 F7]IOAESRLND -11 0.0001 a].1 AESRLNDNv IF6I K Start IISubsequence]i cr~ 1 -4 DGEESLLSK 1125001 rI5 GEESLLSKY ]r2.250 I7[ALDGEESLL II 0.500 IZj[ ESLLSKYNS-11 0.030 F1 J SLLSKYNSN 0-0o10i1 LiiRAL.DGEESL 110.0101 [YLDGEESLLSjI0-o3f ESLSYN f d~i~ ITable IX-Vl-LAA-0mers.

1 93PIElB Each peptide is a portion of SEQ ID NO: 3; 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.I r§~!iII Subsqec ]oe 11219J1 KLEHFGISEY J 500 16RLENQEG"DFIFW] ['331 ESDFEDYPMR l~ho [1341 I[LTDPSSPTIS 112.500] EflSMDIMKIREY 12.00 DW[GEESOFEDY 11.20 139011 78 1[ LSEJPPVASSC ]1 7.5] F 1-73[ EQEAINSD)NY If 75 r-6 FEDYPMRILY .5 j 3231] EVEDRTSLVL jT I 1 i.153I IREYFQKYGY II4.500I 123211 LNEDYTMGLKI] .0 IPELSNCENF I .5 [1573 DTESRLNDNV I .5 277 DAEYTNSPLVJf180 27 KSEEAIDTES IIi~ 367_ -IPEDILQLLS I F357i RTPTPPEVTK11.0 r i30-f LVSTNYPLSK[ 1.000 r-6-72[ ELSNCENFQK 19 ][DCETARLQRA]0.01 r321'][ ILEVEDRTSL[0.0 M6] NCENFQKTDV]0.0 [-3T1[ NSIANSTNY 57W 333I NSDTCFENLTIFos 178 I NSDNYKEEPV .5 225I ISY LNE L10.6751 7171I KIDVKDDLSD F.-625I 7T] TLDCETARLQ ]10.500 Table IX-V-HLA-Al0mers.

-V193PIEIB Each peptide is a porton* of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start sition lus nine.

[Startj Subsequence E(Score I 263 FATPSPIIQQ [I 0.50 0 20MDDFECVTPKIf55- 132~] GIDFIKATKV 1.05005 I .1I TSSYENLLR 05007 2-89 1 FCTPGLKIPS][W F2- 0-j YTNSPLVPTF ][0.50] [97 QLSDFGLERY 13681l PEDILQLLSI< I 1 1901I TPTQLV 0f .250 189 IVTPPTKQSLV 0.505 128 J[ENQEGIDFIK I 251] AIDTESRLND 0.25D] I266] PSPIIQQLEK I io 85 ][SSCISGKSPR [3181 SSNDLEVEDR V~o 1271f QQLEKSDAEY II0.150 48 HSEVQTLKDD .3 l33~ DPSSPTISSY [T J F2587 LNDNVFATPSIf015 233 I NEDYTMGLKN If- 0125] 319i SNDLEVEDRT 0~.i 380 SNLATPIAIK 1010 1188] ivTPPTKQSL F-1 R)0.

185~] EPVIVTPPTk]fT [Ti10 QVLPNPPQAV I:h- 214[ ECVTPKLEHF I0.01 27~] RALDGESFJ 0.100] [382 1 LATPIAIKAV 1.1005 329I SLVLNSDTCF 0[.100] 2151 CVTPKLEHFGIlT i 11~7 IIQAVNLLDKAR J 00 389f KAVPPSKRFL J I [102 IIGLERYIVSQV I1 0.0901 272i QLEKSDAEYT I.00 [337 fCETDSjf00] 3579 NSNLATPIAI I1 0.075 I 2 5 j I DAETNSP 0.0i5l I947[ RSPQLSDFGL Table VIII-V12.HLA.A1.9mers.

193PIElB Eachpeptide 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.

Table IX-VI-HLA-AI-1 Omers- 1 93PIIEIB 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. [Startl Subsequence ]F S-coire] [349 I SSYENLILRT ]F -0-7--i [282 I1 NSPLVPTFCT IF 0.075.

[-279 1f TPGLKIPSTK f~0.050]1 r33111 VLNSDTGFEN If Thi6 1 [-54l1 LKIDDVNIPEL j[Thi05] [3-8 if NLATPIAIkA I1.5 [1671~ STLDCETARL If ~5 L-44]1 LYDLHSEVQT I1O0500 p23 YTMGLKNARN I 0l5 r28 If ALDGEESDFE IF 0.050 FIL[2 LPNPPQAVNLI1 .5 -7 IKDDLSDPPVAI11 0050-1 2586] VPTFCTPGLKIF 0.0-50 I f-1-21]1 LLD KARLENQ 50 I57 fI-- SVHEQEAINS]I .5 If 2 ]f11 CLNEDYTMGL If 0.050 S141-11 VLMEKNSMADI ]F o.oso-1 I iIZ llI VKDDLSDPPV][ -0-0501 I i f[j IMKIREYFQK [I12 jf NLLDKARLEN S2-091 KMODFECVTP ]iI--:570 II CTPGLKIPST ]f 0.05 [FI61 IKATKVLMEK ____057 [jjT11 KEEPVIVTPP 1100-545] F3511 SYENLLRTPT IF OXN55 DFEDYPMRIL]1045 ITable IX-HLA-1 1 Omers- 193PIElBJ Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is amino adds, and the end position for each peptide is the start position plus nine.

ar ISbeuence 1[ Score SSIES r r- T-l ISKPSQ1103 F7 SCSKPSI1.2 2_1 PVASSCISEKI __F572 F311 VASSOISEKS ]~JF--1 F 1EKSPRSPQLSI__ I ASSCISEKSP]I 57 Table IX-V5-HLA Ii93PIEIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the I start position plus nine.

IS-t~rtI Subsequence I[S-c-re-1 LZIII CISEKSPRSP 1I0.001 I PPVASSCISE IF0.-00 M I SEKSPR SPQL [0 0__00 I Table IX-V6-HLA-Al-l0mers- [193PIElB.8 IStart ISubsequence FScore Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

II KSEEAIDAES If2:0 M9I DAFSRLNDNV 31. o9-00 3 4 741 SEEAIDAESR 0.450 IZII ADASRLND]F6727 6 If EAIDAESRLN 0010-1 NNKSEEAIEDA].o 10 j] AESRLNDNVF] oi =1i IDAESRLNDN] I 0.00 1.

lilli EEAIDAESRL 001 FY11 NKSEEAIDAE Table IX-ViO.HLA-A-0mers- Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 10 amino acids, and th end position for each peptide is the start position plus nine.

F-arlj[ Subsequence I cr F13 WIYPTQKLNK 11000 MT] TKIPEDILQK ][F0.500 1Z6][ DILQKFQWIYI~1 Lii[] IPEDILQKFQ ]I 0.225 r 271[ KIPEDILQKF j[T5 rI1][ YPTQKLNKM][ 0025 1 FT]f PED)ILQKFQWIFO 0Th I 10 I1 KFQWIYPTQK]I~T I Til QKFQWIYPTQ F I7 ILQKFQWIYP 11 -000 1 .E1 IYPTQKLNKMI J112~ Fl QWYTKN 000 II IfILQKFWIYPT 0.000 Table IX-V12-HLA-A-i0mers- I193P1 Ell SEach 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 I start position plus nine.

[sta'rtl SubsequenceIF-S-core I M I 31ADGEESLLS 11 .0 LxiI LDGEESLLSK ]j 0050 I GEESLLSKYN II004 ESLLKYNSN I0015 I FT]I SLLSKYNSNL ]5 f 2~ RALDGEESLL I~.i 7]11 EESU..SKYNS ooo 1= 1 QRALDGEESL 0.01 Table X-VI-HLA-A0201-9mers- Each peptide is a portion *of SEQ ID NO: 3; each start. pos ition is specified, the length of peptide is 9 amino acids, and the end position for each peptide. is the start position plus eight.

Sitart Subseqence Scre F2 ]7 RLNDNVFAT 114750 I-11 1-I V[PNPPQAV 118.2381 [-3676I KIPEDILOQL]I6.7 11iE~11 KMDDFEGVT 4-8.131 f-3774 1LSKYNSNL 1136.316I 11340 1 NLTDPSSPT. ]1.30.553 1 F 3-0-4] ALVSTNYPL ][-2T-36] r[220][ TMCLNEDYT ][1T4I7 I ~71 TLDCETA-RL If54 F-07 KLRSLASTL 1[~ F~14-0[ KVLMEKNSM 11i 5.291 F 1-979 KVLKTPKCA ]5.629 YPMRLYOL I1Tj IiiffIi AEYTNSPLV I[4-28I Table X-VI-HLA-A0201 -9mers- 193PI ElB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Subsequence I Sc-]~ [46 I] DLHSEVQTL f[3.685 TI NVFTPSPl F3478 TISSYENLL if2.937 [I_22 1 LEVEDRTSL 112.895 F329]I SLVLNSbTC i 2.434 [2_08 If LKMDDFECV i 21 r383ff ATPIAIKAV [2.222 F3_90hj AVPSK RFL f256 F15105 QVLPPA]11.0 [37L SSYENLLRT I16 [-66 ]I EFK~VI 1.352 I ]VLKTPKCAL I1;271j []IYTNSPVP 1.*09] [283 31SPLVPTFCT 11 1.0441 I 1 IKTNSSSNDL]I r2_077 ALKMDDFEC rj 1.009~ 1 11Il VNLLDKARL IU5_87] I 2_7051 IQQLEKSDA]I 0.856] I 421 LMEKNSMDI 1f 0.820 1 9511j SPQLSDFGL If080 S3-71 1[ILQLLKNi 67 F352 YENLRP][66 IiiILIDFIKATKV ]1 0.608 1 I fKNSMDIMKI 0:548 15IfQTLKDDVN1Ij[56 [i~iJ TTQSL 1][.54j 23-1[CLNEDYTMG ][07458 TF 3 PP I Q S V 16. 5 F1795 QSLVKLK 1 41 F3 7-3 QLLSKYNSN II .1 LFRYISV1142 f3_ KNSIALVST 1j0.392j FI-4][ VLMEKNSMD]1TT F 114 NPPQAVNELW .2 I 265] -TPSPIIQQL 11 0.321 378I NSNLATPIl1 1] 15811I QKYGYSPV II0.309_ i8]07 LATA _[0-252] 0211 KPKCALKM 11 0.242~ Tle X-VI-LA-A0201-9mers- I 93PMEB 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 eicht.

IF!tiIt Subsequence If scorel 1307 11 STNYPLSKT IF 0238] f VPTFCTPGLi037 FGISEYTMCi .2 EVQTLKDDV ]~TT F250-[ MCLNEDYTM1001 1361 QEGiOFIKA ]756.1-8r-321 TSLVLNSDT ]I_0.18 2821] NSPLVPTFC I 017 r -51l YDLHSEVQT]1 .7 Flii.11 LASTLDCET ]11 0176 I IZ77 FCGKLRSLA 104j F-3I6 FEDYPMRIL 10l4 F3_67_1 1PEDI-LOLL IF 0143-11 VLN-SDTCFE 1113 75]I KDDLSDPPVI 03 13111 EGIOFIKAT I hT- -12811 ENQEGIDFI j i _58_ VNIPELSNC I 012 12i1 RSLASTUJC I 120 323]1 EVEDRTSLV I .120 382f LATPIAIKA I .1 ::2D4 Vf VE DRTSL V LJ 1 168] 0.SHE114fii 2 91 I TPGLKIPST If0.112 1i~3 FKATKVLM Ifj~ -106 3 YVSQVLPN 0 108 [Table X-V6-HLA-A0201-9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is' specified, the length of peptide is 9 amino acids, and the end position for each peptide is the tart position plus ight Start Subsequence II coe] 9 AESRLNDNV II0.663] 5 I EAIDAESRL 11 0.091] 1t F NKSEEADA ]1'0.028] 6 if AIDAESRLN ]j 0.00I fIDAESRLND I0 .000 2 IfKSEEAIDAE11h

I

.iIISEEAIDAES ~f0.000I 87f DAESRLNON 5 _j 41EEAJDAESR Table X-VI O-HLA-A0201 49mers- 193PIElB Each peptidleis 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 sart position plus eight.

13411 LTDPSSPTI] I0099 I [i557 KDDVNIPEL J[5§ 250IEA ID TE S RL jF 0.091 1 LTI GKLRSLAST Ii 0.088I F3979I KHGQNIRDV II 0.078 [i IQAVNLLDKA 11 0.078 [_2I1 -STKNSIALV 107j 73207 NDLEVEDRT I .7 72327 LNEDYTMGL I o- [70If QKTDVKDDL 0.060~ EK]I SDFGLERYI I 0.059] 73-47] I LUPPI h] 72377 TMGLNARN §jariI Subsequence j~cr 5 1 DILQKFQWI ]F4.16o 6 1ILQKFQWIY .8 141 PQKLNKM 1[o.i3 ]i1I1I WlYPTQKLN 10.151 1 KFQIYPT 10088 Table X-V1O-HLA-A0201-9mers.

I93PEIB Each peptide is a portion of SEQ ID NO: 21; each start position is specfied, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight [startj[ Subsequence Score F KIPEDILQK 0.068J Fl-1 FQWIYPTFK ]0058-3 11 71 QWIYPTQKL O003 (7 I LQKFQWIYP I OA00 1 [7271 IPEDILQKF 0.000 9 1 KFQWIYPTQ 30-I1 1 4 EDILQKFQW 0.0001 3 i PEDILQKFQ 110.000 PTQKLNKMR )10.000 [13 7 IYPTQKLNK .oo.] Table X-VI 2-HLA-A0201.9mers- I93PEiB Each'peptide is a porion 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 positionplus eight.

Start Subsequence Score F TI ALDGEESLL )8.545 1 RALDGEESL 8 ]j SLLSKYNSN 6 14 I i Ui1 LGEESLLtS [01001 j6 I EESLLSKYN 0.001 GEESLLSKY 1 0.000 r-7j ESLLSKYNS [4JD DGEESLLSK I o:ooo Table XI-VI-HLA-AO201 10 mers-193P1E18 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.

jstart Subsequence Score 199I1L KVLKTPKCAL 24.206 110 ]QVLPNPPQAV] 22.517 102 GLERYtVSQV 10.238 322 LEVEDRTSLV ]_9.4267 355 LLRTPTPPEV 8.986 374 LLSKYNSNLA 8.446 1 13l SLASTLDCETI[ 7.452 194 KQSLVKVLKT iF 8 3 NLATPIAIKA _4.968 [2W YTMCLNEDYT 4.747 L i 11 STLDCETARL 4.

501 1321 GIDFIKATXV ]3.825 382 LATPIAIKAV 3j77_ l2l29 TMCLNEOYTM 3588 188 IVTPPTKQSL 3.178 1-M LVPTFCTPGL J[ 3.178 207 ALKMDDFECV] 2266 127I LENQEGIDFI 2.138 1IYSPRVKKNSV 2.088 118 AVNLLDKARL [1.8691 303 IALVSTNYPL 11 1.866_1 51 1i VQTLKDDVNI 1198 189 VTPPTKQSLV I1642I 206 CALKMDDFEC 1.481 216 VTPKLEHFGI 1.429 261 NVFATPSPII 1.385 fQLEKSDAEYT F--.85 130 QEGIOFIKAT 1.266 45 IA YDLHSEVqh1I 1.161 269 IIQQLEKSOA 1.161 389 KAVPPSKRFL 111.142 1 I FQKYGYSPRV I 1.135 12D NLLDKARLEN IF 1.130 295 KIPSTKNSIA 0.980 332 LNSDTCFENL][ 0.905 94 1 RSPQLSDFGL IF 0.809 Table Xt-VI-HLA-A0201-1 0mers-193P1E1B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

i a j[ Subsequence Score 315 [TNSSSNoLEV 0.454] 2 0 LKMDDFCVT [0.416 3 97 j1 QLSDFGLERY I0344 [8 11 NSPLVPTFCT f0.282 74 ]jVKDDLSDPPV I[0.269 290 I CTPGLKIPST 0.238 5 ][RSFGKLRSLJ 0.237 112 ][LPNPPQAVNL[ 0.237 [296]f IPSTKNSIAL '0.237 [Fl[2 KIREYFQKYG ]0.234 i SDEDYPMR3 -4 T jj LKDDVNPEL 10.1901 306 VSTNYPLSKT 0.190 349 ISSYENLLRT 0.190 142 LMEKNSMDIM 0.180 28 TNSPLVPTFC 0.178 9 ]tFQ69 DVKDDL [0.171 63LSNCENFQKT 0.157 [358] TPTPPEVTKI 110.157I 99 SDFGLERYIV 0.147 371 ILQLLSKYNS 0.127 276 SDAEYTNSPL 0122 1[ KNSVHEQEAI 6[87T [57] RLNDNVFATP] 0.116 271QQLEKSDAEY 0.115 I30411 ALVSTNYPLS 0.112 9I GKLRASTL 0.1101 3271 RTSLVNSDT 0.104 332 DLEVERTS. 0103 I I SPTISSYENL jI0.1021 f 4[ GISEYTMCLN I1O971 178 1NSDNYKEEPV[ 0.089 20j CETARLQRAL20 0.083 F11 IDFIKATKL 31o 39811 LKHGQNIRDV]I 0.076 [i li DVNIPELSNC -0-0-I75 329][ SLVLNSDTCF [0.075 333 NSDTCFENLT 0.074 37211 LQLLSKYNSN 0.071 36531 TKIPEDILQL F0-0687 [79 NSNLATPIAI 1 0.068 E11 ILYDLHSEVQ 0067I Table XI-VI -HLA-A0201 -10mers-i 93P1 ElB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Start[ Subsequence Score [T141 3VLMEKNSMDI 269.051 F3] KIPEDILQLL j 96947 [1 (CLNEDYTMGL jj 87.586 I3731QLLSKYNSNL j(j 7 i 1340 HNLTDPSSPTI 42

I

I 31] VLNSDTCFEN I 0.735 I 264 IIATPSPIIQQL 0.2 _220 LEHFGISEYT 0.664 49 SEVQTLKDDV j0.663 1223 FGISEY IFCL ]0.641 1109 SQVLPNPPQA] 0.504 42 RIYDLHSEV 1 35.385 00 00 Tabe X-V1-H1LA-A0201.10.

mners-193P1E1B Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptde is amino acids, and the end position for each peptide is the start position plus nine.

IStart IISubsequence cr "209 JJKMDDFEC VIP 1~ ROE2 1129] NQEGIDFIKA II0.061.

F3778 I YNSNLATPIA____ F32]8 TSLVLNSDTc IF 0.0659 141 LASTLOCEA] f[007 M354. NLLIRTIPTPPEJ[005 77j 1DLSDPPVASjr0.0531 1i vLENP QAVN rO.052 11 256l D SR NNVFAT]I .5 9 LSDFGLERYI J05 I TabI e XI-V5-HLA-A0201 -1 0mers- 193PI EllB Each.peptide is a portion of SEQ ID NO: 11; each start position is.

specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Start Subscq .uence[Soe 9 SEKSPRSPL11s L7 CISEKSPRSP .0 3~1 VASSCISEkS ][0.001 SCISEK SPRS 0.000 SSCISEKSPR I .0 =4I ASSCISEKSP ~I6 [.21 PVASSCISEK 0.000 ISEKSPRSPQ 1 000i I SjEKSPRSPQS 000 [17J PPASSCISE ETab Ie X I _V6-HLA;.A021.1 Omers] Each peptide is a portion of SEQ [ID NO: 13; each start position is specified, the length of peptide i amino acids, and the end position for each pppfde is the start position plus ne.

Start Subsequence]Sor 1EEAIDAESRL 1[ 0.031 9 DAESRLNDNV 0.005Tj 8 IDAESRLNIJN j[ =002 -i NNKSEEAIDA jj0.00 7 AIDAESRLNDIf001 10o] AESRLNDNVF 3T] KSEEAIDAESj[oo 2I 1t NKSEADE [6 [EAIJAESRLN i .0 [TLrSEEIDAESR ~i Table XI-VlO0-HLA-AO201. 1 1 0mers.193P1 El B Each petd saprin of SEQ ID NO: 21; 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.

Start1I Subsequence ]jScoreJ J~11 FQWIYPTQKL II 826941 [E KIPEDILQKF 11 0.38J [7 I LOKFQWIYP- It023 LL8II[ LQKFQWIYPT 11[0.103 1111 DILQKFQWIYJf5i 1T iWJ[WYPTQKLNK]0X0j [T[EDILQKFOWI j [1_[IYPTQKLNKM ][0.003j F 15][ YPTQKLNKMR 1[ 00 LL3 1 IPEDILQKFQ- JIW5 I9 1j QKFQWIYPTQ]F _05550 1 4 PEDILQKFQW 0.0005~ LiItTKIPEDILQK 0[ .000 K7ifIFQWIYPTQK It 0.000 E 12 I _QWIYPTQKNj I0.000 ITable AM V2-HLA-A0201.

1 0mers-193PIE1B 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.

[tarti Subsequence Score 9i II SLLSKYNSNL It79.041 a I RALDGEESLL tTT F73'[ ALDGEESLLS I .3 I fQRALDGEESL ]f0.00 1 EE]. I GEESLLSYN][ oo 4A LDGEESLLSK]oa 8~I ESLLSI(YNSN 1[0.000 Mhi EESLLSKYNS J[0.0 DGEESLLSKY U~ Iable XII4-I.HLA-A3.9mers.

1I93PIElB Each peptidle is apri 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.

['Start Subeqene Score] '1-239 IGLKNARNNK II60.0001 IF3_I[ NLATPIAIK It 45. 000 F-97[ QLSDFGLER It.00I 152[ KIREYFOKY 116.2001I 12 GIDFIKATK FJ79 NOEGIOFIK _4.05 U~1K HGN] 22 LEKSDAEY II4.000 [387 AIKAVPPSK I300 U AUJGEESDF 3.000 11941 KSVLK 2.700 1 1137 I KATKVLMEK j[2.700 j [3041 ALVSTNYPL ]2.700 [1971 LKLTPK ][2.000 PI4] LLSKYNSNLI 1.80011 10 KLRSLASTL 1180 2271GISYML 14 1LMEKNSMDI lT FiII ILYDLHSEV It.0-0 I §IKMDDFECVT r 9~ 20 VU TPKCAL_ 0.900 S2_57 j[ RLNDNVFAT J[ .0.90 3-66]1 KIPEDILOL ].810 j_267] [SPIIQQLEK 0.600 r[27F[ ALKMD DFEC [.0J [3061 VSThYPLSK']o.0 3[91J VPPSKRFLK [7177~ TLDCETARL

IW

46fl DLHSEVQTL 0. 01 358 TPTPPE-VTK 0.450 2 36 YTMGU NAR II0.450 Mi25 CVTPKLEI A 0.45 rt2i9]j KLEH-FGISE 1f0.6 186s r PVIVTPPT< ]1 0.300 83_ VASSCIGK I 11 VLPNPPQAV 63 LSNEFQ Jr 0300 330 LVLNSDTCF J~0.300 16 STLDCETAR 1~ 0.300 261 NVFATPSPI 0301 228 IYTMCLEDY II T3 0-0 Table XII-VI-HLA-A3-9mers- 193PI EllB 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.

St~art I Subsequence S-core 329 I1 SLVLNSDTC 300 2 J1 DPIRSFCGK I[0.7 102j GLERYIVSQ 0.27 370 IIDILQLLSK-Y ][0.7 1181 AVNIIDKAR IF 0.2-00 116 1 PQAVNLLDK ][18 [401 QNIRDVSNK I[0.8 [-29511 KIPSTKNSI ]r 0.1-80 [3481 TISSYENLL 0.1-80 1541 REYFQKYGY ]I0.8 YGYSPRVKK 0.1-50 271- PTFCTPGLK ][15 07 NLTDPSSPT 0.150 14791 DIMKIREYF .3 211 DOFECvTPK 0.1320 1571 FQKYGY-SPR I[0- 2 3111 GEESOFEDY ][0.108 3879 KAVPPSKRF 0.101 229 1 TMVCLNEDYT o1o0 151 ]1 MKIREYFQK I[ 0.090 14 ~KVLMEKNSM ~s [314 .KTNSSSNDL .9 [2 5 KCALKMDDF 090 7293] GU IPSTKN .9 F-17571 EAINSDNYK 0.090 F 397 YPMRILYDL ][0.6 F 6711 ENFQKTOVK I[000 F 202][ KTPKCAU<M 110 0060 F1 507[ IMKIREYFQ 0.060 [j86]1 SCISGKSPR IF006 [j53]1 TLKODVNIP IF 0060 [-47]1 LHSEVQTLK IF 0.0-45 [1 99[ KVLKTPKOA IFO0O045 F QVLPNPPQA ]F 0045 [T64[ VTIKIPEOILI[ 0.045 [52.]1 QTLKDDVWI -IF 0045 [18931 VTPKQLI005 341 11 LTDSTI10.4 [3493 ISSYNL F 0040 [1~TI LLKARLN 1 0.040 1_ DIK4R 11 0 040 rable XII-VI -HLA-A3-9mers- 193P1E1B 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 191].I PPTKQSLVK II0.040 196 ]1 SLVKVLKTP -I 0034 14611 NSMDIMKIR F 0.0-34 .~211 LKTPKCALK IF 030 23171 CLNEDYTMG_ IF 030 373 1[ QLLSKYNSN ]j 030 34T1[ SDFEDYPMR 1I0.3 24. 1 RLQRALDGE IF 0.0-30 -98 11LSDFGLERY I- .0030 354 11NLLRTPTPP 0I 0030 355 J1 LLRTPTPPE .3 13 11 SASL E FOO0.30 363 1 EVTKIPEDI .2 369 1 DLLSK .2 233]1 NEDYML 0.0-27 319 1 SNOLEVEOR] =I0.024 Table XII-V5-1-LA-A3-9mers- 193M1B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each poptidle is the start position plus eight.

Staf Subsequence 2J j VASSCISEK IF -0.300 5jscis-sp-RI o~ 0.606 6]1l CISEKSPRS 000 1 ]j PVASSCISE F] .0.000 3 ]1 ASSCISEKS 0.000 9 ]1t EKSPRSPQL 0.0-00 81 SEKSPR-SPQ 0.0-00 4 j SSCISEKSP 0-0 0 7711I ISEKSPRSP J[ 0.0-00 Table XII-V6-HLA-A39mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight 11-ail s euence Scorej F 471 EEAIDAESRIt004 F 57 EAIDAESRL 10.003] F271i KSEEAIDAE 1[0.001] F1111 NKSEEAIDA ]1.01 F97I1 AESRLNDNV _IL51 AIDAESRLN 1 0.000 f-731 SEEAIDAES II000 F 8I1 DAESRLNDN fl:j[ IDAESRLND ]r o 000j Tale XII-VIO*HLA-A3-9mers- 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.

FSta usqec JScore [F-1-1 KIPEDILQI( ][127.000 FOWIYPTOK 31 9.0-00 F-57]1 DILQKFQWI ][iT F2 I IPEDILQKF I[~4 F]3I1 IYPTQKLNK 0.040 I]J PTQKLNKMR 0.0101 112 11 WIYPTOKLN 0.0071 [±811 QKFQWIYPT [17411 YPTQKLNKM 0.003- [1111] QWIYPTQKL .0 r17111 LQKFQWIYP 0.001 F471 EDILQKFQW .0 [911l KFQWIYPTQ]fooo1 1I I PEDILQKFQ .0 Table XVIIV2LA-A3-gmers- 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.

FttI Subs[qu cr] 2I ALDGEESLL I[ 0.900]7 IT1l GEESLLSKY 1_[547 8 1 SLLSKYNSN j[000 4 1 DGEESLLSK II0.027] M I RALDGEESL FI 009~ F7-11 ESLLSKYNS I[ 0. 0 00 M I1 LDGEESLLs I 111EESLLSKYN 0.00 [Table XIIIHLA.A3.lmers.

193P1 ElB f Each peplide is a portion of SEQ (D NO: 3; each start position is specified, the length of peptide is amino acids, and (he end position for each peptide is .the star position plus nine.

[Start[ Subsequence IIScore [1501 IMKIRYQ I~ r ~[SLVKVLKTPK I 000 200[ VU<T KCALK j 000 I219 J[KLEHFGISEY if18.000 62 1 ELSNCEFK] 800 [30511 LVSTNYPLSK 200 46~ I DLHSEVQTIJ<[ 390 AVPPSKRFLK 9,00oo0 97 IQLSDFGLERY 6.00 '231l CLNEOYTMGLJ5.0 401. RDSNK 11 5AO Ii16 RLENQEGIOF II4.000] I 329-1 SLVLNSDTCF I I FGLERYIVSQvl =I .700 1373 QLLSKYNSNL] 2.700 11411 VLMEKNSMI]270 357] RTPTPPEVTK ]T5 [348 IITISSYENLLR .0 366 j KIPEDILQLL.U 0.608 207 I ALKMDDFECV J 10 F147 SMDIMKIREY .0 381J NLATPIAIKA JJ0.600 387 AJKAVPPSKR 0.600J 30[NLDSTIj060 229][ TMCLNEDYTM J 386 IAIKAVPPSK [0.460 26 NVFATPSPII 0.450 IKVLKTPKCAL 190f TPPTKQSLVK

AO

82. PVASSCISGK 0. 3 0 291 jjTPGLKIPSTK J0.300 142 IfLMEKNSMDIM 280. YTNSPLVPTF ]1 0.300] 271[ QQLEKSDAEY II0.270 355] LLRTPTPPEV ~f0.200 3741 LLSKYNSNLA 1 0.200 321] DLEVEDRTSL 1 0.180 3801 SNLATPIAIK rI 0.135 143]1 MEKNSDK 0.120 371 1[ILQ LLSKYN S 1 Table i 93PiE18* 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 peptidle is the start position plus nine.

[Start Subsequence iSore 1231 GLKNARNNKS[ F0.120] 9j-6 PQLSDFGLER[ 0-.10O87 13ji] SLASTLDCET ][0.0 43.] ILYDU-ISEVQ 0.0 I 1272] QLEKSDAEYT ]I 010 0 [j159] KYGYSPRVIKI 0.090 j 1361 IKATKVLMEK 1(00 2!1 vTPKLEHFGI L [i88I IV/TPPTKQSL ITW [25 II RLNDNVFATP i__ [Th 1 5 EPVIVTPPTK 0.09 -~51 [128 1ENQEGIDFIK .8 264I ATPSPIIQQL]5

J

I LTiJ] QVLPNP PQV](08 1h MDPIRSFCGK] 0.060 210~] MDDFECVPI(J[ .6 I is[PPQAVNLLDK I 16 304! I ALVSTNYPLS 110.060] 295 J~KIPSKNSIA 0. (060] f293l GLKIPSTKNS [_QT601 53W TLKIDVPEf 7 118l AVNLLDKARL 5] 132 fGIDFIKATKV if0.060 31VLNSDTCFEN ]fE5 17 T O[ EAINSDNYK 66fCENFQKTDVK J 16 I Th][ KLRSLASTLD ][.a060] 286~] VPTFCTPGLK J 16 1 2_8 5 LVPTFCTPGL J F31 SSNDLEVEDR ]j0.060 209 IIKMDECVTP I0.060 1201 NLLDKARLEN 77i~ OLPVS I 0.05 Fi9±I1 KQSLVKVLKT 7x729 NQEGIDFIKA J~T 151 MKIREYFQKY 0.054J 3011 NSIALVSTNY I ~4 NLLRTPTPPE 0045 1 16~ STUJCETARL 3 14 ~f[PTFCTPGU<I 0 (045 397_ FLKI-GQNIRD 0.0I 323]1f EVE DRTSLV 0.0 Ini =Table XIIW-I-HLA-A-lniers.

I I93P1 EllB 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 eadh peptide. is the 'Ltart poition lus nine.

g taRj Subsequence Score 173I1 EQEAINSDNY 0.(0361 13 TKQSL VKVU K1 0030~ 2381 MGLKNARNNK 0.030 28] ALDGEESDFE j 0.030 427] RILYDLHSEV ].'0.030 1 117 QAVNLLDKAR 11F63_] I27 11RALDGEESDF II 0.030 I'll~ VLPNPPQAVN]1~~& 92i SPRSPQLSDF .0.(030] L 1] LLDKARL-EN ]J0.3 [358fl TPTPPETK -11(07 3032~II IALVSTNYPL I:0.027 SDFEDYPMRI .02 [363] EVTKIPEDIL Jf0.027 I fKNSMDIMI(IR '.027]J 6 1 FQTVKDDL F1.07 F36 FEDYPMRILY 0.024j 11 0 1 PMIYDLHS .024 Table XIII-V5-HLA.A3.lomers- I193PIE1B Each peptide is a portion -of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and. the end position for each peplide is the start position Plus nine.

[-Start I Subsequence][ coe 2 il PVASSCISEK 1][.300 M SSISEKSPR I[ (10 =9 SEKSPRSPQLrO02 I~ (SCEKSPRS i .0 [3 IfVASSCISEKS 0.00h1 711 CISEKSPRSP 8 ISEKSPRSPQ I 1j PPVASSCISE

]W~

4 1f ASSCISEKSP II 00 a llI EKSRPQLS I ~o Tal III-V6-HLA.A3.lomers.

I193PIEIB [Eah epide is a portion of SEQj ID NO: 13; each start position is specified, the length of peptide Is amino acids, and the end position for each peptide is the start opsiion plus nine.

Start Subsequence Score 4 SEEAIDAESR 0.012 7-1I AESRLNDNVF 0.006 7] AIDAESRLND 0104] [T-I KSEEAIDAES 1F 0001] [1111 NNKSEEAIDA II 0011 9 DAESRLNDNV 0.001 f-57 EEAIDAESRL 0.001 =8 IDAESRLNDN F0.00 L121.II NKSEEAIDAE 0.000 6 EAIDAESRLN .ooo Table XIII-VO-HLA-A3-lOmers- Ta 93PEB 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 posifion plus nine.

ItarI1 Subsequence i I) r 13 WIYPTKLNK 30.000 2 KIPEDILQKF 11 2.025_ 6 J DILOKFQ'NIY IT627 FjOI[ KFQWIYPTQK 110.180 FT1- TKIPEDILK 0.135.- I11I FQWIYPTQKL 11-0.135 I .i LQKFQWIYPT 10.

T 77 ILQKFQWIYP 0.040 [*Thj[ YPTQKLNK<MR 0.020 -IEDILQKFWI I-0001 F141 IYPTQKLNKM 1f0W LI I PEDILQKFQW II000 9 QKFQWIYPTQ i000 F311 IPEDILQKFQ J 0.000 2 1 QWYPTQKLN II0100 1 FTable EXIIIVI 2HLA-A3-lmers- 193PIE1 Each peptide is a portion of 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.

Start Subsequence Score Mi SLLSKYNSNL 2700 Table XIII-VI2-HLA-A3-l0mersb X 93PIEi B 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.

Startl Subsequence IScore F 3 ALDGEESLLS 0.120 FK j LDGEESLLSK 0.090 7 RALDGEESLL 0.009 1 5 1 DGEESLLSKY][ 0.003 1I QRALDGEESL 0.001 7F EESLLSKYNS 0.000 M I GEESLLSKYN 0.000 I ESLLSKYNSN 01001 Table XIV-V1-HIA-AI101 9mers.

193PIEiB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start I Subsequence I Score F17 ILVKVLKTPK jI 2.000 [IL94] KQSLVKVLK 1.800 129 NQEGIDFI< 1.800 239 GLKNARNNK 1.200 137 KATKVLMEK 1.200 132 GIDFIKATK 1.200 I 29L]l VPPSIRFLI 0.600 Li ISPIIQOLEK 0.600 [i ~AIKAVPPS J 0.400 ]j YTMGLKNAR 0.400 381 NLATPIAIK F0.400 186 PVIVTPPTK I[ 0.300- 16 L STLDCETAR 1V535 118 AVNLLDKAR 0.200 287 PTFCTPGLK 0.200 358 PTPPEVTK 11 0.200 [83 11 VASSCISGK I 0.200 fable XIV-VI-fLA-AI 01-9mers.

193P1ElB 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 eit Start I Subsequence Score 397I FLKGNIR 0.080 402 QNIRDVSN( 1 0.060 1 I LSNCENFQI ][060] [202 11KTPKCALKM [0.060 [F2 NEDYTMGLK 0.060 [861[ SCISGKSPR II IIjII1 KVLKTPKCA 0.045 I YGYSPRVKK F-0.040 19I 1 PPTKQSLVK II0.040 I 2. I VSTNYPLSK II0.040 L2§i] NVFATPSPI I 0.040 1 3 ]0 LNSDTOF 0.030 II0 T QjVLPNPPQA 11[ (030 f Ii] KTNSSSNDL] .0.030 F67[ ENFQKTDK 0.024 I i4i GISEYTMOL 110.024 I9 ]I DCETARLQR If0.024 Lu6I KIPEDILOL 147 If LHSEVTLK ~[0.020 2281 YTMCLNEDY J 0.020 311 CVTPKLEHF 0.020 [25 II LKTPKCALK ][0.020 [iE EDILQLLSK ]Wi18 QTLKDDVNI ][0.015 I29.. KIPSTKNSI 01FWi 11411 EKNSMDIMK II.h0iT 125 DDFECVTPK Lu9 ALVSTNYPL ~I0.012 F-5 2F KLRSLASTL if 0.012 1 T KIREYFQKY 7.0012.1 298 STKNSIALV 0.010 189 VTPPTKQSL 0.010 1341 LTDPSSPTI F 0. 010 F364-1 VTKIPEDIL [7001-0 r396 RFLKHGQNI 10.009 142 EKNS[DI 0.008 ISSYENLLR 0.008 34[ SDEDYPMR[ 0.008 [319] SNDLEVEDR[ 0.008 4T if ILYDLHSEV If0108] 139 ~fYPMRILYOL if .oos I 54I! REYFKYGY 0.007 9711 QLSDFGLER F 0.160 11] PQAVNLLK (F 0.120 159 KYGYSPRVK If 0120 1571 FQKYGYSPR 0.120 151 if MKIREYFQK ]f 0.090 140 KVLMEKNSM if 0.090 175 I EAINSDNYK 1 0.090 DPIRSFCGK j 0.090 00 00 able XIV-VI-ILA.AIIOI.9mers] 193PIEIB 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.

ta Subsequence Scare [323 EVEDRTSLV ]j_0.006 ios][ RVKKNSVHE 0.006 EVQTLKDDV 0.006 1 363 EVIKIPEDI f 0.006 205 IKCALKMDDF 0.006 21711 TPKLEHFGI 0.006 95 II SPQLSDFGL 110.006 I I 270 i IQQLEKSDA 11 0.006 I 230 fMCLEDYTM 0.006 F363 FATPIAIAV 0.005 r389 if KAVPPSKRF 0.005 F200] VLKTPKCAL IF 0.004 I I146 IiNSMDIMKIR if0.004 LATPIAIKA If 0.004 382] I~tA .0 222[ HFGISEYTM J[ 0.004 27][ QLEKSDAEY J[ 550 26 ALDGESDF J[ 0.004 Th][l VLNppAV 01(004 111 0.004 8 j[q TFCTPGLKI ][00 135 FIKAKVLIf 0.004 388 0.004 7[ TLDCETARL 0.004 Table XIV-V5-HLAAI1OI-9m ers- 193PIEl Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

I srfltl subseuenceJ[ Score 2 VASSCISEKf 0.200 F57 1 SCISEKSPR][ 0.060 [T1 Jf PVASSCISE] 0.000 CISEKSPRS] 0.000 LT]EKSPRSPQL] 0.000 LA[SEKSPRSPQ][ 0.000 ASSCISEKS 0.000 [7 i SSCISEKSP II0.000 [7 ifISEKSPRSP if0.000 XIV*V6-HLA-A1101-9mers- Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start j[Subseuencef Score 75:] EAIOAESRL jf 0.001 I9IIAESRLNDNV1[ 0.001 F j NKSEEAIDA[ o.ooo 2 I[KSEEAIDAE~J 0.000 8f[DAESRLNDNf 0.000 3f SEEAIDAES~J 0.000 f Tj IDAESRLND[ o.ooo I6l AIDAESRLN] 0.000J Table XIV.V 0-HLA.A 101- 9mers-193P1 El B 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 sto lsegt Start ifSubsequence J[or] I i KIPEDILQK ][.4fl 10 ff FQWIYPTQK T250 13 if IYPTQKLNK 00 Th7 IPTQKLNKMR[ 5.0j 6 i ILQKFQWIY J 0.008 14 I YPTQKLNKM] 0.002] Lj4iI LLSKYNSNL 1 0.004 TISSYENLL F -0.0 0 47.

181_ NYKEEPVIV 0.004 302][ SIALVSTNY 0.004 [T7. i RLNDNVFAT 11 0.004 49 EEAIDTESR 0.004 [92 PGLKIPSTK 1 0.003 717 KTDVKDDLS 0.003 357-1 RTPTPPEV 0.003 117 1 QAVNLLOKA 1 0.003 :K7] RTSLVLNSJ ]F 0.003 Table XIV-V5-HLA-AI 101 -9mers 193PIElB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Start] Subsequence Score Table XIV-VI2-HLA-AII101- 9mers-i93PIEIB Each peptide is a portion of SEQ ID NO: 25; each sta 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 J Subsequence :Score I7] DGEESLLSK 0.012 I'l7 RALDGEESL 11 .0.0091 [2]I ALDGEESLL *0.004 5 iGEESLLSKY II '0.002 FF1 SLLSKYNSN ][o001] 1311 LDGEESLLS 7 ESLLSKYNS FO00 [6 .EESLLSKYN 11 -0.000 Table XV-VI-H1LA-Al1-1 Omersa 93P1E1B 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 startposition plus nine.

[tarfSubse uehce -Score I 39071 AVPPSKRFLK 6.000 LVSTNYPLSK 14.000 [37 RTPTPPEVTK] I 401 GQNIRDVSNK]1.800 -59 KYGYSPRVKK] j 1h] IMKIREYFQK ][T2J 7196 SLVKVLKTPK[a600I 200 1 VL14TPKCALK 00 1907 TPPTKQSLVK [OAO0 621 (ELSNCENFQKI 0.360 38671 ~IAKAVPPSK 10300 291 TPGLKIPSTK 1I200 286 VPTFCTPGLX1I5 0I 82 1 PVASSCISG- 02070 _39 RFLKHGQNIR 0.180 00 00 Table XV-VI-HLA-AII-lOmers- I93PIEIB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Start Subsuen Score 348 TISSYENLLR 0.160 46 DLH-SEVQTL( 0.120 143 MEKNSMDIMK 0.120 199 KVLTPKCAL 0.090 185 EPVIVTPPTK 0.090 I AIKAVPPSKR 0.080 F-38T]SNLATPIAIK 0.060 174 OEAINSDNYK 0.060 66 CENFQKTOVK 0.060 136 IKATKVLMEK 0.040 115 PPQAVNLLDK 0.040 156 YFQKYGYSPR 0.040 261 NVFATPSPII 0.040 232 LNEDYTMGLK 0.040 796 PQLSDFGLER 0.036 128 ENQEGIDFIK 0.036 238 0.030 .110 QVLPNPPQAV 0.030 117 QAVNLLDKAR 0.030 216 VPKLEHFGI 0.030 235 DYTMGLKNAR 0.024 F126 RLENQEGIDF 0.024 188 IVfPPTKQSL 0.020 193 TKQSLVKVLK 0.020 E MDPIRSFCGK 0.020 210 MDDFECVTPK 0.020 118 AVNLDKARL 0.020 285 LVPTFCTPGL 0.020 42 I(RILYDLHSEV 0.018 141 VLMEKNSMOI 0.016 16 STLDCETARL 0.015 145 KNSMDIMKIR 0.012 129 NQEGIDFIKA 0.012 219 KLEHFGISEY 0.012 132 GIOFIKATKV 0.012 .368 PEDILQLLSK 0.012 366 KIPEDILOLL 0012 295 KIPSTKNSIA 0.012 323 EVEDRTSLVL 0012 248 SEEAIDTESR 0.012 102 GLERYIVSQV 0.012 377 KYNSNLATPI 0.012 Table XV-VI-HLA-AI-l-Omers- 193PIE1B Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

FS Subsequence]Jf Score 264 ]1 ATPSPIIQQL 0.010 280 11 YTNSPLVPTF j 0.010 189 7 TPPTKQSLV 0.010 27 1 RALOGEESOF I 0.009 13171 EGIDFIKATK 0.009 F140 0.009 109 ISQVLPNPPQAII 0.W9 271 IQQLEKSDAEY 0.009 F231 CLNEDYTMGL 0.008 F-18 ]ILDCETARLQR 0.008 F38] NLATPAIKA 0.008 II 9 1[MLEYM] 0.008 F51 11 VQTLKDDVNI 0.006 373 QLLSKYNSNL I 0.006 [9 ][FQKTDVKDDLj 0.oo6 F-303 IALVSTNYPL 0.006 i] SLVLNSDTCF 0.0o FT2 I KARLENEGI jf0.006 1363 EVTKIPEDIL 0.006 F71 IKroVKoDSo 0.006 F157 FQKYGYSPRVI aoos 1165 IIRVKKNSVHEQJ 0A306I I5 T~]QKYGYSPRVKI 0.004] 126611 PSPIIQQLEK 0.004 1 269 IIQQLEKSDA I0.004 F28I PTFCTPGLKI 0 04 SIfSSOISGKSPR[ 0.004] I 3 I0.YPMRILYDL[ 004 F37LLS47 NSNLA 0.004 f55 LLRTPTPPEV 0.004 F--If PIRSFCGKLR 0.004 -296 IPSTKNSIAL 0.004 1J5 ASTLDCETAR 9I F IfQLSDFGIERY[ 0004 [207 [ALKMDDECV 0.004 F70]I SVHEQEAINS 0.004 318 SSNDLEVEDR 0004] L142 ]LMEKNSMDIM]J 0004 391 VPPSKRFLKH j 0.04 NLTDPSSPTI I0.004] [F KQSLVKVLKT 0004 T5D RYIVSQVLPN 0T004 Table X'-VI -ILA-AI1i Omers.

193PIElB 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 314 KTNSSSNDLE 0.003 Table XV-V5-HLA-A1IO1.

i0mers-193P1E1B Each peptide is a portion of SEQ ID NO: 11. each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Start ISubseuence Score 2 ]7 PVASSCISEK.] 5 1 SSCISEKSPR1 0.004 9 ][SEKSPRSPQLI 0.001 I 6 ][SCISEKSPRS 0.000 3] VASSCISEKS o.oo 1 PPVASSCISE 0.000 L7 CISEKSPRSP 0.000 1 8 ISEKSPRPQ 0.000 ASSCSEKSP If .O550 S10 EKSPRSPQLSJ[ 0.000] Table XV-V6-HLA-A101l0mers-193P1E1B Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Start H uencel Score 4 SEEAIDAESR 0.012 EI NNKSEEAIDA 0.001 7 AIDAESRLND 0.001 9 IIDAESRLNDN 0.001 10 AESRLNDNVFJ[ 0.001 5 EEAIDAESRL J.555] 3 KSEEAIDAES][0.000] I 8 ]FIDAESRLNDN 050* 2 I NKSEEADAEIf 0.000 6 EAIDAESRLN IM Table XV-VIO.HLA-AI 101lOmers.193P1E1B 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.

Start Subse uene Score] 13]WIYPTQKLNK 1.6007 KFQWIYPTQK 0.600] 1 IITKIPEDILQK 0.060 IIYPTQKLNKMRII 0.020 2 ]KIPEILQKF 0.012 11: FWIYPTQKL 0.012] 1i4] IYPTQKLNKM [0A304] T7[DILQKFQWIY If0X004] 8 ]LQKFQWIYPT] W.5T] 7[ ILQKFQWIYP 05T [4][PEDILQKFQ 0.000 =E QKFQWIYPTQ Q- W7 727 1QWIYPTQKLN 0.000 Table XV-V12-HLA-A1101l0mers-I93PIElB Each. peptide Is a portion of 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.

SStaF Subsequence Score 14] LDGEESLLSK 0.040 1 2 RALDGEESLLI 0.009 I SLLSKYNSNL 0.006 3. ALDGEESLLSI 0.001 1' IQRALDGEESLI 0.000 I 11 GEESLLSKYNI 0.000] DGEESLSKY 0.000 I7 EESLLSKYNS 0.000] !III ESLLSKYNSN 0.000] ITable XVI.VI.HLA.A24.9rers..

Each peptide is a portion of SEQ ID NO: 3; each start position is seiedthe lengh of peptide 1s9 amino acids, and the end position for: each peptide is the start I~trt ubs uece Score [212 DFECVTPKL 1146.200 (134 DFIKATKVL 30.000 Table XVI-VI -HLA-A24-9mers- 193PIE1B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end position for each peptide is the start position plus eight.

Startfl Subsequence Jj Score SFCGKLRSL 20.000 3 RFLKHGQNI II18.000 366 KIPEDILOL 14.400 314 KTNSSSNDL 14.400 367 IPEDILQLL 12.096 10 JKLRSLASTL 9.600 -35 Jj DFEDYPMRI ]9.000 18911 VTPPTKQSL I 8.640 39 YPMRILYDL 8.400 L265] PSPIIQQL ]I8.064 309] NYPLSKTS 7.500 [1Ti NPPQAVNLL ][7.200 L2 I EAIDTESRL ][7.200] KAVPPSKRF lii7.2001 AVPPSI RFL ][7.2001 [232] LNEDYTMGL ]f7.200 [161 ][GYSPRVKKN If6.600 [I9 1 SPOSOFOL 1 6.000 [Ti1] VNLIDKARL 116.000 ALVSTNYPL II6.000 [17Z] DAEYTNSPL (6.000 11811 NYKEEPVIV 16.000 288 IITFCPGLKI I 5.500 1 3 I DYTMGLI(NA! 5.000 [262 j[VFATPSPII ~j5.0001 1 i I EYFQKYGYS ~j5.000 224 ~(GISEYTMCL ]j4.800 I 4.1 DLHSEVQTL ]4.800 I2lJ11 ETARLRAL ]I4.800 I NSDTCFENL 114.800 3 ]I TISSYENLL ][4.800 [1491 DIMKIREYF ][4.200 VU(TPKCAL ][4.000 12][ VPTFCTPGL 114.000 f64[ VTKIPEDIL ][4.000 [17 I[TLDCETARL I 4.000] I 05] KCALKMDDF 1 4.0001 1174] IISKYNSNL II4.000 2951 KIPSTKNSI ~(3.600 330[ LVLNISDTCF II3.000 i 4I RNNKSEEAI ~[3.000 L?8i1 TNSPLVPTF 2.880 Table XVI-VI-HLA-A24-9mors 193PIElB Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight FSt~artl Subsequence Score r222 1 HFGISEYTM 1 2.500 1215 CVTPKLEHF 2.400 1255 ESRLNDNVF 2.400 KNSMDIMKI 2.200 [28 ALDGEESDF 2.000 [J12 ENQEGIFI 1.800 [140 KVLMEKNSM 1.800 [202 [KTPKCALKM 1.650 [F SNL3TP8I0 1.500 RYIVSQVLP ILV500I [142 LMEKNSMDI I .i0 [169 ]INSVHEQEAI IIto [80 DPPVASSCI 1.500 52 QTLDDVNI 1.500 377 KYNSNLATP 1.500 363 EVTKIPEDI 1.400 [341 LTDPSSPTI 1.200.

.137[ YNSNIATPI 1.200 1180]1 DNYKEEPVI 1.000 2171 TPKLEHFGI 1.000 1159 KYGYSPRVK 1.000 26[ NVFATPSPI 1.000 I W3 CFENLTDPS If0.900 351 SYENLLRTP 0.900 55 KDDVNIPEL 0.880 f MCLNEDY20] 0.750 I 38 11 DYPMRILYDIF 0.7507 I322 11LEVEDRTSL ][0.720 F3I TKQSLVKVL 0.720 1i PNPPQAVNL 110.720I rT ERYIVSQVL 0.672 F70 QKTDVKDDL 0.672 227 MCLNED 0.660 347 PTISSYENL 0.600 I33f ESEDYPM 0.500 [744 LYDLHSEVQ 0.500_] FIKATKVLM 1 0.500 2779 ENSPLVP 0.500 100 DFGLERYIV 0.500 1111 GKSPRSPQL 0.480 1 r PIRSFCGKL 0.440 VEDRTSLVL Table XVI-VI -HLA-A24-9mers- 193P1E1B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the start position plus eight.

start[ Subsequence ore [71 PSTKNSIAL[ 0.400 7 [1 EDYPMRIL ][0.400 152 11 KIREYFKII 0.380 91 KSPRSPQLS 0.360 2571 RLNDNVFAT 1 0.360 1199 1 'KVLKTPKCA 0.300 F357 RTPTPPEVT 0.300 f11 RSLASTLDC I1 0.300 r127 LENQEGIOF 0.300 F16781 KNSVHEQEA] 0.264 209- 1- KMDDFECVT IF 0.240 [185 EPVIVTPPT 0.210 1 287 21NSPLVPTFC 0.210 [4II HGQNIRDVS II0.210 1 Table XVI-V5-HLA-A24-9mprs.- 193P1EIB Each peptide is a'portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.__ Fstart IStart Iubse uence Score II( EKSPRSPQL 0.480 F-3 ASSCISEKSI 0.154 6 CISEKSPRS 0.120 [liii ISEKSPRSP][ 0.015 [II] SCISEKSPR[ 0.015 F2 3VASSCISEK 0.011 SSCISEKSP ]0.010 F 78SEKSPRSPQ1 El IPVASSCISE 0.001 Table XVI-V6-HLA-A24-9mers- 193P1E18 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start osition lus eight.

Start jSubsequence Score SEAIDAESRL 7.200 Table XVI-V6-HLA-A24-9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start FSubseguencej Score 8I1DAESRLNDN r 0.180 7[ AIDAESRLN F 0.100 1 2 7 KSEEAIDAE I 0.036 3 F SEEAIDAES 0.023 IIIAESRLNDNVI 0.0121 il NKSEEAIDA If0.012 [71 IDAESRLND I 0.001 7 EEAIDAESR 0.001 Table XVI-VIO-HLA-A24-9mers- [93PIElB 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 pepideis the start position plus eight [Start [Subsequencej[ Score I 1T If QWIYPTQKL If7.920 2 I IPEDILQKF If6.653 5 DILQKFQWI I 2.1603 I13 11 IYPTQKLNK[ 0.750] [14 II YPTQKLNKM If0.6601 [91 KFQWIYPTQ II0.210 SII ILQKFQWIY I 0.150 1211 WIYPTQKLN 0.120 F1 I KIPEDILK 0.036 LT 1[EDILQKFQW I 0.0151 FTI LQKQWIYP 310.010 [8[o QWlWYPr3 0.010 IZ I][FQWIYPTQK] 0.010 FiflPTQKLNKMRI 0.002 I 3 PEDILQKFQ I 0.000 Tb XVI-VI 2-H LA.A24-9 mers- F al 93PEB Each peptide is a portion of SEQ ID NO: 25; each1 start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start psto lsegt [Start lSubse uencel Score Table XVI-Vi 2-HLA-A24-9mers- I93P1E1B 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 Start ISubsequenceJI Score 1 RALDGEESL 14.400 72 ALDGEESU.. 31 4.000 1 8 1SLLSKYNSN]II 0.1807 7 ESLLSKYNS F 0.150-1 F5 GEESuLS 0.020 [II]DGEESU~SKI 0.018 E63EESLLSKYNI 0.012 K IfIDGEESLIS 3 0.012 [Table XVl-V1-HLA-A240mers- 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 pstoplsnine.

Start ]subsequence Score] 38 ]fDYPMRILYDL 1420.000] I377 ][KYNSNLATPI 180.000] 135 ][DFEDYPMRIL 36.000] [I67i KIPEDILOLL .24.192] [IiI I RYIVSQVLPN 11.15.0001 38 I KAVPPSKRFL 31 4i4o 194 IRSPQLSDFGL 120003 f199 [KVLKTPKCAL3120001 [2643 ATPSPIIQQL 31 .08o [3V 1 SYENLLRTPT[ .9QJ I [31 NYPLSKNSS[ *955 I 1161 GYPVKS[8.400 3] RSFCGKLRSL[ 8.000 I231 CLEDTGLI200I I16 SILOCETARLf 7.200] 112 LPNPPQAVNL[7200] [323 EVEDRTSLVL 7.200 27 RALOGEESF 7.200 F DPIRSFCGKL 6.600 [227 EYMCLNEDY 6.000 T181 NYKEEPVIVT 1 6.000 321 IIDLEVEDRTSLI 6.000 [TT I AVNLLDKARL 6.000 373 31QLLSKYNSNL 6.000 11 RLENQEGIDFN; 6.000 Table XVII-VI -HLA-A24-1lOmersi93PIE1B 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 startyosibon plus nine.

Start IIsubsequen Score [285 LVPTFCTPGL 6 .00 223 jj FGISEYTMCL7F 6.000 303 j IALVSTNYPL 56.000 F 188 IVTPPTKQSL 1 567601 32 LNSDTCFENL 77 FQKTDVKDDL 5.600 44 TILYDLHSEVQT1F55] [2T I EYTNSPLVPT ]f5.000 1 296 IPSTKNSIAL 4.000 F3673 EVTKIPEDIL 4.000 89' ISGKSPRSPQL 4.000] F4If SPTISSYENL ][i0] 134 DFIKATKVLM T7IT] 280 YTNSPLVPTF 3.600.1 JfIPELSNCENF ]L001 I214.1 ECVTPKLEHF ]3 .050j 329- SLVLNSDTCF .000 -24] ARLENQEGi2.000] 92 -]SPRSPQLSDF [.65] KNSVHEQEAI 2 141- VLMENSMDI .800] 260 DNVFATPSPI 1.500 1379 NSNLTPIAI 1.500 216 VTPKLEHFGI IFT0 F TPPEVm ][T2 NLTDPSSPTI 301.200 F 8] LSDFGLERYI 1.200 F si VQTLKDDVNI 1.000 __61 NVFATPSPII 11.000 S13 PNPPQAVNLL 0.864 S]337 CENLTDPSS[ 0750 142 LMEKNSMDIM 0.750 2-1 iT]1oFECV]TPL I I F9 7 GKLRSLASTL 7 20 V 7 TKIPEILQL [0.720 34 I PTISSYENLL 0.720 1- YDLHSEVQTL 457W F DYTMGKNAR 0.720 [103 LERYIVSQVL 0.672 L.6 SFCGKLRSLA 0.600 TI KSEEAIDTES 0.554 Table XVI -VI -HLA-A24.lOmers1 193P1ElE 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.

jtart Fs ubsequencelj Score 54 LKDDVNIPEL IF .W] 155 EIYFQKYGYPII 0.500 222 IHFGISEYTMCI 0.5001 Th1 0 DFGLERYIVSII f.500 229 JIILEMI asooi 276 SAENSPLII 0480 20 fCETARLQRALIj 0480 192 IIPTKQSLVKVL f 0.480 313 IfS NSSNDLI 0480] 148_ MDIMKIREYF 133 IDFIKATKVL 0.400 249 EEAIDTESRL 0.400 42 RILYDLHSEV 0396 219 KLEFGISEY 0.330 295 ]7 KIPSThNSIA II 0.300 137IlcArKv1EKN]I 0.264 300 KNSIALVSTN II 0.240 1 254 ]ITESRLNDNVF] 0.240 395 KRFU(HGQNI 0.240 327 RTSLVLNSDT 0.240 63 [6LSNCENFQKT 0.238 194 KQSLVKVLKT 0.220 294 LKIPSTKNSI 0.216 110 QVLPNPPQAV][ 0 216 367 IPEDILQLLS 0.216 30 ][DGEESDFEDYIf 0.216 102 GLERYIVSQV 0.210 301 NSlALVSTNY 0.210 388 IKAVPPSKRF 0.200 271QQLEKSDAEY][ 0.198 [9 ][NIPaLSNCEN] 0.198 ~1f NQEGDFI198 H2o i NLLDKARLEN 0.198 ble XVIIV5-HLA.A24- mers- T 93PE1B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

SStart ISubs quence Score ITable XVII-V5-HLA-A24-1 Omers] I93P1ElB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Start Subsequence Score F9]SEKSPRSPQL]D r040] T lVASSCISEKS][5 [6 I SCISEKSPRS7S 0.150 8 IIISEKSPRSPQII[ 0.015 7Th IIEKSPRSPQLSI 0.014] 7 I CISEKSPRSP 0.012 [4

T

jAsscISEKSP] 0.010 L..SJSSCISEKSPR[ 0.010 [Z I PPVASSCISE] 0.002 PVASSCISEK][10.001] Tb XVII-V6-HLAA24-0mers- 1 Each peplide is a portion of SEQ) ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nin.

Start ]f Subse uence fj Sco] 13 ]IKSEEAIDAES 1 0.554 5 ]fEEAIDAESRL .00 io ]AESRLNDNVF 0.240 6 EAIDAESRLN 0.180 9 ]DAESRLNDNVj[ 0.1801 1 ]NNKSEEAIAI[.105] 8 IDAESRLNDN j[0.014 I7 ]AIDAESRLNDj[ 0.010 I4 ]SEEAIDAESR II 0.002] I2 ]NKSEEAIDAE II Table XVII-VIO-HLA-A24- I Omers-193P1 El B 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.

[tart Subsequence Score [14 IYPTQKLNKM][49.500 [F KIPEDILQKF 1153 6 [11 IFQWIYPTQKL[ 5.280 [1111 EDILQKFQWI[ 0.216 Table XVII-VIO-HLA-A24- 10mers-193P1ElB 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.

Start Subsequence Score jY PTQK 0.150 671 DILQKFQWIY I 0.150 12QW1YPTQI LN 0.150 87 ILQKFQWIYPT 10.100 3 11 IPEDILQKFQ 0.022 II ILKFWIYP 1[ 0.015 Th [IYPTKLNKMR 1 0.012 13 IWIYPTKLNK 0.012 11 I TKIPEDILQK 1 0.002 9 QIKFQWIYPTQ1 1 0.001 4 PEDILQKFQW 0.001.

Table XVII-V12-HLA-A24l.10mers-I93PIEIB Each peplide is a portion of 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. Start. Subsequence [Score 2 IRALDGEESLLI[ 14.400 91 ISLLSKYNSNLIJ[ 6.000 RALDGEESL F 0.400 7 DGEESLLSKY 0.238 .ESLLSKYNSN I 0.180 .j IALGEESLSI 0.100 G GEESLLSKYNI 0.018 I7 IEESLLSKYNSI 0.010 .4 ILGEESLLSK 0.001 Table XVII-V-HLA-B79mers- 193P1E18 Each peptide is a portion of SEQ ID NO: 3; each start position is specfied, the length of peptide Is 9 amino acids, and the end position for each peplide is the start position plus eight.

Table XVIII-VI-fLA-B7-9mers- 193PIlB 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 FSubseguen Score 286 VPTFCTPGL 80.000 390 AVPPSKRFL 60.000 [i J KLRSLASTL ][40.000 V1 SPRVKKNSV P 000 367 1IPEIF2L4 0-00 304 11 ALVSTNYPL 12.000 2 ]0 EAIDTESRL ][12.000 7 TPKLEHFG ][8A2000 [21I VLKTPKCAL][.000 364 VTKIPEDIL 6.000 189 IIVTPPTKQSL[ PIRSFCGKL[ 4.000 S314 ]KTNSSSNDL][4.000] S374 I LLSKYNSNL[ 4.000 I GLSErMCL][4A00I [Th TPPTK<QSLV] T.00 =11 VNDKARL[4.000 [366 KIPEDILOL E S46 jI DLHSEVQTL] 4.000 [348] TISSYENLL] 4.000 [1 1l ETARLQRAL][4X00] S277 IIDAEYNSPL][3.600] 2 ]I SPRSPQLSD] 3. 1 S283 ]ISPLVPFCT j3.000I [185 IIEPVIVTPPT ]2.000I [26F] NVFATPSPI11 2.000 IPSTKNSIA[ 2.000 [3W 1 EVTKIPEOI 12.000 S291 ][TPGLKIPST ~[2.000 12T][LNEDYTMGL 1.2oo 17] TLDCETARL 11.200 333 ]NSDTCFENL 1.200] I 0 s ]EVQTLKDDV [1.000] fl3][FIKTKVLM L.0] I3 ~W ]MCLNEDM 1.000] j 0T ]KrPKCAIKM [I1.000] ATPIAIKAV j[0.600] 343 ][DPSSPTSS II0.600] I1 TT]LPNPPQAVN II0.600] [52 LEEDRTSL j[0.600 Table XVIII-VI.HLA.B7-9mers.

I93P1E1B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.__ Fstart Start jSbeuneScore 1 110J~ QLPNPQAJ[0.5007 S199 I KVLKTPKCA 0.500 22 J[ TARLQRALD]I0.450] 113 PNPPQAVNL][ LDK 170 jj QKTDVKDDL[ 0ii0] 297 11PS'IhNSIAL ][51] 145 J[KNsMDIMKI][I 0.4001 [169 11 NSVHEQEAI ][-0i4] 347 IfPTISSYENL J[0.400 1 90 II GKSPRSPQL][ 0.4 1134 11DFIKATKVL 310 JYPLSKNSS][0.400] 346 IISPTISSYEN ][401 378 ~[YNSNLATPI 6 I2SFCGKLRSL] 0 0.400 193 J[ TKSLVKVL [180 11DNYKEEPVI ][*5.55J 1295 11KIPSTKNSI ][0.400 [244 11RNNKSEEAI I 104 J[ERYIVSQVL] 0.400 1128 11ENQEGIOFI ][10.400I 380 ][SNLATPIAI 0.400 152 11 QTLKDDVNI [0.400] 1323 11 EVEDRTSLV] 0.300 117 1IAVNLL][W3I I15 JjASTLDCETA[ 0.3001 I33 IESDFEDYPM] 0.300 I382 J~LAPIAIKA ][0.300 242 JNARNNKSEE] 0.35] S207 Jl ALKMDFEC][ 0.300 I VLPNPPQAV~J 0.301 391 ]j VPPSKFLK] 0.3001 124 KARLENQEG 0.300 I358 ]I TPTPPEVTK]IjO.300] I14 ]~LASTLCET 110.300 I384 ]ITPIAIKAVP '020 I 360 ]1 TPPEVTKIP 11 0.200] I2 ]fDPlRSFCGK[10200] I22J STKNSIALV] 0.200] I j2 KIREYFQKY][0 200] I43 11ILYDLHSEV ]j0.200 203 TPKCALKMD] 0.200 Start ]ISubsequenIc score III YPMRILYDL 11240.000 75 ]SPIQL ]180.000 ThT NPPQAVNLL ]80000 SPQLSDFGL II80.000 Table XVIII-V-HLA.B7-9mers- 193P1ElB 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.

SStart IjlubsequenceJ Scre Start jcou 316 NSSSNDLEV 267 11 SPIIQQLEK 0.200 103 LERYIVSQV 00 [255 ESRLNDNVF [0.

[3F If1FEDYPMRIL 1 LLRTPTPPE 0:150 [280j YTNSPLVPT ]j0.150 IDVNIPELSN I 0.150 Eac pe NLTDPSSPT i 0150 7 1 FCGKLRSLA I 0.150 IDO I AVNLLDKAR 1t0150 STNYPLSKT I0.150 Table XVII I-V5-H LA-87-9me rs- I93PEIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Start Subsequence Score 9 EKSPRSPQL I 0.400 T711 ASSOISEKS II0.060 VASSOISEK 10.030 67[ CISEKSPRS 0.020 5_1 SCISEKSPR 0.010 411 SSCISEKSP 0.010 1 PVASSCISE 0.005 7ISEKSPRSP 0.003 81 SEKSPRSPQ 0.002 Table XVIII-V6.HLA-B7-9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start positon plus eight.

Start Subsequence Score EADAESRL 12.000 AESRLNDNV 0.060 T 1 AIDAESRLN 110.018 Table XVIII-V6-HLA-B7-9mers- 193P121B Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight [Startll Subsequence If Score 87 DAESRLNDN jj 018 F NKSEEAIDA 0010 F-2] KSEEAIDAE 0.003 7 IDAESRLND I0.002 14_] EEAIDAESR If oo T SEEAIDAES f 0.001 STable XVIII-VI 0-HLA-B7-9 mers- I93IEIB 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.

taj Subsequence 31 r []1i4 YPTQKLNKM 320 00 11[ _QWIYPTQKL T6[0iL] DILQKFQWI (400 M IPEDILQKF 110.120 WIYPTQKLN73 00-20 6 ILQKFQWIY ][07' E1_ QKFQWIYPT 0:010 LQKFQWIYP 31 0.010 FQWIYPTQK 0.010 1 KIPEDILQK 10(0010 F14-][ EDILQKFQW 3-0002 EI] YPTQILNK I[00T L 15 PTQKLNKMR[ 0.001 Ii]_ KFQWIYPTQ 3[ 7: PEDILQKFQ ][000 Table XVIII-V12-HLA-B7-9mers- IT 193P1E18 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 osition lus eight.

Start Subsequence Score f RALGEESL 12000 2 ALGEESLL (3.600 EL] ESLLSKYNS (0.020 SLLSKYNSN 10.020 SDGEESLSK If0.003 LDGEESLLS j[:0.002 6 i3[ EESLLSKYN 1f 0.002 F5[ GEESLLSKY If0.00 1 Table XIX-VI-HLA-B7-Omers- 193PE18 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 SubsequenceScore 296 IPSTKNSIAL j 80.000] 2 I DPIRSFCGKL j80.000 112]LPNPPQAVNL 346 SPTISSYENL 80.000 118 AVNLLDKARL .60.000 3631 EVTKIPEDIL J30.000] 199f KVLKTPKCAL 1 30.000 18781 IVTPPTKQSL -1 20.000 2851 LVPTFCTPGL ]F 20.000] 303 1 IALVSTNYPL 12.000 389 KAVPPSKRFL I112.000 264I ATPSPIIQQL 1112.0001 1241KARLENQEGI 1112.000 358 TPTPPEVTKI j '8.000 323 EVEDRTSLVL I 6.000 231 CLNEDYTMGL 4.000 366 KIPEDILQLL I4.000 M I LERYIVSQVL' ].4.000 I _FQKTDVKDDL 4.000] 92 1[ SPRSPQLSDF A4.000 94 If RSPQLSDFGL jI4.000 51 RSFCGKLRSL] 4.000 223 FGISEYTMCL 4.000 332]I LNSDTCFENL IF '4.000 16 1 STLDCETARL 4.000 89 If SGKSPRSPQL 114.0001 373 I QLLSKYNSNLII 4.000 261 NVFATPSPII 3.000 242 I NARNNKSEEA ]F 3.000 355 LLRTPTPPEV I 0001 163 ISPRVKKNSVH ]1 2.000 32 I DLEVEDRTSL ii F1800 110 QVLPNPPQAV I1.50 14 VLMEKNSMDI 1 255 ESRLNDNVFA 111.000] 229 TMCLNEDYTM 0 0 Table XIX-VI-HL-A-B7-Irmers.

193PI El6 Each peptide is a portion of SEQ ID NO: 3; each start position i specified, the length of peptideis amino acids, and the end position for each peptide is the start position plus nine.

Farial Subsequence ]jcr [2-7 If ALKMVDDFECV ]j0.600 -3fiI LATPIAIKAV .F0.600] 1 39-11 YPMRILYDLH IL 0.600 F-5771 DVNIPELSNC I__ I 97i1 LVKVLKTPKC 07 ]f IDFIKATKVL 0.4700 379-11 NSNLATPIAI T__407 F2- ll TPKLEHFGIS 19 111I PPTKQSLVKV ]F 0.400] lRE-11 TKIPEDILQL 1[] r 34 31 DPSSPTISSY II040 GKLRSLASTL ]F-.4-00 1l6-8]1 I NSVHEQEAl If0.400] I -76]l SDAEYTNSPL j .40 F DYPMRILYDL IFI00 L-21-]f DDFECVTPKL ]F 0.4001 45]1 YDLHSEVQTL IF 0.400 1 VQTLKDDVNI ]F-0.400 [F CETARLORAL 0.400 [2-60 II DNVFATPSPI .0 192 If PTKQSLVKVL II 0.400 1 F313 SKTNSSSNDL J 0.400 1 F340 if NLTDPS-SPTI ]F0.400 I 267II SPIIQOLEKS ]j 4-00 1113 1] PNPPQAVNLLI]. 0.400 124911l EEAIDTESRL ][0.400 1.801 DPPVASSCIS[ 0.400 1 S3-47]11 PTISSYENLL ][F0.400 1 F3-10]l YPLS KTN SSS ]f 0.400- S228 ]1 YTMCLNEDYT ]f20 F-14 ILATLDCETA If 0.300] f-722[TARLQRALDG jo30 [j-4]l LMEKNSMDIM 11KODE 0.300~ F3-97 AVPPSKRFLK .2 F2-8 If SPLVPTFCTP .0 LQRALDGEES ]~0.20 1 291 I1 TPGLKIPSTK J 0.200 1 [265 II TPSPIIQQLE ]F0.200 Fh jf 5 EPVIVTPPTK Ij020 U0_ DYEPI 110200]1 Table XIX-VI-HLA-87-1 Omers- 193P1EIB 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.

[S ta-rtI Subsequence Scor [2-073 TPKCALKMD][020 [2-876 VPTFCTPGLK I [40]1 NIRDVSNKEN F37 1VPPSKRFLKH .307 I-891 VTPPTKQSLV If55 F1174[ NPPQAVNLLD IFO-- 0-~ 3874 I[ TPIAIKAP w] 0 1 42 ]f RILYOLHSEV .0 F-9571 SPQLSDFGLE 1 .0 3151 TNSSSNDLEV If 0.200 1 1l901 TPPTKQSLVK If 0.200 [M1T FQKYGYSPRV If_._ F367 TPPEVTKIPE I .0 Ii~f YPRKKNSV I .0 I 35 I DFEDYMI IF0. 1-80 12-7 7 1f DAEYTNSPLV If.8 1306 If VSTNYPLSKT If 0-150- [3-351 ENLTDPSP I 0.1-50.

[2-8211 NSPLVPTFCT1 0150- [i6-011 IPELSNCENFII 0.1-20-1 [-2-4-31 ARNNKSEEAI ]F 0.120 [[361 -IPEDILOQLLS .2 Table XIX.V5HLAB7-0mers.] I93PIEIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the satposition plus nine.

I t~arf S ueene If cr [191] SEKSPRSQ It0.400 I VASSCISEKS If .6 IETLt ASSCISEKSP [if 003 1 66I1 SCISEKSPRS II 0.020 FF11 PPVASSCISE II .2 F511 SSCISEKSPR 11 .1 F-7]1 CISEKSPRSPI .1 I7 IISEKSPRSPQ If 0.007 2 IPVASSCISEK I .0 f~IEKSPRSPQLS II 0002 ITable XIX.V6HLA-B7-l0mers.1 I193P1El B Each peptide is a portion of SEQ ID NO: 13; each start position is secified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[S-a rtJ[ Subsequence][cr [T5I1 EEAIOAES-RL I[ 0.400 DAESRLNDNV][ 0.1l8-0j [T I NNKSEEAIDA ciioo F-671 EAIDAESRLN 0060 [T771 AIOAESRLND 0.013~ D ]1 AESRLNDNVF FT]I KSEEAIDAES If 0006- W fIDAESRLNDN I[T002- 2 fNKSEEAIDAE I[00 4ET] SEEAIDAESR]I ooI Table XIX.VIO-HLA-B7-1 Omers-] Each peptide is a portion of SE Q ID NO: 21; each start position is specified, the length of peptide is 10 amino acids, and the end position for each pepide is the start position plus nine. I SSt a rt Ij Subsequence If Score I I-1 ilj FQWIlYPTQKL If-66566I II DYTKLK F1 TOO I F18][ LQ KIFQ WIY PT If F o 0-00 MI[ IPEDILQKFQ .11100601 EDILQ FQW O- 0040 IfDILQ FQWY If0.020 T fKIPEDILQKF F. 0-0-2 0 1=3 1 WIYPTQKLNK1 .1 MT ]fLQKFQWIYP 12][l QWIYPTQKLN 1[ 0.002 r-9 QKFQWIYPTQ IF 0.001 [1 IKFQWIYPTQK ZIfTKIPEDILQK 11 0.00 M4PEDILQKFQW ][51~ Table XIX-VI 2-H LA-B7-10mers- 193PiEIBI Each peptide is a portion of SEQ1 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. I 00 00 l~tartl Suse 1ec 7ral [1271 RALOGEESLL 1112A7001 SLSYNN 9 174000I F1I1 QRALDGEESL ]FO0.400 F 8 SLSYS 10.2 F371 ALIDGEESILLS ]F0.018 F- 5.1 DGEESLL SKY jF 0006-1 F771] EESLLSKYNS j0.2 F 4I1 LDGEESLLSK GEESLLSKYN oai Table XX-I-1-L1A-B33501 .9mers-1 1 93P1E1B Each peptide is a portion of SEQ NO: 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.

[F!St 7r Subsequence ][--co7r 1 391 YPMRILYDL1] 000 1 '95~ I SPQLSDFGL F20.000] [TT]j NPPQAVNLL 20-I00 [286] VPTFCTPGL I20] [7267f TPSPIIQQL ]I2.00 F551ESRLNDNVF ]I 5A00] 1-16 3 1 SPRVKKNSV I1.0 I j I IPEDILQLL IIT5o L-85I1 DPPVASSCI ]F-8-050] I EAJDTESRL I100 -61 KIPEDILQL 11&00 F-14 KVLMEKNSM I[AJO L!ji FIKATKVLM ]F-6.000] 9JJ1 KAVPPSKRF 1 1 1 KLRSLASTL 6.000 [-3731 ESDFEDYPM II 4.500 F-2072 KTPKCALKM II 4.000] F1901I TPPTKQSLV II 4.000 I1 F--2730 MCLNEDYTM 3.000 F3_4I] VTKIPEDIL 3.-000 200] V lKTKA I 169][ NSVHEQEA l[ 3001 F307][ SIALVSTNY 1 F2976 IPSTKNSIA 550] F12[LPNPPQAVN ]2[0-070 L DILQLLSKY If~ Table XX-Vl.HLA-B3 501 .mr.

193P1EIB 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 I start position plus eight. [st a RtI Subsequence I S-co-re] [2-271 GISEYTMCL 1 [.000] EPVIVTPPT 2.000 YThiOLNEOY If [321741 KTNSSSNOL [3461[ SPTISSYEN [20 KCALKMDDF 0 I [291] TPGLKIPST 2.0-003 [343][ DPSSPTISS 2.00 0 [-312[ LSKTNSSSN ][T-00 DLHSEVQTL 1f 1.500 VNLLDKARL ][T15070 NSDTCFENL ][TI5] [3j75]1 LSKYNSNLA I.1.500 [1545] KNSMDIMKI 1.0] RSFCGKLS I1000-] F-21i]I CVTPKLEHF7 I 1.0-00] [3_9fl AVPPSKRFL 1.T0001 304 AVTPL 1.000 Fl-891 VPKQLFt 1.000-6 LiF-1 SSELI[. 1.000 F-14 9 DK IRE FT 1000- F374 jLLSKYNSNL 11.000 71W NFNlEV 1000 L3-3 -0 LVLNSDT F 1.00 0.

F3-474 PSSPTIS 1.000 F3-41 TISSYENLLIl.0 1 FilKSRPQS -IA000_ [281 TNSPLVPTF r[27][ ETARLQRAL I[i.5 [2i7 DAEYTNSPL 1[~W F24]4 RNNKSEEAI J o F2i5- IPNQII ]1 0.800 F-15- ASILDOETA [0.75 [272][ QLEKSDAEY 070] IPELSNCEN 0I 0 r-43 MEKNSMDIM 0.0 18 I[ DNYKEEPVI I[ 0.600I 1 iI QTLKDDVNI JF-060] I[T] SPRSPQLSD Table XX-VI -HLA-B3501 -9mers-j I93PIEIB_ 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 eiht Start I Subsequence] 7232:] LNEDYTMGL_ 203 tTKAKD] T797 1tNSLAPI I0.500] 328]1TLL O I F0. 500] 297]1t PSTKNSIAL 1f050 -3701] NSIALVSTh IF- 0-5o0] 2_8 fj NSPLVPTFC G so 7195 QSLVKVLKT ]F -05]0 17l1 TLDCETARL 1. :28]1 ALOGEESDF 1f0.5 207]} ALKMDDFEC L.s 275 ]1 KSDAEYTNS: 0.450W 261 II NVFATPSPI II 75.*50- [lT4 1 REYFOKYGY 11 -0.400 [331 EVTKIPEDI -t0.400 fjTPPETIPI040 [EY 11 ILYDLHSEV II0.0 [38] YNSNLATPI. 11-0.400 [27] RLNDNVFAT J 0.4-00 38 1IAIKAVPPS: II 0.300 CGKLRSLAS_1030 117[QAVNLLDKA I030 Fl-4-1 LASTLDCET 030 ETjPIRSFCGKL JI 0.300 ITable XM--.A35011:9mers- Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide'is the start position plus eight.

I--arl Susqe c oe [T 1 1ASIEK If.5-00 LW](1 CISEKSPRS IFO 0200 911 EKSPRSPQL ft 0.100 L 141 SSCISEKSP IF 0050 F-2-11 VASSCISEK II 0030 [E11] ISEKSP RSP ft 0.15 00 00 Tlable XX-V5-HLA.B3501.gmers1 I93P El B Each peptide is a portion of SEQ ID NO: 11;- each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight._j [Starij[ Subsequenc-e TS-core 1 [ili]j SCISEKSPR h O0015] SGKSPRSPQ jj000 PVASSGISE I .0 f Table XXV6-HLA-B3501k9mers.] Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position -plus eight.

[Start I Subsequence Score 1 -5i EADAESL ][6500 8 DAESRLNDN II 090] KSEEAIDAE Ij006.

6_ AIDAESRLN 05 F-1ll NKSEEAI.DA I .3 F-91 AESRLNDNv UIoAJ2O F-7371j SEEAIDAES ]I 0.003 I 7 j IDAESRLND 1 .0 f IEEAIDAESR .0 Table XX-VIO-HLA-B3501- 9imers-1193P11l Each peptide is a portion of SEQ ID NO: 21; each s itart 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.

[S-taIl Subsequence 1 cr F141 YPTQKLNKM 1140.0001 F2]l IPEDILQKF 12.0-00] F 6I1 ILQKFQWIY 2A00] 1 51 D[IILQKFQW1 0.400 F 1[_WIYPTQKLN 10 KIPEDILOK 110.060] 4T] EDILQKFQW 0.050 LQKFQWIYP ]1 0.030] FOWIYPTOK 155-17] 8 QKFQWIYPT 0.OX10] 1311 KFQWIYPTQ 1>02 [1 13 1 IYPTQKLNK 0.0 F1571 PTQKLNKMR .0 L~l PEDILQKFQ p j FTable XX-V12-HILA-B3501- 9mers.I 93P1E1 B Each peptide is a portion of SEQ ID N0r 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peplide Is the start position plus eight.

Istart:1 Subsequence lsoe I-171 RALDGEE-SL 11.0 I 7 1 ESLILSKYNS F 0.500 I ALDGEESLL7II 0450 EL~] SILLSKYNSN II 0.100 I II5i1 GEESLLSKY If0.060 I 37 ]I LIDGEESLLS ]f~3 a]1 EESLLSKYN IF 0. 010 I IED1 DGEESLLSK F 0.006 [Table XXI-VI-HLA-B3501i0mers-I93PIEiB 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 TS-core [92 ]1 SPRSPQLSOF[I 60.000 [34731 DIPSSPTISSY 000 F346[ SPTISSYENL I2-0.0001 1I 2[ DPIRSFCK 1200] [-112I[ LPNPPQAVNL j [0.00 [i LI RALOGEESOF ]Fi-80-]0 13-85[ TPTPPEVTrK~i [5o [F i If NSIALS FN -10.000] [94]I RSPLDG 10.000 1 5l1 RSFCGKRLI F1 411 KARILENQEGI 1 7.2001 f389 11 KAVPPSKRFL f -00 217 I TPKLEHFGIS 16.000] q IPELSNCE F 16000-1 F271 I QQLEKSDAEYIF5] F36611 KIPEDILQLL ]F.40001 r97-11 QLSOFGLERY I .0 I 303 I~ ALVSNP F 3.0-00 189 ]1SKPSQLI .0 169 I FQKTDVKDDL 1300 Table XXI-V1.HLA.B33501.1 I Omers-193P1 El B 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.

Stat Sbeuence

I[

22] STWDCETARLl ][.000 2551 ESRLNDN F 2250] :1:0]1 YPLSKTNSSS F T1 KVLKTPKCAL 11200 231][ CLNEDYTMVGL 2000 1 3321 LNSDTCFENL 2.-000 I 37911 NSNLATPIAI 2.000 8011 DPPVASI 267 SPIIQOLEKS 1 .0 3:0f[ DGEESDFEDY]1 .0 37751I LSKYNSNLAT 11500_I AVNLLDKARLI 11.500 I 1681 KNSVHEQEAI 111.200-1 367]1f IPEDILQLLS 151.200 1 219 KLEHFGISEY 11201 718781 IVTPPTKQL] .0 28011 YTNSPLVPTF~J 00 32 1i SLVLNSDTCF 11 {.507] 264] If ATPIIL 11.0001 7223:] FOISEYTO I[ -1 -0 0 373]1 QLLSKYNSNL Ii.

2851I LVPTFCTPGL 1100 i:1E]1 ECVTPKLEHF j[ i-oo 9 0 1261I RLENQEGIDF _I090 2071 IIALKMDDFECV If 0.900 242T] NARNNKSEEA 11 0.900 I 201EAIDTESL IF0 (9 00 I 3 40[1 NLTDPSSPTI I .0 1141111 VLMEKNSMDi TIoF .00 F21-6][ VTPKLEHFGI If 0.600 1 F32311 EVEIDRTSLVIL IF- 0.600 1 [T47 11 SMDIMKIREY If060 [iT 1 EESDFEDYPMJ 0.600] [1571I FQKYGYSPRV][0.0 F2o03]1 TPIKCALKMDD] 0.6001 F571 ]IVQTLKDDVNI ]L [14721 LMEKNSMDIMl 0.600 1 Table XXI1-VI-HLA-B3501- I Omers-193P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

[Start] Sbeune ][Score 403-I NIRDVSNKEN 0.600 247_ KSEEAJDTES F -6655 98 )[LSDFGLERYI 0600 173[ EQEA N SONY 0r 0600 355][ LLRTPTPPEV ]~0.600 163] SPRVKKNSVH If0.600 306_ VSTNYPLSKT If0.500 28211 NSPLVPTFCT If 050 0 345I SSPTISSYEN 1050 1169 JjNSVHEQEAIN jj0.500 [3278 TSLVLNSDTC 0:500o [49. ISSYENLLRT jL5-0o] [63 LSNCENFQKT]050 LQRALDGEES [77 14 1LASTLOCETA 206]1 CALKMDDFEC] 0.450 31DLEVEDRTSL]I4 5[ 0.45 12601] DNVFATPSPI NVFATPSPII f0.0 ][471 RLYDLHSEV I .0 [191 IfPPTKQSLVKV 1 .0 360.PPVTIP 0.400 [1381 ATKVWMEKNS [0.30 298: STKNSIALVSJI000J [180 j[DNYKEEP.VIV ][0.300 192-[ PTKQSLVKVL ]0.3005 17 SVIIEQEAINS 0.300 I197][ LVKVLKTPKC ]1 0.300 2391 GLKNARNNKS][ CGKLRSLATi ~o F Table XXI.V5-HLA-B3501- I I mers-l 93P1 El B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

1[i!J[ SubseqIencre SEKSPRSPQL[ 0.300 FT3[ VASSCISEKS][ _6 SCISEKSPRS VT~ [s SSCISEKSPR ]F 07-51 [T4j ASSOISEKSP 1I 0.056o [F7]1 CISEKSPRSPI f2 11 1 IF PPVASSCISE I .2 jIi] ISEKSPRSPQl .1 0:0:11 EKSPRSPQLS 15T L VASSCISEK Table XXI-VG-HLA-B3501..

I Omers.193P1 El B Each peptide is a portion of SEQ ID NO: 13; each start'position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

[L§!rt Subseuence MT EAIDAESRLN fo oI L J NNKSEEAIDA II-9I DAESRLNDNV If0.180 I~IEEAIOAESRL 11 .0 II1AESRLNDNVFj5] [j87[ DAESRLNDN

F]K

Eli I AIDAESRLN-D 0.003] Ii2iI1 NKSEEAIDAE d TM[ SEEAIDAESR7 W Table XX(I-VI 2-H LA-B3 501l0mers-193P1E18 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 L start position plus nine.

Fsj7tatSbuece S1 core.

F-27 RALDGEESLL 11.18.000 151DGEESLLSKYIf1.20 1a SLLSKYNS-NL .0 EDJ ESLLSKYNSN]j 0.500 1111 QRALDGEESL] 0I 0100 ALDGEESLLS 0.045.

EESLLSKYNS

0.010 IMI[ GEESLLSKN0.3 [-41i LDGEESLLSK 11[ 0.002- ITable XXIVIO-HLA-B3501.

l0mers-193P1 El B 139 TKVLMEKNSM II0.300 103 IfLERYIVSQVL If0.300 178 ]f NSDNYKEEPV If0.300 i fVASSCSKS FO 300 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 :s the -start position plus nine.

Start Subseque-nce S-core IISPQLSDFGLE 1f 0.300 293 GUISTKNS IO 0 Tables XXII-XLIX: Table(Il-VIM-HA-AI 9mers-193PIEIB 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 98 LSDFGLERY 31 31 GEESOFEDY 28 37 EDYPMRILY 26 272 QLEKSDAEY 26 48 H-SEVQTLKD 24 228 YTMCLNEDY 24 344 PSSPTISSY 23 152 KIREYFQKY 21 78 LSDPPVASS .20 .341 LTDPSSPTI 20 182 YKEEPVIVr 19 302 SIALVSTNY 19 71 KTDVKDDLS 18 121 LLDKARLEN 18 324 VEDRTSLVL 18 368 5EDlLQLLS .18 19 OCETARLOR .17 54 LKDDVNIPE 17 154 REYFQKYGY 17 220 LEHFGISEY 17 -333 NSDTCFENL 17 370 D!LQLLSKY 17 147 SMDIMKIRE 16 183 KEEPVIVTP 16 219 KLEHFGISE 16 225 ISEYTMCLN 16 253 DTESRIINDN 16 148 MDIMKIREY 15 171 VHEOFAINS 15 174 QEAINSDNY 15 178 NSDNYKEEP 15 258 LNDNVFATP 15 TableXXII.VS-HLA-AI- 9mers-l 93P1 El B Each peptide is a portion of SEQ ID) NO: 11: each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 7 ISEKSPRSP 14 3 ASSCISEI S 7 4 SSCISEKSP 6 Table)O(H-V6HLA-AI- 9mers-l 93P El B Each peptide is a portion of SEQ tD NO: 13; each start position is specified, the length of peptide is 9 amino acids, .and the end position for each peptide is the start position plus eight.

Pos 123456789 score 2 KSEEAIDAE 14 6 A!1DAESRLN 13 3 SEEAIDAES 12 8 DAESRLNIJN 10 71IDAESRLND 7 Tab teXXII-VI 0-H LA-Al 9mers-l 93P1 El B 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 score 6 tLQKFQWIY 16 2 IPEDILOKE 12 3 PEDILQKFQ 10 13 IYPTQKLNK 8 15 PTQKLNKMR 8 TabteXXII-V12-HLA-AI- 9mers-l 93P1 El B 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 5 GEESLISKY 27 4 DGEESLLSK 16 2 ALDGEESLL 15 TableXXlI.VI-HLA- A02Ol.Smers-193PI18 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 TableXXIII-VI-HLA- A0201 -9mers-l93Pl El 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 plus eight.

Pos 123456789 score 43 ILYDLHSEV 27 366 KIPEDILQL 26 46 DLI-SEVQTL 10 KLRSLASTL 24 17 TLDCETARL 24 224 GISEYTMCL 24 111 VLPNPPQAV 23 304 ALVSTNYPL *23 374 LLSKYNSNL 23 200 VLKTPKCAL .22 6 SFCGKLRSL. 21 257 RLNDNVFAT 21 298 STKNSIALV 21 295 KIPSTKNSI 348 TISSYENLL 383 ATPtAIKAV 102 GLERY!VSQ 19 189 VTPPTKQSL 19 341 LTOPSSPTI 19 381 NLATPIAIK 19 3 PIRSFCGKL 18 13 SLASTLOCE 18 39 YPMRILYDL 18 55 KDDVN!PEL '18 192 PTKQSLVKV 18 280 YTNSPLVPT 18 390 AVPPSKRFL 18 142 LMEKNSMDI 17 196 SLVKVLKTP 17 307 STNYPLSKT 17 314 KTNSSSNDL 17 359 PTPPEVTKI 17 42 RILYDLHSE 16 52 QTLKDDVNI 16 133 IDFIKATKV 16 163 SPRVKKNSV 16 232 LNEDYTMGL 16 265 TPSPI!QQL 16 322 LEVEDRTSL -16 355 LLRTPTPPE 16 356 LRTPTPPEV 16 367 IPEDILQLL 16 370 DILQLLSKY 16 373 QLLSKYNSN 16 9 GKLRSLAST 21 ETARLQRAL 24 RLQRALDGE 53 TLKDDVNIP 97 QLSDFGLER TableXXIl.VI-HLA.

A0201 -9mers-l 93 P1El 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 plus eight.

Pos 1 23456789 score 103 LERYIVSQV 15 106 YIVSQVLPN 15 114 NPPQAVNLL 15 117 QAVNLLDKA 15 119 VNLLDKARL 15 -121 LLDKARLEN 15 125 ARLENQEGI 15 141 VLMEKNSMD 15 145 KNSMDIMKI 15 158 QIKYGYSPRV 15 209 KMDDFECVT 15 340 NLTDPSSPT 15 364 VTKIPEDIL 15 399 KHGQN!RDV 15 28 ALDGEESDF 14 *77 DLSDPPVAS 14 GKSPRSPQL 14 99 SDFGLERYI 14 120 NLLDKARLE 14 135 FIKATKVLM 14 140 KVLMEKNSM 14 152 KIREYFOKY 14 176 AINSDNYKE 14 193 TKQSLVKVL 14 195 OSLVKVLKT 14 208 LKMDDFECV 14 250 EAIDTESRL 14 278 AEYTNSPLV 14 329 SLVLNSDTC 14 331 VLNSDTCFE 14 350 SSYENLLRT 14 14 LASTIDOET 13 78 LSDPPVASS 13 SPQLSDFGL 13 1 10 QVLPNFPQA 13 128 ENQEGIDFI 13 179 SDNYKEEPV 13 181 NYKEEPVIV 13 .207 ALI MDDFEC 13 .212 DFECVTPKL 13 219 KLEHFGISE 13 231 CLNEDYTMG 13 261 NVFATPSPI 13 262 VFATP5SPII 13 268 PIIQQLEKS 13 272 QLEKSDAEY 13 286 VPTFCTPGL 13 293 GLKIPSTKN 13 300 KNSALVST 13 316 NSSSNDLEV 13 323 EVEDRTSLV 13 347 PTISSYENL 13 TableXXIlI-VI .HLA- A0201-9mers-193P1 El B Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 380 SNLATPIAI 13 382 LATPIAIKA 13 386 IAIKAVPPS 13 397 FLKHGQNIR 13 50 EVQITLKDDV 12 59 NIPELSNCE 12 75 KDDLSDPPV 12 87 CISGKSPRS 12 132 GIDFIKATK 12 134 DFIKATKVL 12 182 YKEEPVIVT 12 187 VIVTPPTKQ 12 202 KTPKCALKM 12 229 TMCLNEDYT 12 236 YTMGLKNAR 12 269 IIQQLEKSD 12 276 SDAEYTNSP 12 288 TFCTPGLKI 12 291 TPGLKIPST 12 302 SIALVSTNY 12 324 VEDRTSLVL 12 354 NLLRTPTPP 12 371 ILQLLSKYN 12 387 AIKAVPPSK 12 403 NIRDVSNKE 12 TableXXIII.V5-HLA- A0201 -9mers- I93PIEIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 6 CISEKSPRS 12 2 VASSOISEK 11 9EKSPRSPQL 1 PVASSCISE 5 TableXXIIl-V6-HLA- A0201-9mers.193P1E1

B

Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 5 EAIDAESRL 14 9 AESRLNDNV 13 6 AIDAESRLN 2 KSEEADAE 7 71IDAESRLND 7 8 DAESRjLNDN 7 1 NKSEEAIDA 6 TableXXIII-VI O.HLA- A0201-9mors-193P1E1

B

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 score 5 DILQKFQWI 18 1 KIPEDI-LQK 16 11 QWIYP-TQKL .16 6 ILQKFQWIY .13 14 YPTQKLNKM 13 8 QKFQWIYPT. 12 WIYPTQKLN 2 IPEOILQKF 8 TableXXlll-VI 2-H LA- A0201 -9mers- 193PIEIB 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 2 ALDGEESLL 24 1 RALDGEESL 21 8 SLLSKYNSN -18 TableXXIV.VI-HLA- A0203-9mers- 193PiEIB Pos 123456789 score NoResultsFound-.

A0203-9mers- 193PIElB Pos 123456789 score NoResultsFound.

TableXXIV-V6-HLA- A0203-9mers- 193P1 El B Pos 123456789 scojre NoResultsFound.

00 TableXX(IV-VI 0-HLA- Aa2o3-9mers- 193PIElB Pos 123456789 score NoResultsFound.

TableX.alV-Vi 2-HLA- A0203-9mers- 193PI ElB Pos 123456789 score NoResultsFound.

TableXXV-Vl.HLA-A3- 9mers-193P1El 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 plus eight.

Pos; 123456789 score 186 PVIVTPPTK 28 387 AIkAVPPSK 28 KLRSLASTL 26 132 GIDFIKATK 25 381 NLATPIAIK 24 97 QLSDFGLER 23 239 GLI NARNNK 23 28 ALDGEESDF 22 197 LVKVLKTPK 22 110 QVLPNPPQA 21 152 KIREYFOKY 21 272 QLEKSDAEY 21 358 TPTPPEVTK 21 402 QNIRDVSNI( 21 43 ILYOLHSEV 20 292 PGLKIPSTI( 20 102 GLERYIVSQ 19 118 AVNLLDKAR 19 151 MKIREYFQK 19 160 YGYSPRVKK 19 165 RVKKNSVHE 19 194 KQSL-V VLK 19 302 SIALVSTNY 19 369 EDILQLLSI( 19 370 DILQLLSKY 19 159 KYGYSPRVK 18 188 IVTPPTKQS 18 215 CVTPKLEHF 18 219 KLEHFGISE 18 267 SPIIQQLEK 18 330 LVLNSDTCF 18 366 KIPEDILOL 18 385 PIAIKAVPP 18 24 RLORALDGE 17 77 DLSDPPVAS 17 120 NLLDKARLE 17 140 KVLMEKNSM 17 191 PPTKQiSLVK 17 201 LKTPKCALK 17 TableXXV-VI -HLA-A3- 9mers-l 93P El B Each peptide is a portion of SEQ ID N0. 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.

Pos 123456789 score 284 PLVPTFCTP '17 306 VSTNYPLSK 17 354 NLLRTPTPP 17 373 QLLSKYNSN 17 397 FLKHGo-NIR 17 2 DPIRSFCGK 16 42 RILYDLHSE 16 57 DVNIPELSN 16 126 RLENQE-GID 16 141 VLMEKN-SMD 16 196 SLVKVLKTP 16 199 KVLKTPKCA 16 257 RLNDNVFAT :16 261 NVFATPSPI 16 323 EVEDRTSLV .16 329 SLVLNSDTC -16 390 AVIPSKRFL -16 67 ENFQKTDVK 15 73 DVKDDLSDP .15 135 FIKATKVLM 137 KATKVLMEK 15 170 SVHEQEAIN 15 183 KEEPvivrP -15 311 PLSKTNSSS 15 37 EDYPMRILY 14 46 DLHSEQQTL 14 116 PQAVNLLDK 14 121 LLD15KARLEN 14 154 REYFOKYGY 14 175 EAINSDNYK 14 207 ALKMDDFEC 14 321 DLEVEDRTS 14 340 NLTDPSSPT 14 344 PSSPTISSY 14 17 TLDCETARL 13 23 ARLORALOG 13 47 LHSEVQTLK 13 63 LSNCENFQK 13 79 SDPPVASSC 13 82 PVASSCISG 13 83 VASSCISGK 13 86 SC!SGKSPR 13 107 IVSQVLPNP 13 I1I1 VL-3PPQV 13 149 DIMKIREYF 13 231 CLNEDYTMG 13 293 GLKIPSTKNf 13 295 KIPSTKNSI 13 304 ALVSTNYPL 13 355 LLRTPTPPE 13 371 ILQLLSKYN 13 374 LLSKYNSNL 13 TableXXV-Vi -HLA-A3- 9mers-l 93P1 El B 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 pepfide is the start position plus eight.

Pos 123456789 score 403 NIRDVSNKE 13 TabloXXV-V5-HLA-A3- 9mers-l 93P1 El B Each peptidle is a portion of SEQ I D NO: 11; each start position is specified, the length of peptide Is 9 amino acids, and the end position for each peptidle is the start position plus eight.

Pos 123456789 score 1 PVASSCISE 13 2 VASSCISE( 13 5 SCISEKSPR 11 6 CISEKSPRS 9 EKSPRSPQL 8 8 SEKSPRSPQ 6 TableXXV-V6-HLA-A3- 9mers.I93PIEIB* Each peptidle is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptidle is the start position plus eight.

Pos 123456789 score 6 AIDAESRLN 13 4 EEAIDAESR 11 71IDAESRLND 8 5 EAIDAESRL 7 3 SEEAIDAES 6 9 AESRLNDNV 6 TableXXV-VI 0-H LA-A3- 9mers-193P1 El B Each peptidle 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 start position plus eight.

Pos 123456789 score 1 KIPEDILQK 28 6 ILQKFQWIY 18 10 FQWIYPTQK 16 12 WIYPTQKLN 15 131IYPTQKLNK 15 TableXXV-VI 2-HLA-A3- 9mers-193P1 l 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 2 ALDGEESLL 18 4 DGEESLLSK 16 8 SLLSKYNSNI 16 I RALDGEESL 11 GEESLLSKY 9 TableXXVl-VI-HLA-A26- 9mers-11931 El 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 plus eight.

Pos 123456789 score 370 DILQLLSI Y 29 21 ETARLQRAL 28 215 CVTPI LEHF 25 73 DVKDDLSDP 24 250 EAIDTESRL 24 363 EVTKIPEDI 23 37 EDYPMRILY 22 46 DLHSEVQTL 22 149 DIMKIREYF 22 323 EVEDRTSLV 22 EVQTLKDDV 21 253 DTESRLNDN 21 347 PTISSYENL 21 57 DVNIPELSN 20 131 EGIOFIKAT 20 134 DFIKATKVL 20 366 KIPEDILQL 20 369 EDILQLLSK 20 148 MDIMKIREY 19 228 YTMCLNEDY 19 255 ESRLNDNVF 19 390 AVPPSKRFL- 19 104 ERYIVSQVL 18 189 VTPPTKQSL 18 211 DDFECVTPK 18 330 LVLNSDTCF 18 335 DTCFENLTD 18 152 KIREYFQKY 17 212 DFECVTPKL 17 265 TPSPIIQQL 17 277 DAEYTNSPL 17 314 KTNSSSNDL 17 TableXX(VI-VI -tiLA-A26- 9mers-l 93P1 El B Each peptide is a portion of SEQ I D NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each pepfide is the start position plus eight.

Pos 123456789 score 344 PSSPTISSY 17 128 ENQEGIOFI 16 155 EYFQKYGYS 16 184 EEPV1VTPP 16 221 EHFGISEYT 16 281 TNSPLVPTF 16 326 DRTSLVLNS 16 364 VTKIPEDIL 16 3 PIRSFCGKL 15 6 SFCGKLRSL 15 67 ENFQKTDVK 15 186 PVIVTPPTK 15 193 TKQSLVKVL 15 214 ECVTPKLEH 15 220 LEHFGISEY 15 227 EYTMCLNED 15 261 NVFATPSPI 15 264 ATPSPIIQQ 15 302 SIALVSTNY 15 307 STNYPLSKT 15 322 LEVEDRTSL 15 367 IPEDILQLL 39 YPMRILYDL 14 56 DDVNIPELS 14 93 PRSPQLSDF 14 98 LSDFGLERY 14 106 YIVSQVLPN 14 107 IVSQVLPNP 14 175 EAINSDNYK 14 185 EPVIVTPPT 14 224 GISEYTMCL 14 325 EDRTSLVLN 14 348 TISSYENLL 14 359 PTPPEVTKI 14 TabeXXVl-VS-HLA-A26- 9mers-193P1 El B Each peptidle is a portion of SEQ ID NO: 11; each start position is specified, (he length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 9EKSPRSPQL 20 1 PVASSCISE 13 TableXXV1-V6-HLA-A26- 9mers-l 93P1 El B Each peptidle is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 5 EAIDAESRL 24 8 DAESRLNDN 13 4 EEAIDAESR 111 TableXXVI-VIU-HLA.

A26-9mers-1 93P1 El B Each peptidle is a portion of SEQ ID NO: 21; 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.

Pos 123456789 score 2 IPEDILQKF 16 4 EDILQKFQW 5 DILQKFQWI 13 11 QWIYPTQKL 13 1 KIPEDILQK 12 6 ILQKFQWIY 15 PTQKLNKMR 8 QKFQVVflYPT 8 14 YPTQKLNKM 7 TableXXVI-V12-HLA- A26-9mers-193P1 EIB 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 5 GEESLLSKY 18 4 DGEESLLSK 16 6 EESLLSKN 11 1 RALDGEESL 7 ESLLSKYNS 2 ALDGEESLL 9 TableXXVII-VI -H LA- 80702.9mers-193P18 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 265 TPSPIIQQL 23 7 TableXXVII-VI -HLA- B0702-9mers.193P1 El 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 plus eight.

Pos 123456789 score 286 VPTFCTPGL 22 39 YPMRILYDL 21 114 NPPQAVNLL 21 367 IPEDILOLL 21 SPOLSDFGL 20 185 EPVlVTPPT 20 283 SPLVPTFCT 20 296 IPSTKNSIA 19 163 SPRVKKNSV 18 291 TPGLKIPST 18 92 SPRSPQLSD 17 DPPVASSCI 16 112 LPNPPQAVN 16 190 TPPTKQSLV 16 217 TPKLEHFGI 16 343 DPSSPTISS 16 358 TPTPPEVTK 16 392 PPSKRFLKH 16 36 FEDYPMRIL 14 GKSPRSPQL 14 191- PPTKQSLVK 14 200 VLKTPKCAL 14 324 VEDRTSLVL 14 390 AVPPSKRFL 14 KILRSLASTIL 13 17 TLDCETARL 13 21 ETARLORAL 13 KDDVNIPEL 13 113 PNPPQAVNL 13 115 PPQAVNLLD 13 134 DFIKATKVIL 13 224 GISEYTMCL 13 304 ALVSTNYPL 13 364. VIKIPEDIL 13 366 KIPEDILQL 13 374 LLSKYNSNL 13 384 TPIAIKAVP 13 3 PIRSFCGKIL 12 6 SFCGKLRSL 12 104 ERYIVSQVL 12 193 TKQSLVKVL 12 212 DFECVTPKL 12 267 SPiIQQLEK 12 280 YTNSPLVPT 12 288 TFCTPGLKI 12 297 PSTKNSIAL 12 322 LEVEDRTSL 12 333 NSDTCFENL 12 348 TISSYENLL 12 2 DPIRSFCGI( 11 28ALDGEESOF 11 46 DLHSEVQTL 11 TableXXVII-VI-HLA- B0702-9mers-193P1 El 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 plus eight.

Pos 123456789 score 60 IPELSNCEN 11 81 PPVASSCIS 11 119 VNLLDKARL 11 182 YKEEPVIVT 11 189 VTPPTKQSL 11 232 LNEDYTMGL 11 250 EAIDTESRL 11 262 VFATPSPII 11 277 DAEYTNSPL 111 281 TNSPLVPTF 11 300 KNSIALVST 11 310 YPLSKThSS 11 314 KTNSSSNDL 11 357 RTPTPPEVT 11 360 TPPEVTKIP 11 381 PPEVTKIPE 11 389 KAVPPSKRF 11 391 VPPSKRFU( 11 TableXXV1I-V5-HLA- B30702-9mers- 193PIE1B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 9EKSPRSPQL 15 TableXXV1I-V6-HLA- B0702-9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of pepide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 5 EAIDAESRL 11 9 AESRLNDNV 10 1 NKSEEAIDA 8 71IDAESRLND 5 TableXXVII-VIO-HLA- B0702-9mers-193P1 El B 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 start position plus eight.

Pos 123456789 score 2 IPEDILQKF 17 14 YPTQKLNKM 16 11 QWIYPTQKL 14 TableXXVII-VI 2.HLA- B0702-9mersi93PIEIB 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 2 ALDGEESLL I RALDGEESIL 11 TableXXV1II-VI-HLA- B08-9mers-1 93P1 El 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 plus eight.

Pos 123456789 score 200 VLKTPKCAL 28 163 SPRVKKNSV 217 TPKLEHFGI 23 6 SFCGKLRSL 22 10 KLRSLASTL 22 364 VTKIPEDIL 21 3 PlRSFCGKL 8 CGKLRSLAS 141 VLMEKNSMD 90 GKSPRSPQL 19 95 SPQLSDFGL 19.

150 IMKIREYFQ 19 122 LDIKARLENQ 18 291 TPGLKIPST 18 296 IPSTKNSIA 18 46 DLHSEVQTL 17 53 TLKDDVNIP 17 114 NPPQAVNLL 17 203 TPKCALKMD -17 205 KCALKMDDF 17- 207 ALKMDDFEC 17 224 GISEYTMOL 17 239 GILKNARNNIK 17 265 TPSPIIQQL 17 00 TableXXVIII-VI-HLA- B08-9mers-1 93P1 El B Each pepfide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight Pos 123456789 score 286 VPTFCTPGL 17 293 GLKIPSTKN 17 304 ALVSTNYPL 17 366 KIPEDILQL 17 367 IPEOILQLL 17 374 LLSKYNSNL 17 391 VPPSKRFLK 17 397 FLKHGQNIR 17 17 TLDCETARL 16 39 YPMRILYDL 16 120 NLLDKARLE 16 135 FIKATKVLM 16 190 rPPTKQSLV 16 215 CVTPKLEHF 16 250 EAIDTESRL 16 277 DAEYTNSPL 16 310 YPLSKTNSS 16 373 QLLSKYNSN 16 255 ESRLNDNVF 15 385 *PIAIKAVPP 15 87 CISGKSPRS 14 92 SPRSPQLSD 14 348 TISSYENLL 14 387 AIKAVPPSK 14 392 PPSKRFLKH- 14 21 ETARLQRAL 13 KDDVNIPEL 13 69 FQKTDVKDD 13 DPPVASSCI 13 124 KARLENQEG 13 166 VKKNSVHEQ 13 179 SDNYKEEPV 13 181 NYKEEPVIV 13 271 QQLEKSDAE 13 298 STKNSIALV 13 B08.9mers.193P1 El B Each peptide is a portion of SEQIDt'NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptideis the start position plus .eight.

Pos 123456789 score 9EKSPRSPQL 20 6 CISEKSPRS 16 8SEKSPRSPQ 12 TableXXVIII-V6-HLA- B08-9mers-193P1 El B Each peptide is a portion of SEQ ID NO: 13; 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.

Pos 123456789 score 5 EAIDAESRL 16 8DAESRLNDN 12 TableXXVIII-VIO.HLA- B08-9mers-1 93P1 El B 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 score 5 DILQKFQWI 20 14 YPTQKLNKM 16 2 IPEDILQKF 13 11 QVVIYPTQKL 11 7 LQKFQWIYP 10 TableXXVIII-V12-HLA- B08-9mers-1 93P1 El B 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 8 SLLSKYNSN 18 2 ALDGEESLIL 16 1 RALDGEESL 14 Tab~eXXIX-V1.HLA.

B1510-9mers-193P1 El 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 plus eight.

Pos 123456789 score 21 ETARLQRAL 15 55 KDDVNIPEL 15 90OGKSPRSPQL 15 265 TPSPIIQQL 15 390 AVPPSKRFL 15 399 KHGQNIROV 15 36 FEDYPMRIL 14 TableXXiX-VI -lILA- BI 51 O-9mers-l 93P El 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 plus eight Pos 123456789 score 113 PNPPQAVNL 14 193 TKQSLVKVL 14 250 EAIDTESRL 14 367 IPEDILOII 14 6 SFCGI LRSL 13 17 TLDCETARL 13 104 ERYIVSQVL 13 119 VNLLDKARL .13 134 DFIKATKVL .13 189 VTPPTKQSL .13 200 VLKTPKCAL 13 2241 GISEYTMCL .13 281 .TNSPLVPTF I13 297 PSTKNSIAL 13 46 DLHSEVQTL 12 47 LHSEVQTLK 12 70 QKTDVKDDL 12 114 NPPQAVNLL 12 171 VHEQEAINS .12 212 DFECVTPKL 12 221 EHFGISEYT 12 232 LNEDYTMGL 12 322 LEVEDRTSL 12 324 VEDRTSLVL 12i 348 TISSYENLL 12' 366 KIPEDILOL 12 374 LLSKYNSNL 12 10 KLRSLASTL 11 39 YPM~RILYDL 11 277 DAEYTNSPL '11 286 VPTFCTPGL 11 364 VTKIPEDIL 11 389 KAVPPSKRF 11 3 PIRSFCGKL 95 SPQLSDFGL 255 ESRLNDNVF 304 ALVSTNYPL 314 KTNSSSNDL 333 NSDTCFENL 347 PTISSYENL 93 PRSPQLSDF 9 135 FIKATKVILM 9.

149 DIMKIREYF 8.

205 KCALKMDDF 8 215 CVTPKLEHF 8 28 ALDGEESDF 7 33 ESDFEDYPM 7 61 PELSNCENF 7 77 DLSDPPVAS 7 1127 LENQEGIDF 7 140 KVLMEKNSM 7 143 MEKNSMIDIM 7 TableXXIX-Vi-HLA- 81510O9mers.1 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peplidle is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 182 YKEEPVIVT 7 183 KEEPVIVTP 7 202 KTPKCALKM 7 222. HFGISEYTM 7 230 MCLNEDYTM 7 358 TPTPPEVTI( 7 8151 0-9mers- 193PIEIB Each peptide is a portion of SEQ ID NO: 11; 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.

Pos 123456789 score 9EKSPRSPQL 15 71ISEKSPRSP 7 TableXXIX-V6-HLA- 8151 0-9mers- 193PiIB Each peptide is a portion of SEQ ID Na.

13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score EAIDAESRL 14 TableXXlX-VI0-H LA- 8151 0-9mers.1 93P1 El B Each peptidle 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 score 11 QWIYPTQKL 11 2 IPEDILQKF 10 14 YPTQKLNI M 8 TableXXIX-VI 2-HLA- 8151 O-9mers- I93PI EllB 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 1 RALDGEESL 12 2 ALDGEESLL 11 TableXXX-VI-HLA- B32705-9mners-l 93P1 l Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 104 ERYIVSQVL 25 164 PRVKKNSVH 25 93 PRSPQLSDF 23 125 ARLENQEGI 22 4 IRSFCGKLR 21 137 KATKVLMEK 18 366 KIPEDILQL 18 389 KAVPPSKRF 18 395 KRFLKHGQN 18 23 ARLQRALDG 17 55 KDDVNIPEL 17 67 ENFQKTDVK 17 90 GKSPRSPQL 17 119 VNLLDI ARL 17 132 GIDFIKATK 17 211 DDFECVTPK 17 292 PGLKIPSTK 17 370 DILQLLSKY 17 10 KLRSLASTL 16 47 LHSEVQTLK 16 86 SCISGI SPR 16 140 KVLMEKNSM 16 154 REYFQKYGY 16 160 YGYSPRVK< 16 194 KQSLVKVLK 16 202 KrPKCALKM 16 239 GLKNARNNK 16 265 TPSPIIQQL 16 267 SPIIQOLEK 16 330 LVLNSDTCF 16 369 EDILQLLSK 16 374 LLSKYNSNL 16 396 RFLKHGQNI 16 402 QNIRDVSNK 16 6 SFCGKLRSL 15 28 ALDGEESDF .15 34 SDFEDYPMR 15 TableXXX-VI .HLA- B2705.9mers-193P1E1 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 die end position for each peptide is the start position plus eight.

Pos 123456789 score 41 MRILYDLHS 113 PNPPQAVNL 134 DFIKATKVL 148 MDIMKIREY 175 EAINSDNYK 191 PPTKQSLVI( 220 LEHFGISEY 224 GISEYTMCL 236 YTMGLKNAR 250 EAIDTESRL 281 TNSPLVPTF 302 SIALVSTNY 314 KTNSSSNDL 322 LEVEDRTSL 326 DRTSLVLNS 347 PTISSYENL 381 NLATPIAIK 388 IKAVPPSKR 397 FLKHGQNIR 11 LRSLASTLD 14 16 STLDCETAR 14 17 TLDCETARL 14 52 QTLKDDVNI 14 61 PELSNCENF 14 83 VASSCISGK 14 114 NPPQAVNLL 14- 118 AVNLLDKAR 14 127 LENQEGIDF 14 129 NQEGIDFIK 14 145 KNSMDIMKI 14 151 MKIREYFQK 14 159 KYGYSPRVK 14.

186 PVIVTPPTK 14 197 LVKVLKVPK 14 205 KCALKMDDF 14 212 DFECVTPKL 14 230 MCLNEDYTM 14 243 ARNNKSEEA 14.

255 ESRLNDNVF 14 256 SRLNDNVFA 14 272 QLEKSDAEY 14 297 PSTKNSIAL 14 304 ALVSTNYPL 14 344 PSSPTISSY 14 349 ISSYENLLR 14 358 TPTPPEVTI( 14 387 AIKAVPPSK 14 390 AVPPSKRFL 14 4041IRDVSNKEN 14 2 DPIRSFCGK 13 5 RSFCGKLRS 13 21 ETARLQRAL 13 TableXXX-V1 .HLA- B2705-9mers.193P1 El B 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.

Pos 123456789 score 39 YPMRILYDL 13 46 DLHSEVQTL 13 63 LSNCENFQK 13 SPQLSDFGL 13 98 LSDFGLERY 13 149 DIMKIREYF 13 152 KtREYFQKY 13 153, REYFQKYG 13 157 FQKYGYSPR 13 180. DNYKEEPVI 13 189 VTPPTKQSL. 13 193 TI QSLVKVL 13 201 :LKTPKCALK 13 214 iECVPKLEH* 13 215 CVTPKLEHF 13 244 RNNKSEEAI 13 287 PTFCTPGLK 13 319 SNDLEVEDR. 13 324 VEDRTSLVL 13 356 LRTPTPPEV 13 359 .PTPPEVTKI 13 367. IPEDILQLL 13 392 PPSKRFLKH 13 3 PIRSFCGKL 12 19 DCETARLQR 12 26 QRALDGEES 12 37 EDYPMRILY 12 QKTDVKDDL 12 97: QLSDFGLER 12 99 SDFGLERYI 12 116 PQAVNLLDK 12 128 ENQEGIDFI 12 144 EKNSMDIMK 12 200 VLKTPKCAL 12 277 DAEYTNSPL 12 306 VSTNYPLSK 12 333 .NSDTCFENL 12 TableXXX-V5.B2705.

9mers-193P1 El B Each peptidle is a portion of SEQ ID NO: 11; each start position is specified, the length of peplide is 9 amino acids, and the end position for each peptidle is the start position plus eight.

Pos 123456789 score 2 VASSOISEK 15 SCISEKSPR 15 9EKSPRSPQL 14 TableXXX-V6-B2705- 9mers-i93P1IB Each peptidle is a portion of SEQ ID NO: 13; each start position is specified, the length of peptidle is 9 amino acids, and the end position for each pepide is the start position plus eight Pos 123456789 score 5 EAIDAESRL 15 4 EEAIDAESR 12 TableXXX.VI O-B2705- 9mers.I93PIEIB Each pepfide 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 start position plus eight.

Pos 123456789 score 1 KIPEDILOK 18 2 IPEDILQKF 15 11 QWIYPTQKL 15 13 IYPTQKLNK 15 14 YPTQKLNI(M 15 6 ILOKFOWIY 14 15 PTQK<LNKMR 14 10 FQWlYPTQK 13 5 DILQKFQWI 11 8 QKFQWIYPT- 9 TableXXX-VI 2.B2705- 9mers-193P1 El B 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 I RALDGEESL 19 5 GEESLLSKY 16 2 ALDGEESLL 15 4 DGEESLLSK 15 TableXXXI-V1 -H LA- B2709-9mers-l 93P1 El 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 plus eight.

Pos 123456789 score 104 ERYIVSQVL 22 125 ARLENQEGI 21 356 LRTPTPPEV 21 93 PRSPQLSDF 19 90 GKSPRSPQL 16 23 ARLQRALDG 326 DRTSLVLNS 366 KIPEDILOL 395 KRFLKHGQN 396 RFLKHGQNI 10 KLRSLASTL 14 113 PNPPQAVNL .14 119 VNLLDKARL 14 256. SRLNDNVFA 14 304 ALVSTNYPL 14 52 QTLKDDVNI 13 55 KDDVNIPEL 13 224 GISEYTMOL 13.

265 TPSPIIQQL 13 314 KTNSSSNDL 13 347 PTISSYENL 13 389 KAVPPSKRF 13 39 YPMRILYDL 12 41 MRILYDLHS -12 46 DLHSEVQTL 12.

61 PELSNCENF 12.

133 IDFIKATKV 12 140 KVLMEKNSM 12 158 QKYGYSPRV 12 193 TKQSLVKVL 12 202 KTPKCAL M 12 244 RNNKSEEAI 12 250 EAIDTESRL 12 278 AEYTNSPLV 12 286 VPTFCTPGL 12 322 LEVEDRTSL 12 367 IPEDILQLL 12 390 AVPPSKRFL 12 3 PIRSFCGKL 11 41IRSFCGKLR 11 17 TLDCETARL 11 26 QRALDGEES 11 43 ILYDLHSEV 11 70 QKTDVKDDL 11 75 KDDLSDPPV 11 103 LERYIVSQV 11 114 NPPQAVNLL 11 134 DFIKATIWL 11 145 KNSMDIMKI 11 1531IREYFQKYG 11 164 PRVKKNSVH .11 180 DNYKEEPVI 11 189 VTPPTKQSL 11 212 DFECVTPKL 11 230 MCLNEDYTM 11 243 ARNNKSEEA 11 281 TNSPLVPTF 11 295 KIPSTKNSI 11 297 PSTKNSIAL 11 324 VEDRTSLVL 11 TableXXXI-VI-HLA- B2709-9mers-193P1 El 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 plus eight Pos 123456789- score 333 NSDTCFENL 11 348 TISSYENLL 11 374 LLSKYNSNL 11 404 IRDVSNKEN 11 6 SFCGKLRSL. 10 11 LRSLASThD 10 21 ETARLORAL- 10 36 FEDYPMRIL 10 SPQLSDFGL, 10 99 SDEGLERYI* 10 200 VLKTPKCAL:* 10 205 KOALKMDDF 10 215 *CVTPKLEHF 10 232 LNEDYTMGL'. 10 261 NVFATPSPI 10 277 DAEYTNSPL: 10 316 NSSSNIJLEV -10 330 LVLNSDTCF 10 341 LTOPSSPTI .10 359 PTPPEVTKI 10 363 EVTKIPEDI 10 364 VTKIPEDIL 10 380 SNLATPIAI 10 399 KHGQNIRDV -10 B2709-9mers- I93PIEIB Each' peptide is a p ortion of SEQ I D NO: 1 1;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.

Pos 123456789 score 9 EKSPRSPQL 13 TableXXXJI-V6-HLA- B2709-9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; 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.

Pos 123456789 score EAIDAESRL 12 9 AESRLNDNV 10 TableXXXI-VI 0-HLA- B2709-9mers-l 93P1 El B Each peptidle is a portion of SEQ ID NO: 21; each start posiion is specified, the length of peptidle is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 11 QWIYPTQKL 11 2 IPEDILQKF 10 5 DILQKFQWI 10 14 YPTQKLNKM 9 1 KIPEDILOK 5 Tab IeXXXI-VI 2.HLA- B2709-9mers-1 93P1 El B Each peptidle 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 1 RALDGEESL 16 2 ALDGEESLL 11 TableXXXII-VI -HLA- 84402-9mers-193P1 El B Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 324 VEDRTSLVL 24 220 LEHFGISEY 23 36 FEDYPMRIL 22 61 PELSNGENF 22 31 GEESOFEDY 21 127 LENQEGIDF 21 174 QEAINSDNY 21 322 LEVEDRTSL 21 154 REYFQKYGY 20 183 KEEPVIVTP 19 265 TPSPIIQQL 19 37 EDYPMRILY 18 390 AVPPSKRFL 18 134 DFIKATKVL 17 148 MDIMKIREY 17 344 PSSPTISSY 17 366 KIPEDILOL 17 21 ETARLQRAL 16 55 KDDVNIPEL 16 TableXXXII-VI.HLA- B4402.9mers.1 93P1 El B Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino adds, and the end posibon for each peptidle is the start position plus eight.

Pos 123456789 score 90 GKSPRSPQL 16 113 PNPPQAVNL 16 250 EAIDTESRL 16 255 ESRLNDNVF 16 278 AEYTNSPLV 16 281 TNSPLVPTF '16 389 KAVPPSKRF 16 28 ALDGEESDF 93 PRSPQLSQF 114 NPPQAVNLL 184 EEPVIVTPP 6 SFCGKLRSL :14 32 EESDFEDYP 14 39 YPMRILYDL 14 49 SEVQTLKDD 14 145 KNSMDIMKI 14 172 HEQEAINSD .14 189 VTPPTKQSL 14 193 TKQSLVKVL 14 213 FECVTPKLE 14 215 CVTPKLEHF 14 297 PSIENSIAL 14 333 NSDTCFENL 14 359 PTPPEVK .14 362 PEVTKIPED 14 367 IPEDILOLL 14 380 SNLATPIAI 14 10 KLRSLASTL 13 99 SDFGLERYI 13 103 LERYlVSQV 13 104 ERYIVSQVL 13.

125 ARLENQEGI 13 128 ENQEGIDEI 13 130 QEGIOFIKA 13 131 EGIDFIKAT 13 149 DIMKIREYF 13 152 KIREYFOKY 13 200 VLKTPKCAL 13 226 SEYTMOLNE 13 233 NEDYTMGLK 13 249 EEAIDTESR 13 304 ALVSTNYPL 13 341 LTDPSSPT 13 347 PTISSYENL 13 348 TISSYENLL 13 368 FEDILQLLS 13 370 DILQLLSKY 13 17 TLDCETARL 12 20 CETARLQRA 12 46 DLHSEVQTL 12 95 SPQLSDFGL 12 98 LSDFGLERY 12 TableXXXII-Vi-HLA.

B4402-9mers.193P1 El 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 plus eight.

Pos 123456789 score 119 VNLLDKARL 12 205 KCALKMDDF 12 212 DFECVTPKL 12 224 GISEYTMOL 12 232 LNEDYTMGL 12 248 SEEAIDTES 12 254 TESRLNDNV 12 261 NVFATPSPI 12 302 SIALVSTNY 12 314 KTNSSSNDL 12Q 330 LVLNSDTCF 12 352 YENLLRTPT 12 363 EVTKIPEOI 12 3 PIRSFCGKL 11 QKTDVKDDL 11 143 MEKNSMDIM 11 169 NSVI-EQEAl 11 228 YTMCLNEDY 11 273 LEKSDAEYT 11 286 VPTFCTPGL 11 288 TFGTPGLKI 11 295 KIPSTKNSI 11 338 FENLTDPSS 11 374 LLSKYNSNL 11 378 YNSNLATPI 11 383 ATPIAIKAV i1 B4402-9mers.193P1 El B Each peplide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos '123456789 score 9 EKSPRSPQL 18 8SEKSPRSPQ 12 TableXXXII-V6-HLA.

B4402-9mers- A93PIEIB3 Each peptide is a portion of SEQ I D NO: 13; 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 Pos 123456789 score 5 EAIDAESRL 16 9AESRWDNV 15 4 EEAIDAESR 13 3 SEEAIDAES 12 TabeXXXll-V1 0-lILA- B4402-9mers.

193P1EIB 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 score 4 EDILQKFQW 17 11 QWIYPTQKL 15 2 IPEDILQKF 14 3 PEDILQKFQ 13 5 DILQKFQWI 10 6 ILQKFQWIY 10 TableXXXII-V124ILA- B4402.9mers- I93PIEIB Each peptide is a portion of SEQ ID NO:.25; each start position is specified, the length of pep tide is 9 amino acids, and the end position for each peplide is the start position pius eight Pos 123456789 score 5 GEESLLS Y 22 2 ALDGEESLL 16 6 EESLLSKYN 16 1RALDGEESL 12 TableXXXIII-VI .HLA.

B51 01-9mors.1 93P1 El 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 peplide is the start position plus eight.

Pos 123456789 score 80 DPPVASSCI 25 180 DNYKEEPVI 22 190 TPPTKQSLV 22 277 DAEYTNSPL 22 114 NPPQAVNLL 21 217 TPKLEHFGI 21 163 SPRVKKNSV 20 367 IPEDILQLL 20 39 YPMRILYDL 19 250 EAIDTESRL 19 TableXXXIII..Vl.HLA- B51 01 -9mers-193P1 El 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 plus eight.

Pos 123456789 score 35 DFEDYPMRI 18 265 TPSPIIQQL 18 95 SPQLSDFGL 17 286 VPTFCTPGL 17 360 TPPEVTKIP 17 2 DPIRSFCGK 16 133 IDFIKATKV 16- 303 IALVSTNYP 16 310 YPLSKTNSS 16 359 PTPPEVTKI 16 380 SNLATPIAI .16 382 LATPIAIKA 16 43 ILYDLHSEV 101 FGLERYIVS 104 ERYIVSQVL 112 LPNPPQAVN 115 PPQAVNLLD 134 DFIKATKVL 158 QKYGYSPRV 160 YGYSPRVIKK 191 P-PTKQSLVK 261 NVFATPSPI 288 TFCTPGLKI 296 IPSTKNSIA 341 LTDPSSPTI 343 DPS SPTISS 384 TPIAIKAVP 386 IAIKAVPPS 392 PPSKRFLKH 46 DLHSEVQTL 14.

52 QTLKDDVNI 14.

60 IPELSNCEN 14.- 125 ARLENQEGI 14.

137 KATKVLMEK 14.

206 CALKMDDFE -14.

212 DFEOVTPKL 14 263 FATPSPIIQ 14 358 TPTPPEVTK 14 -378 YNSNLATPI 14 14 LASTLOCET 13 27 RALDGEESD 13 30ODGEESOFED 13 99 SDFGLERYI 13 117 QAVNLLDKA 13 128 ENQEGIDFI 13 142 LMEKNSMDI 13 145 KNSMDIMKI 13 181 NYKEEPVIV 13 192 PTKQSLVKV 13 193 TKQSLVKVL 13 203 TPKCALKMD 13 278 AEYTNSPLV 13 TableXXXlIIl-HLA- B51Di1-9rners-i 93P1E1 B Each peptide Is a portion of SEQ 10 NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.

Pos 123456789 score 291 TPGLKIPST 13 295 KIPSTKNSI 13 391 VPPSKRFLK 13 396 RFLKHGQNI 13 81 PPVASSCIS 12 83 VASSCISGK. 12 100 OFGLERYIV 12 103 I.ERYIVSQV: 12 175 EAINSDNYK 12 185 EPVIVTPPT 12 208 LKMDDFECV 12 238 MGLKNARNN 12 244 RNNKSEEAI 12 262 VFATPSPll 12 283 SPLVPTFGT. 12 292 PGLKIPSTK 12 324 VEDRTSLVL 12 356 LRTPTPPEV 12 361 PPEVTKIPE 12 363 EVTI(IPEDI 12 389 KAVPPSKRF 12 B51 01 -9mers- 193PIElB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each'peptide is the start position plus eight. Pos 123456789 score 2 VASSOISEK 12- 71ISEKSPRSP 6 9EKSPRSPQL 6 Tab~eXXXIII-V6-HLA- B51 01 -9mers-l 93P1 El B Each peptide is a portion of SEQ ID NO: 13: each start position is specified, the length of peptide is 9 amino acids, and'the end position for each peptide is the start position plus eight.

Pos 123456789 score EA1DAESRL 18 8 DAESRLNDN 16 9AESRLNDNV 9 TableXXXIII-VI 0-lLA.

B51 01 .9mers4193P1 ElB 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 score 5 DILQKFQWI 18 14 YPTQKU'4KM 17 2 IPEDILQKF 16 11 QWIYPTQKL 8 TableXXXIII-VI 2-HIA- B5101 -9mers- 193P1 ElB 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 I RALDGEESL 19 4 DGEESLLSK 15 TableXXXIV-VI -HLA-AIiomers-193PIEIB Each peptide is a por-tion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide, is the start position plus nine.

Pos 1234567890 score 36 FEDYPMRILY 33 30 DGEESDFEDY 28 147 SMDIMKIREY 27 219 KLEIF-IGSEY 26 153 IREYFQKYGY 25 173 EQEAINSDNY 25 71 KfDVKDDLSD 22 129 NQEGIDFIKA 20 151 MKIREYfFQKY 20 225 ISEYTMGLNE 20 .341 CTDPSS-PTIS 20 233 NEDYTMGLKN 19 301 NSIALVSTNY 19 78 LSDPPVASSC 18 251 AiDTESRLND 18 367 IPEDILOLLS 18 253 DTESRLNDNV 17 323 EVEDRTSLVL 17 369 EOILQLLSKY 17 97 QLSDFGLERY 16 126 RLENQEGIDF 16 TableXXXJV-VI-HLA-AI- I Omers*1 93PE11B Each peplide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 227 EYTMCLNEDY 16 333 NSDTCFENLT 16 368 PEOILQLLSK 16 TableXXXIV-V5-HLA-Al 10mers-I93P1EIB Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is amino acids, and the end..

position for each peptide is the start position plus nine.

Pos 1234567890 score 8 ISEKSPRSPO TableXXXIV-V6-HLA-AI- I0mers-193P1E1B Each peptide is a portion of SEQ ID NO: 13; each~ start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 AIDAESRLND 18 3 KSEEAIDAES 14 4 SEEAIDAESR 12 9 DAESRLNDNV 11 TabIeXXXIV-VI 0.HLA- Al-I Omers-l 93P11l 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 score 6 DILQKFQWIY I TKIPEDILQK 3 IPEDILQKFQ 4 PEDILQKFQW 13 WIYPTQ5KLNK TableXXXIV-VI 2-HLA- Al -10mers-193P1 El B Each peplide 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 peptidle is the start position plus nine.

Pos 1234567890 score DGEESLLSKY 27 3 ALDGEESLLS 21 TableXXXV-V1-HLA.

AO2O-10mers.1 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 355 LLRTPTPPEV 25 366 KtPED!LQLL 25 102 GLERVIVSQV 24 231 CLNEDYTMGL -24 141 VLMEKNSMDI 22 373 QLLSKYNSNL 22 16 STLDCETARL 21 42 RI[YDLHSEV 21 207 ALKMDDFECV 21 340 NLTDPSSPTI 21 382 LATPIAIKAV 21 13 SLASTLDCET 20 YDLHSEVOTL 20 54 LKDDVNIPEL 20 132 GIDFIKATKV 20 264 ATPSP!IQQL 20 321 DLEVEDRTSL 20 110 QVLPNPPQAV 19 118 AVNLLDKARL 19 188 IVTPPTKQSL 19 303 IALVSTNYPL 19 365 IKIPEDILOL 19 77 DLSDPPVASS 18 199 KVLKTPKCAL 18 285 LVPTFCTPGL 18 381 NLATPIAIKA 18 398 LKHGQNIRDV 18 RSFCGKLRSL 17 112 LPNPPQAVNL 17 189 VTPPTKQSLV 17 290 CTPGLKIPST 17 294 LKIPSTKNSI 17 374 LLSKYNSNLA 17 389 KAVPPSKRFL 17 99 SDFGLERYIV 16 120 NLLDKARLEN 16 121 LLDKARLENQ 16 147 SMDIMKIREY 16 219 KLEHFG1SEY 18 257 RLNDNVFATP 16 276 SDAEYTNSPL 16 9 GKLRSLASTL 15 97 QLSDFGLERY 15 106 YIVSQVLPNP 15 TableXXXV-VI -HLA- A0201-l0mers-1 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and. the end position for each peptide is the start position plus nine.

Pos 1234567890 score 127 LENQEGIDFI 15 162 YSPRVKKNSV 15 211 DDFECVTPKL 15 229 TMCLNEDYTM 15 358 TPTPPEVTKI 15 28 ALDGEESDFE 14 38 DYPMRILYDL 14 43 ILYDLHSEVQ 14 113 PNPPQAVNLL 14 124 KARLENQEGI 14 191 PPTKQSLVKV 14 209 KMDDFECVTP 14 216 VTPKLEHFGI 14 261 NVFATPSPII 14 269 IIQQLEKSDA 14 280 YTNSPLVPTF 14 322 LEVEDRTSLV 14 331 VLNSDTCFEN 14 347 PTISSYENLL 14 2 DPIRSFCGKL 13 34 SDFEDYPMRI 13 74 VKDDLSDPPV 13 101 FGLERYIVSQ 13 111 VLPNPPQAVN 13 135 FIKATKVLME 13 142 LMEKNSMDIM 13 192 PTKQSLVKVL 13 223 FGISEYTMCL 13 239 GLKNARNNKS 13 256 SRLNDNVFAT 13 272 QLEKSDAEYT 13 287 PTFOTPGLKI 13 295 KIPST~kNSIA 13 296 IPSTKNSIAL 13 299 TKNSIALVST 13 302 SIALVSTNYP 13 304 ALVSTNYPLS 13 315 TNSSSNDLEV 13 332 LNSDTCFENL 13 354 NLLRTPtPPE 13 371 ILQLLSKYNS 13 14 LASTLOCETA 12 24 RLQRALDGEE 12 49 SEVQTLKDDV 12 59 NIPELSNCEN 12 79 SDPPVASSGI 12 87 CISGKSPRSP 12 89 SGKSPRSPQL 12 133 IDFIKATKVL 12 144 EKt4SMDIMKI 12 176 AINSONYKEE 12 180 DNYKEEPVIV 12 194 KQSLVKVLKT 12 TableXXXV-VI -HLA- A0201-l mers-I93PIEIB3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score.

196 SLVKVLKTPK 12 200 VLKTPKOALK 12 243 ARNNKSEEAI 12 253 DTESRLNDNV 12 277 DAEYTNSPLV 12 297 PSTKNSIALV 12.

313 -SKTNSS-SNDL 12 323 EVEDRTSILVL- 12 329 SLVLNSDTCF 12 385 PIAIKAVPPS 12.

A0201-l0mers.193P1 El B Each peptide is a portion: of SEQ ID N9. 11; each start position is specified, the length of peptidle is amino acids, and the end position for each peptide Is the start position plus nine.

Pos 1234567890 score 7 CISEKSPRSP 12 9SEIKSPRSPQL 12 2 PVASSCISEK 9 3 VASSC!SEKS 7- 6 SCISEKSPRS Ta bleXXXV-V6-H LA- A0201-1 Omers-1 93P1 El B Each peptide is a portion of SEQ ID N~Yr 13; each start position is specified, the length of peptide is amino acids, and the end.

position for each peptide Is the start position plus nine.

Pos 1234567890 score 9 DAESRLNDNV 12 7 AIDAESRLND 11.

81IDAESRLNDN 11.

5 EEAIDAESRL 9 2 NKSEEAIDAE 7.

3 KSEEAIDAES 6 TabIeXXXV-VI 0-H LA.

A0201 -l1mers- 193P1El B 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 peptidle is the start position plus nine.

Pos 1234567890 score 2 KIPEDILQKF 17~ 11 FQWIYPTQKL 14 14 IYPTQKLNKM 13 7 ILQKFQWIYP 12 13 WIYPTQKLNK 12 TableXXXV/-V1 2-HLA- A0201-l1mers-193P1 El B Each peolide is a portion of SEQ ID NO: 25; 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.

Pos 1234567890 score 9 SLLSKYNSNL 24 2 RALDGEESLL 17 3 ALDGEESLLS 16 1 QRALDGEESL 14 4 LDGEESLLSK 11 Tab~eXXXVI-V1 -H LA- A0203-l0mers-1 93P1 El B 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 stari position plus nine.

Pos 1234567890 score 6 SFCGKLRSLA 10 14 LASThDCETA 10 19 DCETARLQRA 10 KDDLSDPPVA 10 109'SQVLPNPPQA 10 116 PQAVNLLDKA 10 129 NOEGIDFIKA 10 167 KKNSVHEQEA 10 198 VKVLKTPKCA 10 234 EDYTMGLKNA 10 242 NARNNKSEEA 10 255 ESRLNDNVFA. 10 269 IIQQLEKSDA 10 295 KIPSTKNSIA 10 374 LLSKYNSNLA 10 378 YNSNLATPIA 10 381 NLATPIAIKA 10 7 FCGKLRSLAS 9 ASTLOCETAR 9 CETARLORAL 9 76 DDLSDPPVAS 9 110 QVLPNPPQAV 9 117 QAVNLLDKAR 9 130 QEGIDFIKAT 9 168 KNSVHEQEAI 9 199 KVLKTPKCAL 9 235 DYTMGLKNAR 9 TableXXXV1-VI-HLA- A0203-l0mers-193P1E1 B Each peptide Is a portion of SEQ ID N~r 3; each start position is specified, the length of peptidle is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 243 ARNNKSEEAI 9 256 SRLNDNVFAT 9 270 IQQLEI SDAE 9 296 IPSTKNSIAL 9 375 LSKYNSNLAT 9 379 kSNLATPIAI 9 382 LATPIAIKAV 9 8 CGKLRSLAST 8 16 STLDCETARL 8 21 ETARLORALD 8 77 DLSDPPVASS 8 111 VLPNPPQAVN 8 118 AVNLLDKARL 8 131 EGIDFIKATK 8 169 NSVH-EQEAIN 8 200 VLI TPKCALI( 8 236 YTMGLKNARN 8 244 RNNKSEEAID 8 257 RLNDNVFATP 8 271 QbLEKSDAEY 8 297 PSTKNSIALV 8 376 SKY-NSNLATP 8 380 SNLATPAIK 8 383 ATPIAIKAVP 8 TableXX(XVI-V5-HLA- A0203-l Omers- 193PIElB Pos 1234567890 score .NoResuftsFound.

TableXXXVI-V6-HLA- A0203-1 Omers- 193PIElB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptidle is the start position plus nine.

Pos 1234567890 score 1 NNKSEEAIDA 10 2 NKSEEADAE 9 3 KSEEAIDAES 8 Tab IeXXXVI-VI 0-HIA- A0203.1 Omers- 193P1E1B Pos 1234567890 score NoResultsFound.

TableXXXVIMV2-LA- A0203-l0mers- I93PIEIB Pos 1234567890 score NoResultsFound.

TableXXXVII-VI -HLA-A3- 1 Omers-193P1 El B Each peptide is a porton of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 390 AVPPSKRFLK *27 305 LVSTNYPILSK .26 62 ELSNCENFQK 24 82 PVASSCISGK 24 200 VLKTPKCALI *24 158 QKYGYSPRVK 23 126 RLENQEGIDF 22 196 SLVKVLKTPK 22 219 KLEHFGISEY -22 257 RLNDNVFATP 22 357 RTPTPPEVTK 22 387 AIKAVPPSKR 22 43 ILVDLHSEVQ 21 46 DLHSEVQTLK 21- 131 EGIDFIKATK 21 97 QLSDFGLERY 19 102 GLERYIVSQV 19 110 QVCPNP-PQAV 19 140 KVLMEKNSMD 19 291 TPGLKIPSTK 19 3 PIRSFCGKLR 18 24 RLQRALDGEE 18 111 VLPNPPQAVN 18 159 KYGYSPRVKK 18 323 EVEDRTSLVL 18.

380 SNLATPIAIK 18 386 IAIKAVPPSK 18 10 KLRSLASTLID 17 42 RILYDLHSEV 17 118 AVNLLDKARL 17 120 NLLDKARLEN 17 150 IMKIREYFQK 17 188 IVTPPTKQSL 17 190 TPPTKQSLVK 17 329 SLVLNSDTCF 17 373 QLLSKYNSNL 17.

77 DLSDPPVASS 16 185 EPVIVTPPTK 16 199 KVLKT:PKCAL 16, 368 PEDILQLLSK 16 9 GKLRSLASTL 28 ALDGEESDFE 115 PPQAVNLLDK 135 FIKATKVLME 152 KIREYFQKG 163 SPRVKKNSVH 165 RVKKNSVHEQ 00 00 TableXVII-VI -lLA-A3- I Omers-193P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 170 SVHEQEAINS 15 186 PVIVTPPTKQ 15 251 AIDTESRLND 15 272 QLEKSDAEYT 15 278 AEYTNSPLVP 15 311 PLSKTNSSSN 15 340 NLTDPSSPTI 15 348 TISSYEfNLLR 15 354 NLLRTP-TPPE 15 27 RALOGEESOF 14 EVQTLKDDVN 14 57 DVN IPELSNC 14 66 CENFQiKTDVK 14 136 IKATKV[LMEK 14 174 OEAINSDNYK.: 14 193 TKQSLVKVLK 14 207 ALK4MDDFECV 14 266 PSPIIQLEK 14 271 QQLEKSDAEY 14 284 PLVPTFCTPG 14 295 KIPSTKNSIA 14 321 OLEVEDRTSL- 14 355 LLRTPTPPEV 14 381 NLATPIAIKA 14 401 GQNIRDVSNK 14 17 TLDCETARLQ 13 87 CISGI(SPRSP 13 107 IVSQVLPNPP 13 143 MEKNSMDIMK 13 215 CVTPKLEHFG 13 238 MGLKNARNNK 13 268 PIIQQLEKSD 13 269 IIQQLEKSDA 13 298 STKNSIALVS 13 304 ALVSTNYPLS 13 330 LVLNSDTCFE *13 369 EDILQLLSKY 13 371 ILQLLSKYNS 13 376 SKYNSNLATP 13 A3-l0mers-193P1E1 B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 2 PVASSCISEK 24 7 CISEKSPRSP 12 TableXXXVII-V6-HLA-A3l0mers-193P1 El B Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 7 AIDAESRLND 17 10OAESRLN-DNVF 14 4 SEEAIDAESR 12 3 KSEEAIDAES TableXXXVII-VlO-HL-A- A3-l Omers.193P1E1 B 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 pepflde is the start position plus nine.

Pos 1234567890 score 13 WIYPTQKLNK 28 1 TKIPEDILK 22 10 KFQWIYPTQK 18 2 KIPEDILQKF 16 6 DILQKFQWIY 16 TableXXXVII-VI 2-H LA- A3-1 Omers-193P1 El B 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 score 3 ALDGEESLLS 18 4 LDGEESLLSK 16 9 SLLSKYNSNL 16 2 RALDGEESLL 10 5 DGEESLLSKY 10 TableXXXVIII-VI-HLA- A26-lomers-1 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 323 EVEDRTSLVL 30 369 EDILQLLSKYf 30 363 EVTKIPEDIL 29 214 ECVTPKLEHF 26 TableXXXVIII-VI-HLA- A26-1 Omers-1 93P1E1 B Each peptide is a portion of SEQ ID NO: 3; each start position is specified. the length of peptide is amino acids, and the end position for each peptide is the start position Plus nine.

Pos 1234567890 score 343 DPSSPTISSY 57 DVNIPELSNC 23 211 DDFECVTPKL 23 2 DPIRSFCGKL *22 264 ATPSPIIQQL 22 280 YTNSPLVPTF 22 188 IVTPPTKQSL 21 227 EYTMCLNEDY -21 347 PTISSYENLL -21 30 DGEESDFEDY 38 DYPMRILYOL 50 EVQTLKDDVN 173 EQEAINSDNY 192 PTKQSLVKVL 335 DTCFENLTDP 365 TKIPEDILQL 73 DVKDDLSDPP 19 199 KVLKTPKCAL 19, 249 EEAIDTESRL 19 16 STLDCETARL 18 21 ETARLQRALD 18 118 AVNLLDKARL 18 285 LVPTFCTPGL 18 366 KIPEDILQLL 18 5 RSFCGKLRSL 17 35 DFEDYPMRIL 17 253 DTESRLNDNV 17 321 DLEVEDRTSL 17 37 EDYPMRILYD 16 82 PVASSCISGK 16- 131 EGIDFIKATK 16 144 EKNSMDIMKI 16 155 EYFQKYGYSP 16 67 ENFQKTDVKD 151' 92 SPRSPQLSDF 97 QLSDFGLERY 147 SMDIMKIREY 151 MKIREYFQKY 175 EAINSDNYKE 185 EPVIVTPPTK 186 PVIVTPPTKG 219 KLEHFGISEY 221 EHFGISEYTM 250 EAIDTESRLN 279 EYTNSPLVPT 287 PTFCTPGLKI 325 EDRTSLVLNS 326 DRTSLVLNSD 56 DDVNIPELSN 14 77 DLSDPPVASS 14 165 RVKKNSVH-EQ 14 170 SVHEQEAINS 14 234 EDYTMGLKNA 14 TableXXXVIII-VI -HLA- A26-l Omers-193P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 261 NVFATPSPIl 14 301 NSIALVSTNY 14 A26-1 Omers-l 93P1 El B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, [fhe length of peptide is 10 amino acids, and the end position for each peptide is the start position pius nine.

Pos 1234567890. score 2 PVASSCISEK 16 9 SEKSPRSPQL 11 11 7 CISEKSPRSP* 7 TableXX(XVIII-V6-HLA- A26.l0mers-193P1E1 B Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each pepfide is the start position plus nine.

Pos 1234567890 score EEAIDAESRL 19 6 EAIDAESRLN 15 9 DAESRLNDNV 9 TableXXXVll-V1 0-H LA- A26-l Omers-1193P E'l B Each peptide is a portion of SEQ ID NO: 21; each start position is specified, the length of peptide is 10 amino adids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 6 DILQKFQWIY '22 2 KIPEDILQKF 19 EDILQKFQWI 14 1 TKIPEDILQK 12 TableXXXVIII-VI 2-HLA- A26-l0mers-193P1 El B Each peplide 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 score 5 DGEESLLSKY 26 TableXXXIX-VI -H LA- B0702-l0mers-l 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 296 IPSTKNSIAL 24 112 LPNPPQAVNL 23 2 DPIRSFCGKL 20 346 SPTISSYENL 20 191 PPTKQSLVKV 19 358 TPTPPEVTKI 19 92 SPRSPQLSDF 18 601IPELSNCENF 17 323 EVEDRTSLVL 14 384 TPIAIKAVPP 14 103 LERYIVSQVL 13 115 PPQAVNLLDK 13 118 AVNLLDKARL 13 133 IDFIKATKVL 13 163 SPRVK KNSVH 13 190 TPPTKQSLVK 13 199 KVLKTPKCAL 13 265 TPSPIIQQLE 13 332 LNSDTCFENL 13 365 .TKIPEDILQL 13 367 IPEDILOLLS 13 389 KAVPPSKRFL 13 391 VPPSKRFLKH 13 392 PPSI RFLKHG 13 16 STLDCETARL 12 20 CETARLQRAL .12 54 LKDDVNIPEL 12 113 PNPPQAVNLL 12 114 NPPQAVNLLD 12 185 EPVIVTPPTK 12 188 IVTPPTKQSL 12 192 PTKQSLVKVL 12 -194 KQSLVKVLKT 12 211 DDFECVTPKL 12 249 EEAIDTESRL 12 255 ESRLNDNVFA 12 264 ATPSPIIQQL 12 276 SIJAE'YTNSPL 12 285 LVPTFCTPGL 12 303 IALVSTNYPL 12 321 DLEVEDRTSL 12 343 DPSSPTISSY 12 363 EVTKIPEDIL 12 5 RSFCGKLRSL 11 TableXXXIX-VI .HLA- 80702-1 Omers-l 93P1 El B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 39 YPMRILYDLH 11 45 YDLHSEVQTL 11 81 PPVASSCISG 11 89 SGKSPRSPQL 11 94 RSPQLSDFGL 11 95 SPQLSDFGLE 11- 217 TPKLEHFGIS 11 223 FGISEY'TMCL 11 231 CLNEDYTMGL 11 283 SPLVPTFCTP .11 291 TPGLKIPSTK 11 349 ISSYENLLRT 11 360 TPPEVTKIPE 11 361 PPEVTKIPED 11 366 KIPEDI LOLL 11 80702-1 Omers-193P1 El B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 1 PPVASSCISE 11 9SEKSPRSPQL 11 TableXXXIX-V6-HL.A- B0702-1 Omners-11 93P1 El B Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 5 EEAJDAESRL 12 1OAESRLNDNVF 11 7 AIDAESRLND 7 I1 NNKSEEAIDA 6 9 DAESRLNDNV '6 TableXXXIX-Vl 0-HIA- B0702-10mers-193P1E1 B 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 00 I00 the start position plus nine.

Pos 1234567890 score 3 IPEOILQKFQ 12 11 FQWIYPTQKL 11 YPTQKLNKMR 10 EDILQKFQWI 8 2 KIPEDILQKF 7 8 LQKFQWlYPT 7 141IYPTQKLNKM 7 Tab~eXXXIX-V1 2-H LA- B0702-1 Omers-1 93P1 El B 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 pepfide is the start position plus nine.

Pos 1234567890 score 1 QRALDGEESL 11 2 RALDGEESLL 11 9 SLLSKY~NSNL 10 3 ALDGEESLLS 7 TableXL-VI -I-LA-B08- 10mers-193PIElB Pos 1234567890 score NoResultsFound.

TableXL-V5-HLA-B08- 10mers-193PlE1 B Pos 1234567890 score *NoResultsFound.

TableXL.V6-HLA-BOB8- I Omers-1 93P1 El B Pos 1234567890 score NoResultsFound.

TableXL-VI 0-HLA- BO-6mers-193P1 ElB Pos 1234567890 score NoResultsFound.

TableXL-V1 2-HLA- B08-l0mers-1193P1 l Pos 1234567890 score NoResultsFound.

TableXLI-VI-H-LA- Bi510-10Omers- I193PIE1B Pos 1234567890 score NoResultsFound.

81510-1 Omers- 193PIEIB Pos 1234567890 score NoResultsFound.

TableXU-V6-HLA- 81 510-10mers- 193PI EiB Pos 1234567890 score NoResultsFaund.

TableXLI-VI 0.HLA- B1510-l0mersi93PIEIB Pos 1234567890 score NoResultsFound.

TableXLI-V1 2-HLA- 81 510-j1iners- 193P1E1B Pos 1234567890 score NoResultsFound.

TableXLIl-VI -HLA- B2705-j1iners- 193PIEIB Pos 1234567890 score NoResultsFound.

B2705-10mets- 193P1E1 B Pos 1234567890 score NoResultsFound.

TableXLII-V6-HLA- B2705-10mers- 193P El B Pos 1234567890 score NoResultsFound.

TableXLli-V1 O-HLA- B2705-1 Omers- I93PIEIB Pos 1234567890 score NoResultsFound.

TableXLII-V1 2-HLA- 82705-1 Omers- 193P1E18 Pos 1234567890 score NoResultsFound.

TabteX~llI-VI-HIA- B2709-10mers- 193PIElB Pos 1234567890 score NoResultsFound.

TableXLIII-V5-HLA- 52709-10mers- 193P1El B Pos 1234567890 score NoResultsFound.

TableXLIII-V6-HLA- B2709-j1iners- I93PIEIB Pos 1234567890 score NoResultsFound.

Tab IeXLIII-V1 O-HLA- B2709-1 Diners- 193P1 EiB Pos 1234567890 score tNoResultsFound.

TableXiLII-Vi 2-H LA.

B2709-10Diers- I93PIEIB Pos 1234567890 score* !4oResultsFound.

TableXLIV-Vi -HIA- 84402-l0mors-193PIEIB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 36 FEDYPMRILY 24.

254 TESRLNDNVF 24 20 CETAFRLQRAL 23 249 EEAIDTESRL 22 103 LERYIVSQVL 21 127 LENQEGIOFI 21 365 TKIPEDILQL 21.

362 PEVTKIPEDI 264 ATPSPIIQQL 18 369 tD)LQLLSKY 18 113 PNPPQAVNLL 17 130 QEGIOFIKAT 17 278 AEYTN'SPLVP 17 133 IDFIKATKVL 16 147 SMDIMKIREY 16 151 MKIREYFQKY 16 2 DPIRSFCGKL 54 LKDDVNIPEL 112 LPNPPQAVNL 183 KEEPVIVTPP 294 LKIPSTKNSI 296 IPSTKNSIAL 323 EVEDRTSLVL 324 VEDRTSLVLN 347 PTISSYENLL 389 KAVPPSKRFL 5 RSFCGI LRSL 14 9 GK'LRSLASTL 14 16 STLDCETARL 14.

32 EESDFEDYPM 14 118 AVNLLDKARL 14 144 EKNSMDIMKI 14 148 MDIMKIREYF 14 184 EEPVIVTPPT 14 192 PTKQSLVKVL 14 199 KVLJ(TPKCAL 14 TableXUV.VI -HLA- B4402-1lOmers-193P1 B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 211 DDFECVTPKL 14 214 ECVTPKLEHF 14 219 KLEHFGISEY 14 223 FGISEYTMCL 14 226 SEYTMOLNED 14 233 NEDYTMGLI N 14 243 ARNNKSEEAI 14 301 NSIALVSTNY 14 343 DPSSPTISSY 14 366 KIPEDILQLL 14 379 NSNLATPIAI 14 YDLHSEVQTL 13 79 SDPPVASSCI 13 97 QLSDFGLERY 13 188 IVTPPTKQSL 13 285 LVPTFCTPGL 13 313 SKTNSSSNDL 13 332 LNSDTCFENL 13 352 YENLLRTPTP 13 358 TPTPPEVIKI 13 368 PEDILQLLSK 13 27 RALOGEESOF 12 34 SDFEDYPMRI 12 DFEDYPMRIL 12 38 DYPMRILYDL 12 61 PELSNGENFQ 12 66 CENFQKTDVK 12 89 SGKSPRSPQL 12 92 SPRSPQLSDF 12 126 RLENQEGIDF 12 143 MEKNSMDIMK 12 168 KNSVHEQEAI 12 173 EQEAINSDNY. 12 227 EYTMCLNEDY 12 248 SEEAIDTESR 12 280 YTNSPLVPTF 12 287 PTFCTPGLKI 12 322-LEVEDRTSLV 12 329 SLVLNSDTCF 12 338 FENLTDPSSP 12 363 EVTKIPEDIL 12 388 IKAVPPSKRF 12 395 KRFLKHGQNI 12 DGEESDFEDY 11 49 SEVQTLKDDV 11 IPELSNCENF 11 69 FQKTDVKDDL 11 94 RSPQLSDFGL 11 174 QEAINSDNY< 11 213 FECVTPKLEH 11 231 CLNEDYTMGL 11 261 NVFATPSPII 11 271 QQLEKSDAEY 11 Tab~eXLIV-VI -H LA- 84402-1 Omers-1 93PI Ell B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 273 LEKSDAEYTN 11 276 SDAEYTNSPL 11 303 IALVSTNYPL 11 321 DLEVEDRTSL 11 340 NLTDPSSPTI 11 346 SPTISSYENL 11 373 QLLSKYNSNL 11 TableXLIV-V5-IILA- B4402-l Omers-193P1 El B Each peptidle is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 9SEKSPRSPQL 22 TableXLIV-V6-HLA- B4402-l0mers-193P1 El B Each peptide is a portion of SEQ ID NO-. 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine.

Pos 1234567890 score 10 AESRLNDNVF 27 5 EEAIDAESRL 22 4 SEEAIDAESR 12 TableXLIV-VIO-HLA- B4402-l Diers- 193PIElB Each peptide is a porttion of SEQ ID MY 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.

Pos 1234567890 score 4 PEDILOKFQW 22 5 EDILQKFQWI 15 2 KIPEDILQKF 14 11 FQWIYPTQKL 12 1 TKIPEDILQK 11 6 DILQKFQWIY 11 TableXLIV-VI 2-H LA- B4402.l0mers-1 93P1 El B Each peptide is a portion of 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.

Pos 1234567890 score 7 EESLLSKYWS 14 2 RALDGEESLL 13 5 DGEESILLSKY 12 6 GEESLLSKYN 12 9 SLLSKYNSNL 12 1 QRALDGEESL 11 3 ALDGEESLLS 7 TableXLV-V1 -HLA-B51 01- I Omors-193P1 El B Pos 123456789012345 score NoResultsFound.

TableXLV-VS-HLA-B51 01- I Omers-l 93P1 El B Pos 123456789012345 score NoResultsFound.

TableXLV-V6-HLA-B51 01- 10mers-193P1 EIB Pos 123456789012345 score NoResultsFound.

TableXLV.VI 0-H LA-BSl 01- I Omers-1 93P1 El B Pos 123456789012345 score NoResultsFound.

TableXLV-VI 2-HLA-851 01- I Omers-1 93P1 El B Pos 123456789012345 score NoResultsFound.

TableXILVI-VI-HLA-DIRBI -0101- I 5mers-1 93P1 ElB B Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 130 QEGIDFIKATKVLME 33 124 KARLENQEGIDFIKA 31 168 KNSVIIEQEAINSDNY 31 38 DYPMRILYDLHSEVQ 29 300 KNSIALVSTNYPLSK 29 105 RYIVSQVLPNPPQAV 28 375 LSKYNSNLATPIAIK 28 291 TPGLKIPSTKNSIAL 27 23 ARLQRALDGEESDFE 26 116 PQAVNLLDKARLENQ 26 1531IREYFQKYGYSPRVK 26 TableXiVi-VI -H-LA-DRBI -0101 I 5mers-193P1 El B Each peptide is a portion of SEQ ID NO: 3: each start position is specified, the length of peptidle is amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 335 DTCFENLTDPSSPTI 26 8 CGKLRSLASTLDCET 25 229 TMCLNEDYTMGLKNA 25 267 SPIIQQLEKSDAEYT 25 307 STNYPLSKTNSSSNO 25 361 PPEVTKIPEDILQLL 25 369 EDILQLLSKYNSNLA 25 379 NSNLATPIAIKAVPP 25 RSFCGKLRSLASTID 24 185 EPVIVTPPTKQSLVK 24 195 QSLVKVLKTPKCAL< 24l 283 SPLVPTFCTPGLKIP 24 350 SSYENLLRTPTPPEV 24 353 ENLLRTPTPPEVKi 24 372 LQLLSKYNSNLATPI 24 71 KTDVKDDLSDPPVAS 23 82 PVASSCISGKSPRSP 23 SSCISGKSPRSPQLS 23 104 ERYIVSQVLPNPPQA 23 210 MDDFECVTPK[EHFG 23 256 SRLNDNVFATPSPII 23 338. FENLTDPSSPTISSY 23 368 PEOILQLLSKYNSNL 23 41 MRILYDLHSEVQTLK 22 101 FGLERYIVSQVLPNP 22 108 VSQVLPNPPQAVNLL 22 139 TKVLMEI NSMDIMKI 22 259 NDNVFATPSPIIQQL 22 321 DLEVEDRTSLVLNSD 22 376 SKYNSNLATPIAIKA 22 382 LATPIAIKAVPPSKR 22 385 PIAIKAVPPSKRFLK 22 214. ECVTPKLEHFGISEY 21 77 DLSDPPVASSCISGK 20 132 GIDFIKATKVLMEKN 20 286- VPTFCTPGLKIPSTK 20 1 MDPIRSFCGKLRSLA 19 33 ESDFEDYPMRILYDL 19 36 FEDYPMRILYDLHSE 19 98 LSDFGLERYIVSQVL 19 159 KYGYSPRVKKNSVHE 19 327 RTSLVLNSDTCFENL 19 349 ISSYENLLRTPTPPE. 19 383 ATPIAIKAVPPSKRF 19 4 IRSFCGKLRSLASTL* 18 92 SPRSPQLSDFGLERY 18 131 EGIDFIKATKVLMEK 18 136 IKATKVLMEKNSMDI 18 140 KVLMEKNSMDIMKIR 18 145 KNSMDIMKIREYFQK 18 189 VTPPTKQSLVKVLKT 18 194 l(QSLVKVLKTPKCAL 18 197 LVl VLKTPKCALKMD 18 199 KVLI(TPKOALKMIJDF 18 TableX-VII-VII-HLA-DRBI-01101.- I5mers.I93PIEIB Each peptidle is a portion of SEQ ID NO: 3: each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 205 KCALKMDDFECVTPK 18 227 EYTMCLNEDYTMGLK 18 235 DYTMGLKNARNNKSE 18 237 TMGLKNARNNKSEEA 18 265 TPSPIIQQLEKSDAE 18- 270 IQQLEKSDAEYTNSP 18 15 ASTLDCETARLQRAL 17 20 CETARLQRALDGEES 17 97 QLSDFGLERYIVSQV 17 102 GLERYIVSQVLPNPP 17.

110 QVLPNPPQAVNLLDK 17 137 KATKVLMEKNSMDIM 17 178 NSDNYKEEPVIVTPP 17 186 PVIVTPPTKQSLVKV 17 196 SLVKVLKTPKCALKM 17 207 ALKMDDFECVTPKLE 17 255 ESRLNDNVFATPSPI 17 285 LVPTFCTPGLKIPST 17 296 IPSTKNSIALVSTNY 17 302 SIALVSTNYPLSKTN 17 306 VSTNYPLSKTNSSSN 17 319 SNDLEVEDRTSLVLN 17 393 PSKRFLKHGQNIRDV 17 7 FCGKLRSLASTLDCE 16 12 RSLASTLDCETARLQ 16 40 PMRILYDLHSEVQTL 16 48 HSEVQTLKDDVNIPE 16.

57 DVNIPELSNCENFQK 16 72 TOVKODLSDPPVASS 16 ,75 KDDLSDPPVASSCIS 16 100 DFGLERYIVSQVLPN 16 107 IVSQVLPNPPQAVNL 16 142 LMEKNSMDIMKIREY 16, 147 SMDIMKIREYFQKYG 16 183 KEEPVIVTPPTKQSL 16 184 EEPVIVTPPTKQSLV 16 202 KTPKCALKMDDFECV 16 232 LNEDYTMGLKNARNN 16 240 LKNARNNKSEEAIDT 16 252 IDTESRLNDNVFATP 16 258 LNDNVFATPSPIIQQ 16 260 DNVFATPSPIIQQLE 16 290 CTPGLKIPSTKNSIA 16 293 GLKIPSTKNSIALVS 16 309 NYPLSKTNSSSNDLE 16 318 SSNDLEVEDRTSLVL 16 336 TCFENLTDPSSPTIS 16 351 SYENLLRTPTPPEVT 16 364 VTKIPEDILQLLSKY 16 371 ILQLLSKYNSNLATP 16 377 KYNSNLATPIAIKAV 16 380 SNLATPIAIKAVPPS 16 TableXLVl-V5-HLA-DRBI -0101.

I 5mers-11931El B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptidle is the start position plus fourteen.

Pos 123456789012345 score 10 SSOISEI SPRSPQLS* 23 2 DLSDPPVASSCISEK 6 PPVASSCISEKSPRS 7 PVASSCISEKSPRSP 12 CISEKSPRSPQLSDF 3 LSDPPVASSCISEKS: 14 9 ASSOISEKSPRSPOL .14 13 ISEKSPRSPQLSDFG .14 TableXLV-V6-HLA-DRBI -0101- I5mers-I93PIEIB Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 15 amino acids, and the*.end position for each peptidle is: the start position plus fourteen.: Pos 123456789012345 -score 4 ARNNKSEEAIDAESR .18 1 LKNARNNKSEEAIDA 16 131IDAESRLNDNVFATP .16 7 NKSEEAIDAESRLND 14 8 KSEEAIDAESRLNDN 111 14 DAESRLNDNVFATPS .11 2 KNARNNKSEEAIDAE 9 6 NNKSEEAIDAESRLN 8 9 SEEAIDAESRLNDNV -8 10 EEAIDAESRLNDNVF 8 12 AIDAESRLNDNVFAT 8 TableXLVI-ViO-HLA-DRBI-01 01- 15mers-193P1E1B Each peptide is a portion of SEQ ID NO: 21; each start position is.

specified, the length of peptidle it 15 amino acids,' and the end position for each peptidle is the start position plus fourteen. Pos 123456789012345 *score 2 PPEVTKIPEDILQKF 2 13 LQKFQWIYPTQKLNK 23 10 EDILQKFQWIYPTQK -18 14 QKFQWIYPTQKLNKM 18 5 VTKIPEDILQKFQWI 17 1 TPPEVTKIPEDILQK 8 IPEDILQKFQWIYPT TableXLVl-VI 2-HLA-DRBI01-1 I Smers-193P1 ElB B Each peptidle is a portion of SEQ ID NO: 25; each start position is specified, the length of peptidle is 15 amino acids, and the end position for each peptidle is the start position plus fourteen.

00 00 Pos 123456789012345 score 3 ARLQRALDGEESLLS 26 13 ESIISKYNSNLATPI 24 6 QRALDGEESLLSKYN 23 DGEESLILSKYNSNLA 17 12 EESILLSI(YNSINLATIP 16 9 LDGEESLLSKYNSNL 15 TableXLVII..VI-HLA-DRBI .0301 I 5mners-1 93P1 ElB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle is amino acids, and the end position for each peptidle is the start position plus fourteen.

Pos 123456789012345 -score 364 VTKIPEDILQLLSC-' 31 229 TMCLNEDYTMGLKNA 29 197 LVKVLKTPKCALKMD 28 371 ILQLLSI YNSNLATP. 28 51 VQTLKDDVNIPELSN 27 186 PVIVTPPTKQSLVKV 27 116 PQAVNLLDKARLENQ 26 -361 PPEVTKIPEDILQLL 26 67 ENFQKTDVKDDLSDP 25 *247 KSEEAIDTESRLNDN 24 319 SNDLEVEDRTSLVLN 24 PMRILYDLHSEVQTL* 23 205 KCALKMDOFECVTPK 22 174 QEAINSDNYKEEPVI 21 *327 RTSLVLNSDTCFENL 21 329 SLVLNSDTCFENLTD 21 138 ATKVLMEKNSMDIMK 20 346 SPTISSYENLLRTPT 20 13 SLASTLDCETARLQR 19 SPOLSDFGLERYIVS 19 118 AVNLLDKARLENQEG 19 124 KARLENQEGIDFIKA 19 145 KNSMDIMKIREYFQK 19 213 JECVTPKLEHFGISE 19 217 TPKLEHFGISEY-TMC 19 237 TMGLKNARNNKSEEA 19 249 -EEAIDTESRLNDNVF 19 *321 DLEVEDRTSLVLNSD 19 369 EDILQLLSKYNSNLA. 19 388 IKAVPPSKRFLKI-GQ 19 24 RLQRALDGEESDFED 18 44 LYDU-ISEVQTLKDDV 18 57 DVNIPELSNCENFQK 18 109 SQVLPNPPQAVNLLD 18 150 IMKIREYFQKYGYSP 18 194 KQSLVKVLKTPKCAL 18 266 PSPIIQQLEKSDAEY 18 271 QQLEKSDAEYTNSPL 18 283 SPLVPTFCTPGLKIP 18 293 GLKIPSTKNSIALVS 18 315 TNSSSNDLEVEDRTS 18 345 SSPTISSYENLLRTP 18 394 SKRFLKHGQNIRDVS 18 18 LDCETARLORALDGE 17 LQRALDGEESDFEDY 17 TableXLVII-VI -HLA-DRBI -0301l5mers-I93PIElB Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 29 LDGEESDFEDYPMRI 17 33 ESDFEDYPMRILYDL 17 60 IPELSNCENFQKTDV 17 132 GIDFIKATKVLMEKN 17 225 ISEYTMCLNEDYTMG 17 267 SPIIQQLEKSDAEYT 17 387 AIKAVPPSKRFLKHG 17 395 KRFLKHGQNIRDVSN 17 4 IRSFCGKLRSLASTL 16 74 VKDDLSDPPVASSCI 16 108 VSQVLPNPPQAVNLL 16 146 NSMDIMKIREYFQKY 16 147 SMDIMKIREYFQKYG 16 206 CALKMDDFECVTPKL 16 301 NSIALVSTNYPLSKT 16 -34 SDFEDYPMRILYDL- 15 117 QAVNLLDKARLENQE 15 274 EKSDAEYTNSPLVPT 15 TableXLIVIII-V5-HL11A-DRIBI -03011 15mers Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 ainino acids, and the end position for each peptidle is the start position plus [ourteen.

Pos 123456789012345 score .10 SSCISEKSPRSPQLS 12 5 DPPVASSCISEKSPR 11 12 CISEKSPRSPQLSDF 10 15 EKSPRSPQLSDFGLE 10 14 SEKSPRSPQLSDFGL 8 7 PVASSCISEKSPRSP 7 8 VASSCISEKSPRSPQ -7 11 SCISEKSPRSPQLSD 7 TableXLVII-V6-HLA-DRBI-0301 15mers Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 8 KSEEAIDAESRLNDN 24 10 EEAIDAESRLNDNVF 19 15 AESRLNONVFATPSP 13 TableXLVII-VI 0-HLA-DRBI- 0301-1 Smers Each peptidle 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.

Pos 123456789012345 score 5 VTKIPEDILQKFQWI 31 2 PPEVTKIPEDILQKF 26 9 PEIJILQKFQWIYPTQ 26 TableXiVII-VI 2-H LA-DRBI- 0301-1 Each pepUde is a portion of SEQ ID NO: 25; each start position is* specified, the length of peptidle is 15 amino acids, and the end.

posifion for each peptidle is *the start position plus fourteen.- Pos 123456789012345 score 12 EESLLSKYNSNLATP .28 4 RLQRALDGEESLLSK 26 5 LQRALDGEESLLSKY .18 6 QRAUJGEESLLSKYN .13 TableXLVIII-VI -HLA-DRI -0401l5mers-193P1E18 Each peptide is a portion of. SEQ ID NO: 3; each start position is specified, the length of peptiide is 15 amino acids, and the end position for each peptidle is the start position plus fourteen.

Pos 123456789012345 score 349 ISSYENLLRTPTPPE 28 40 PMRILYDLHSEVQTL 26 41 MRILYDLHSEVQTLK 26 44 LYDLHSEVQTLKDDV 26 57 DVNIPELSNCENFQK 26 229 TMCLNEDYTMGLKNA 26 237 TMGLKNARNNKSEEA :26 283 SPLVPTFCTPGLKIP 26 319 SNDLEVEDRTSLVLN 26 368 PEDILQLLSKYNSNL 26 98 LSOFGLERYIVSQVL 22 132 GIDFIKATKVLMEKN 22 157 FQKYGYSPRVKKNSV 22 179 SDNYKEEPVIVTPPT 22 307 STNYPLSKTNSSSND 22 335 DTCFENLTDPSSPTI *22 8 CGKLRSLASTLDCET 15 ASTLDCETARLQRAL 26 QRALDGEESDFEDYP 38 DYPMRILYDLHSEVQ 48 HSEVQTLKDDVNIPE 51 VQTLKDDVNIPELSN 60 IPELSNCENFQKTDV 71 KTDVKDDLSDPPVAS 109 SQVLPNPPQAVNLLD 116 PQAVNLLDKARLENQ 119 VNLLDKARLENQEGI 130 QEGIDFIKATKVLME 138 ATKVLMEKNSMDIMK 147 SMDIMKIREYFQKYG 00 00 TableXLVIII.VI.HLA-DRI .0401.

I Smers.1 93P1 El B Each peptide is a portion of SEQ ID NO: 3; -each start position is specified, the length of peplide is amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 185 EPVIVTPPTKQSLVK 20 '194 KQSLVKVLKTPKCAL 20 195 QSLVKVLKTPKCALK 20 205 KCALKMDDFECVTPK 20 249 EEAIDTESRLNDNVF 20 *259 NDNVFATPSPIIQQL 20 267 SPIIQQLEKSDAEYT 20 291 TPGLI IPSTKNSIAL 20 293 GLKIPSTKNSIALVS 20 300 KNSIALVSTNYPLSK 20 309 NYPLSKTNSSSNDLE 20 329 SLVLNSDTCFENLTD 20 338 FENLTDPSSPTISSY 20 -361 PPEVTKIPEDILQLL 20 364 VTKIPEDILQLLSKY 20 *369 EDILQLLSKYNSNLA. 20 372 LQLLSKYNSNLATPI 20 388 IKAVPPSKRFLKHGQ 20 RSFCGKLR.SLASTLD 18 77 IDLSDPPVASSCISGK 18 78 LSDPPVASSCISGKS 18 *97 QLSDFG3LERYlVSQV 18 *101 FGLERYIVSQVLPNP 18 106 YIVSQVLPNPPQAVN 18 122 LDKARLENQEGIDFI 18 170 SVHEQEAINSDNYKE 18 182 YKEEPVIVrPPTI<QS 18 214 ECVTPKLEHFGISEY 18 221 EHFGISEYTMCLNED 18 234 EDYTMGLKNARNNKS 18.

264' ATPSPIIQQLEKSDA 18 280 YTNSPLVPTFCTPGL 18- 290 CTPGLKIPSTKNSIA 18 320 NDLEVEDRTSLVLNS. 18 325 EDRTSLVLNSDTCFE 18 337 CFENLTDPSSPTISS 18 343 DPSSPTISSYENLLR 18 365 TKIPEDILQLLSKYN 18 376 SKYNSNLATPIAIKA 18 392 PPSKRFLKHGQNIRD 18 4 IRSFCGKLRSLASTL 17 33 *ESDFEDYPMRILYDL 16 *42 RILYDLHSEVQTLKD 16 103 -LERYIVSQVLPNPPQ 16 210 MDDFECVTPKLEHFG 16 225 ISEYTMCLNEDYTMG 16 260 DNVFATPSPIIQQLE. 16 277 -DAEYTNSPLVPTFCT 16 -375 LSKYNSNLATPIAIK 16 394 SKRFLKHGQNIRDVs 16 118 AVNLLDKARLENQEG, 15 139 TKVLMEKNSMDIMKI 15 321 DLEVEDRTSLVLNSD 15 371 ILQLLSKYNSNLATP 15 TableXiVIll-VI .HLA-DRI.0401 l5mers-193P1E1 B Each peptidle 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 peptidle is the start position plus fourteen.

Pas 123456789012345 score 1 MDPOIRSFCGKLRSLA 14 11 LRSLASTLDCETARL 14 22 TARLQRALDGEESDF 14 75 KDDLSDPPVASSCIS 14 80 DPPVASSCISGKSPR 14 95 SPQLSDFGLERYIVS 14 100 DFGLERYIVSQVLPN 14 105 RYIVSQVLPNPPQAV 14 108 VSQVLPNPPQAVNLL 14 140 KVLMEKNSMDIMKIR 14 150 IMKIREYFQ Y.GYSP 14 163 SPRVKKNSVHEQEA 14 168 KNSVHEQEAINSDNY' 14 174 QEAINSDNYKEEPVI 14 184 EEPVIVTPPTKQSLV 14 186 PVIVTPPTKQSLVKV 14 197 LVKVLKTPKCALKMD 14 198 VKVLKTPKCALKMDD 14 207 ALKMDDFECVTPKLE 14 217 TPKLEHFGISEYTMC 14 222 HFGISEYTMCLNEDY 14 227 EYTMCLNEDYTMGLK 14 270 IQQLEKSDAEYTNSP 14 302 SIALVSTNYPLSKTN 14 303 IALVSTNYPLSKTNS 14 328 TSLVLNSDTCFENLT 14 346 SPTISSYENLLRTPT .14 352 YENLLRTPTPPEVTK 14 353 ENLLRTPTPPEVTK 14 379-. NSNLATPIAIKAVPP 14 385 PIAIKAVPPSKRF[K 14 395 KRFLKHGQNIRDVSN 14 TableXLVIII-V5-HLA-DRI-0401 I 5mers-193P1 El B Each pepticle is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 amino acids, and the end position for each pepide is the start position plus fourteen.

Pos 123456789012345 score 2 DLSDPPVASSCISEK 18 3 LSDPPVASSCISEKS 18 9 ASSCISEKSPRSPQL 18 5 DPPVASSCISEKSPR 14 6 PPVASSCISEKSPRS 12 11 SCISEKSPRSPQLSD 12 12 GISEKSPRSPQLSDF 12 TableXLVIII-V6-HLA-DRI-0401 I Smers-1 93P1 El B Each peptidle is a portion of SEQ ID NO: 13: each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 10 EEAIDAESRLNDNVF 9 SEEAIDAESRLNDNV 18 1 LKNARNNKSEEAIOA 12 4 ARNNKSEEAIDAESR 12 6 NNKSEEAIDAESRLN 12 8 KSEEAIDAESRLNDN 12 14 DAESRLNDNVFATPS 12 15 AESRLNONVFATPSP 12 TableXLVIll-VI 0-H-LA-DRI -0401l5mers-I93PIEIB 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.

Pos 123456789012345 score 13 LQKFQWIYPTQKLNK 22 15 KFQWIYPTQKLNKMR .22 2 PPEVTKIPEDILQKF 6 TKIPEDILQI FOWIY -18 5 VTKIPEDILQKFQWI .14 10 EDILQKFQWYPTQK -14 4 EVTKIPEDILQKFQW 12 14 QKFQWIYPTQKLNKM 12 TableXLVIII-VI 2-HLA-DRI -0401l5mers-1193PIEIB3 Each peplide 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 start position plus fourteen.: Pos 123456789012345 score 6 QRALDGEESLLSKYN 26 13 ESLLSKYNSNLATPI 9 LDGEESLLSI(YNSNL 189 12 EESLLSKYNSNLATP 2 TARLORALDGEESLL :14 3 ARLQRALDGEESLLS 12 4 RLQRALDGEESLLSK 12 7 RALDGEESLLSKWNS :12.

10 DGEESLLSKYNSNLA .12 14 SLLSKYNSNLATPIA 12 TableXLX-I LA-DIRBI1 11.'l l5mers-I93PiEIB Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 159 I<YGYSPRVKKNSVHE 26 267 SPIIQQLEKSDAEYT 26 TableXLlX-VI -HLA-DRBI-i 101- 1 5mers.193P1 El B Each peptidle is a portion of SEQ ID NO: 3; each start position is specified, the length of peptidle is amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 369 EDILQLLSKYNSNLA 26 4 IRSFCGKLRSLASTL 25 349 ISSYENLLRTPTPPE 24 116 PQAVNLLDKARLENQ 22 335 DTCFENLTDPSSPTI 22 194 I QSLVKVLKTPKCAL 20.

233 NEDYTMGLKNARNNK 20 306 VSTNYPLSKTNSSSN 20 132 GIDFIKATKVLMEKN 19 157 FQKYGYSPRVKKNSV 19 38 DYPMRILYDLHSEVQ 18 105 RYIVSQVLPNPPQAV 18 300. KNSIALVSTNYPLS( '18 98 LSDFGLERYIVSQVL 17 153 IREYFQKYGYSPRVK 17 191 PPTKQSLVKVLKTPK 17 210 MDDFECVTPKLEHFG 17 286 VPTFCTPGLKIPSTK- 17 2 DPIRSFCGKLRSLAS 16 97 OLSDFGLERYIVSQV 15 144 EKNSMOIMKIREYFQ 16 .186 PVIVTPPTKQSLVKV 16 287 PTFCTPGLKIPSTKN 16 307 STNYPLSKTNSSSND 16 19 DCETARLQRALDGEE 15 68 NFQKTDVKDDLSDPP 15 319 SNDLEVEDRTSLVLN 15 361 PPEVTKIPEDILQLL 15 381 NLATPIAIKAVPPSK 15 388 IKAVPPSKRFLKHGQ 15 397 FLKHGQNIRDVSNKE 15 PMRILYDLHSEVQTL 14 104 ERYIVSQVLPNPPQA 14 118 AVNLLDKARLENQEG 14 137 KATKVLMEKNSMDIM 14 160 YGYSPRVKKNSVHEQ 14 175 EAINSDNYKEEPVIV 14 195 QSLVKVLKTPKCALK 14 -197 LVKVLKUPKCALKMD 14 213 FECVTPKLEHFGISE 14 214 ECVTPKLEHFGISEY 14 249 EEAIDTESRLNDNVF 14 255 ESRLNDNVFATPSPI 14 358 TPTPPEVTKIPEDIL 14 392 PPSKRFLKHGQNIRD 14 8 CGKLRSLASTLDCET 13 41 MRILYDU-ISEVQTLK 13 48 HSEVQTLKDDVNIPE 13 SSCISGKSPRSPQ[S 13 102 GLERYIVSQVLPNPP 13 127 LENQEGIDFII{ATKV 13 130 OEGIDFIKATKVLME 13 136 IKATKVLMEKNSMDI 13 TableXLIX-VI .HLA-DRBI-1 101- I 5mers.193P El B Each peptide is a portion of SEQ ID NO: 3; 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.

Pos 123456789012345 score 145 KNSMIJIMKIREYFQK 13 237 TMGLKNARNNKSEEA 13 293 GLKIPSTKNSIALVS 13 302 SIALVSTNYPLSKTN 13 318 SSNDLEVEDRTSLVL 13 365 TKIPEDILQLLSKYN 13 372 LQLLSKYNSNLATPI 13 376 SKYNSNLATPIAIKA 13 379 NSNLATPIAIKAVPP 13 385 PIAIKAVPPSKRFLK 13 5 RSFCGKLRSLASTLD 12 12 RSLASTLDCETARLQ 12 23 ARLQRALDGEESDFE 12 33 ESDFEDYPMRILYDL 12 57 DVNIPELSNCENFQK 12 -71 KTDVKDDLSDPPVAS 12 75 KDDLSDPPVASSGIS 12 82 PVASSCISGKSPRSP 12 121 LLDKARLENQEGIDF 12 147 SMDIMKIREYFQKYG 1-2 150 IMKIREYFQKYGYSP 12 165 RVI KNSVHEQEAINS 12 168 KNSVHEQEAINSDNY 12 181 NYKEEPVIVTPPTKQ 12 185 EPVIVTPPTKQSLVK 12 207 ALKMDDFECVTPKLE 12 225 ISEYTMCLNEDYTMG 12 232 LNEDYTMGLKNARNN 12 256 SRLNIJNVFATPSPII 12 277 DAEYTNSPILVPTFCT 12 282 NSPLVPTFCTPGLKI 12 291 TPGLKIPSTKNSIAL 12 350 SSYENLLRTPTPPEV 12 368 PEDILQLLSKYNSNL 12 382 LATPIAIKAVPPSKR 12 383 ATPIAIKAVPPSKRF 12 TabteXLlX-V5-HLA-DRB1Il101- I Smers-1 93P1 El B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 10 SSCISEKSPRSPQLS 13 7 PVASSCISEKSPRSP 12 3 LSDPPVASSCISEKS 8 8 VASSCISEKSPRSPQ 8 9 ASSCISEKSPRSPQL 8 11 SCISEKSPRSPQLSD 8 6 PPVASSCISEKSPRS 7 TableXLIX-V5.HLA-DRBI-l 101l5mers-193P1E1 B Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

Pos 123456789012345 score 131ISEKSPRSPQLSDFG 7 2 DLSDPPVASSCISEK 6 5 DPPVASSCISEKSPR* 6 TableXLIX-V6-HLA-DRBI -1101- I Smers-193P1 El B Each peptidle is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 15 amino acids, and the endposition for each peptidle is the start position plus fourteen.

Pos 123456789012345 score 10 EEAIDAESRLNDNVF 14 13 IDAESRLNDNVFATP 7 1 LKNARNNKSEEAIDA '6 4 ARNNKSEEAIDAESR 6 6 NNKSEEAIDAESRLN 6 7 NKSEEAIDAESRLND 6 14 DAESRLNDNVFATPS -6 TableXLIX-V1O-HLA-DR6I-1 101- I Smers-193PlE1 B 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 enoi position for each peptidle is the start position plus fourteen.

Pos 123456789012345 score 13 LQI FQWIYPTQKLNK '16 2 PPEVTKIPEDILQKF 7 KIPEDILQKFQWIYP .14 10 EDILQKFQWIYPTQK 12 15 KFQWIYPTQKLNKMR -11 5 VTKIPEDILQKFQWI 7 9 PEDILQKFQWIYPTQ 7 TableXLIX-VI -ILA-DRBI -1101- 15mers-193PIEIB 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 peptidle is the start position plus fourteen.

Pos 123456789012345 score 10 DGEESLLSKYNSNLA 6 QRALDGEESLLSKYN -13 13 ESLLSKYNSNLATPI 13 3 ARLQRALDGEESLLS 12 00 0 TS PAGE INTENTIONALLY LEFT BLANK

(O

Table L: Properties of 193PI ElB Variants 1, 5, 6 Bioinformatic Program URIL Outcome ORF ORF Finder hhpl/www.ncbinlm.govtgorf 805-2043 Protein.Length n/a nta 412 amino acids Transmembrane region TM Pred http:flwww.ch.embneLorg/ No TM HMMTop http://Www.enzim-hu/hmlmtop/ No TM Sosui http://www.gename.ad.ip/SOSui/ No TM, soluble TMHMM http:llwww.cbs.dtu.dk/serVices/TMHMM No TM Signal Peptide Signal P htp:/Iwww.cbs.dtu.dk/servces/SignalP/ indicates no signal pI p1/MW fool hftp:/www.expasy-chtools/ p1 5.03 Molecular weight pl/MWN tool http://www.expasy.ch/tools/ 46.2 kfla Localization PSORT http://psort.nibb.ac;.jp/ Mitochondrial 48% PSORT 11 http://psort.nibb.ac.jp/ Nuclear 60.9% iPSORT http-.ilpsort.nibb.ac.jp No signal Mott Motifs Pfam http:l/www.sanger.ac.uk/Pfarm/ No motif.

Prints htip:i/www.biochem.ucl.ac.uk/ Rhodopsin Blocks http://wvww.blocks.fhcrC.orgf No motif Prosite http://www.genome.ad.jp/ No mofif Variant 9 Bioinformatic Program URL Outcome ORF ORF Finder http://www.ncbi.nlm.gov/gorf 989-1981 Protein Length n/a n/a 330 amino acids Transmembrane region TM Pred http://www.ch.embnetorg/ No TM HMMTop http://lwvi.enzimi.hu/hmmtop/ No TM Sosui http:/Iwww.genome.ad jp/SOSuiI No TM, soluble TMHMM hltp://www.cbs.dlu.dk/servicesrrMHMM No TM Signal Peptide Signal P hftp://www.cbs.dtu.dk/services/SignalP/ indicates no signal PI p1/MW tool http://www.expasy.ch/tools p] 5.17 Molecular wieight p1/MW tool* hftp://www.expasy.chtoots 16.5 kDa Localization PSORT http://psort~nibb.ac.jp/ Cytoplasmic PSORT 11 httpl/psortnibb.acijp/ Nuclear 60.9% iPSORT httpJ/psort~nibb.ac.jp No signal motif Motifs Pfam httpi/www.sanger.ac.uk/Pfam/ No motif Prints http://www.bioch~em.udl.ac.uk/ No motif Blocks http://www.blocks.thcrc.org/ No motif Prosite http://www.genome.ad.jp! No motif Variant 10 Bioinformatic Program URL Outcome

ORF

Protein Length Transmembrane region .ORF Finder n/a IM Pred HMMTop Sosui

TMHMM

http://www.ncinim.govgort n/a http://www.ch.embnet.org/ http,.//wwvj.enzim.hu/hmmtop/ http:lfwww.genome.ad.jp/SOSui/ http:/twww.cbs.dtu.dk/services(TMIIMM 388 am ino acids No TM No TM No TM, soluble No TM Signal Peptide pi Molecular weight Localization Motifs Variant 12

ORF

Protein Length Transmembrane region Signal P p1/MW tool p1/MW tool

PSORT

PSORT 11 iPSORT Pfam Prints Blocks Prosite Bioinformatic Program O RE Finder n/a TM Pred HMMTop Sosui

TMIHMM

Signal P p1/MW tool p1/MW tool

PSORT

PSORT 11 iPSORT Pfamn Prints Blocks Prosite httP://www.cbs.dtu.dk/services/SignaI http:I/vww.expasy.ch/Itools/ http:/lwww.expasy.ch/tools/ http:/lpsort.nibb.ac.jp/ http:/lpsort.nibb.ac.jo/ http:/lpsort.nibb.acjp http://www.sanger.ac.uk/Pfam/ http://www,.biochem.ucl.acuk/ http://www.blocks.fhcrc.orgi http://www.genome.ad.jp/

URL

http:l/www.ncbi.nlm.gov/gorf n/a htt~p:lvWww.ch.embnet.org/ hfip:/lwww.enzimhu/hmmtop/ http:/Imvw.genome.ad.ip/SOSui/ http:/fwvw.cbs.dtu.dl(/servicesfFMHMM httP://www.cbs.dtu.dk/services/signalPi httpJ/www.expasy.ch/tools/ indicates no signal p1 4.8 34.5 kDa Mitochondrial 48% Nuclear 60.9% No signal motif No motif No motif No motif No motif Outcome 805-1026 73 amino acids No TM No TM No TMV, soluble No TM indicates no signal pi 9.4 Signal Peptide pI Molecular vweight Localization Motifs http://www.expasy.ch/tools/ 8.1 kDa http://psort.nibb.ac.jpl Mitochondrial 48% htlp://psort.nibb.ac.jp/ Nuclear 60.9% http://psort.nibb.ac.jp No signal motif htip://www.sanger.ac.uk/Pfami No motif litip://www.biochemuclacuk/ No motif http://www.blocks.fhcrc.orgi No motif http://www.genomead.jp/ No motif Table LI. Nucleotide sequence of transcript variant 193PIElB v.9 (SEQ ID NO: 93 61 121 181 241 301 361 421 481 541 601 661.

721 781 841 901 961 1021 1081 1141 1201 1261 tatcatctgt caattagact catgtttgtt tttcagttcc ogac tcagga atctatgcg tgcgcaccgc aatggttctc cgaaccgcg 9gagcgcagt gccagcagac 9agacttcgg ccgagattca, gagtccttcc tctctggcca gaaagcggat tgatttcata aagagagtat gcaagaagcc tgtgaaagat acgtagtcca aaaccc tcca 9actgaggaa tttaagtatt tgttttaata aggaccaggt ttaggaccat ggaac ttg ggcgtggccg Cgcctcgatc ccgccggt9c caggaacatg gccgt9gcgc ctctcgcgag aactagtggc ccgctgtgct gcacgc tyga gatgttaata aaggcaacaa ttccagaagt attaactctg gatctgtctg caactttrcag caggcagtga atccctatct ggggggttta aagactggtc aaagatggtc ttcttggtga gaagtggcgg cgctcctgct tCCaggcggc ttcccgatcc gtgcctgccg aagcgcaccc agaggactgc gggaggctgt cagca tgga c ctgcgagacg ttcttcttga aagtactaat atgga ta tag acccagagtt atcct-cctgt attttggact acaactataa tcctatcaga gagc t ctaga caaaggctca agctccgtga cattgagatg ccgCctttat ccggtcat ggatcatgtt actgacggcc cgctgctcaa gtctcgcggg gCCtgcgcag gagctgagcg cctatccgga gcccggc tgc taaagcaaga ggaaaaaaat ticca cgtgtc gtctaattgt tgcaagcagt tgagcggtac gjgaagagccc ctaatgaaac tattcyatat ttttcacaca tccataaaat gtcgagctgg ggcctcgaag gtagggcatg Ctgcttggcg 9ggaatgcgg gactctgCgt; gtctccgggg agccgaggac gtggggtctg gcttctgcgg agcgagcgct ttggaaaatc tcaatggata aagaaaaatt gaaaattttc tgtatttctg atcgtatccc gtaattgtaa cacaggacag gcagactact agctacaytt ccaagggtga tccqcaatga gCCtCCCtCC ctcagccagtcgcaacgaca ccgcgccaga Ctccgcg9cc gcctcggcga gcgtccggcg cgtacgcctg gaagctgcg ggacggagag aagaaggcat ttatgaaaat cagtacacga agaagactiga ggaagtctcc aagttctacc Ccccacctac 1321 1381 1441 Isol 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 264-1 2701 caaacaatca tgagtgtgta tgaagattac tacagaatcc ggaaaaaagt ttztgaaaatt aaaaacaaat ttcagacaca gaatctgctc gcttttatca cagtaaaagg aaattccagt accgatttta aagacaaaca ccttagtctt tgatactaaa aagtaatttc gccagagaga ccacaccttt aacaccagtt tgtgaccatt 9gtgtggact tcacgccttc cgtaaccctg ctagtaaaag actcctaaat acaatgggac aggctcaatg gatgccgaat ccatctacaa agttcatcaa tgc tttgaga agaacaccta aaatacaact ttccttaaac ggatctatcc acattcacat tgaacattaa gttgacatgt ttacccagct aaaaatataa cattgctgcc tacttgctca aaactaatta ggcggttgag tcggtgctct tattcccatt atgtacccct tactaaaaac tagaacactt ttaaaaatgc ataatgtttt ataccaactc agaacagcat atgatttgga atttaacaga cacctccaga caaacctagc atggacagaa aacacagaaa tgccctgcct catcataata agcccagtca taatcaacag aggtgtttgc agccagctct gtgctgtctg ggaaacagag accggtc ttc tccaagtttt ggcgctgaac ctaaaaggtg tccaaaatgt IzggtatCtCt gaggaat aat tgCCaCtCCC tcctttggta agct ttggta agttgaagat tccctcttca agtaac taaa tactccaata catccgagat ctgaacaaaa ctgtccccct tgctttttat ttcactcttt aatggtttaa tactcagatg gccttcccat aagatgcagt ggagatttcc aaccagtgga cacctggggg tCttaaggtC aggggic gcactaaaaa gaatatacta aaaagtgagg agccccatca cctacattct tccacaaatt cgtacttcgt cctacgattt attccagaag gcaattaaag gtcagcaaca tgaga tgaaa ttaaacgtt9 gaagtttcaa aaggactatt gtagtaccag aggccgcccc catctccttt tgctgtttgc aggcctgggt accccgaact gggfgagc taa actctggtc9 tggatgattt tgtgtttaaa aggccataga tccagcagtt gtactcctgg acccattatc tggtt ttaaa cttcttatga atattctcca cagtgccaco aagaaaac tg gccgagctgg acccatttta taaggtttaa agtgtttcat gaagtaggac tgaccttctg caggaccgtc aaacaacagg aactatatac ctgctgtcag ccccctatgt cttgtgaccc Table LII. Nucleotide sequence alignment ofi193Pl El Bv.1 (SEQ ID NO: 94) and 193P1EIII0 v.9 (SEQ ID NO: Score =1744 bits (907), Expect O.Oldentities 907/907 (100%) Strand Plus I Plus V.1: 1 V.9: I- tatcatctgtgactgaggaaatccctatctccatcagactaatjaaaccacaggacag V.1: 61 caattagacttttaagtattgggggtttagagctctagatattcgatatgcagactact 120 V.9: 61 caattag3acttttaagtattgggqggtttagagctctagatattcg3atatgcagactact 120 V.1: 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 V. 9: 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 V.1: 181 tttcagtccaggaccaggtaaagaktggtcagctccgtgatccataaaatccaagggtga 240 V. 9: 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 V. 1: 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V.9: 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V.1: 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360 V.9: 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360 V-1: 361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V.9: 361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatqtagggcatgctcagccagt 420 00 V.1: 421 aagtccgccaccaggcgacttcgtgcccagc 480 V.9: 421 aagtccgccaccaggcgacttcgtgcccagc 480 V. 1: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccg 540- V. 9: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggcccccg 540 rN~ V.1: 541 ggggatagaagtctcgccgtagccggccggc 600 V. 9: 541 ggggatagaagtctcgccgtagccggccggc 600 00 V. 1: 601 gcacgccggctaccccgcccggccgggccgg 660 V. 9: 601 9ccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctcggggctgg 660 V.1: 661 9agacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccgc 720 V.9: 661 gaatcgttgggggatcgcggaacggaggcgc 720 V.1: 721 ccaataatggcgagttggtacgggttctcct 780 V-9: 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtcgtcgc~tg 780 V. 1: 781 gatctcccggtactgccttcggtcgggacgg 840.

V.9: 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.1: 841. tccgcacccgatcaaggcgcgacaccgaga 900 841 tccgcacccgatcaaggcgcgacaccgagaa 900 V.1: 901 gaaagcg 907 1111111 V.9: 901 gaaagcg 907 Score =3519 bits (1830), Expect =0.Oldentifies 1830/1830 (100%) Strand =Plus Plus V.1: 969 ggtagtaattttgtagagttgaacaagcttgattt 102 8 V-9: 907 ggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaag ttgttt 966 V.1: 1029 caaagacaatcatgaaaatatgttagaaagg 1088 V-9: 967 caaa~acaatcatgaaaatatgttagaaagg 1026 V.1: 1089 gtttcgattgttgcagttagaatcgaaggag 1148 V.9: 1027 gtttcgattgttgcagttagaatcgaaggag 1086 00 V.1: 1149 agctaccgccggtttatggaattaagcgtta 1208 V. 9: 1087 agctaccgccggtttatggaattaagcgtta 1146 V. 1: 1209 agatgatctgtctgatcctcctgttgcaagcagttgtatttctggaagtctccacgtag 1268 V.9: 1147 agatgatctgtctgatcctcctgttgcaagcagttgtattctgggaagtctccacgtag 1206 V. 1: 1269 tcaactcgttgatggggaacttcagttcaac 1328 V.9: 1207 tcaactcgttgatggggaacttcagttcaac 1266 V.1; 1329 tcaagatacattagaagcgatgaccacacac 1388 00 ili 1111 1111111111111111111111111111111111111111111111111111 V.9: 1267 tcaagatacattagaagcgatgaccacacac 132 6 V.1: 1389 atcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgattttgagtg 1448 V.:1327 atcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgattttgagtg 1386 V.1: 1449 tgtaactcctaaattagaacactttggtatctctgaatatactatgtgtttaaatgaaga 1509 V.9: 1387 tgtaactcctaaattagaacactttggtatctctgaatatactatgtgtttaaatgaaga 1446 V.1: 1509 ttccaggctaatcagataaagggagctgtcg 1568 1447 ttccaggctaatcagataaagggagctgtcg 1506 V.1: 1569 atcgccagtagttgcctcaccactcgatgaa 1628 1507?Ilillil till till 1111111 II 111111111111111111111 liii II till1lul V.1: 1629 aatagcattcacccttgtetctcgatcgtta 1688 V. 9: 1567 aatagcattcacccttgtctctcgatcgtta 1626 V.1: 1689 aatcttcagaactgttgttccatacattaaa 1748 V.9: 1627 aatcttcagaactgttgttccatacattaaa 1686 V.1: 1749 aatgtacatatgagtaaactctgtgttatcg 1808 V.9: 1687 aaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggttttaaatcaga 1746 V.1: 1809 caagttaattaaaccctcctcatcttaggac 1868 V.9: 1747 caagttaattaaaccctcctcatcttaggac 1806 V.1: 1869 gccgaacaaccaagactaatcgaaattcgtt 1928 V.9: 1807 gccgaacaaccaagactaatcgaaattcgtt 1866 00 V.1: 1929 atcaaaatacaactcaaacctagctactccaatagcaattaaagcagtgccacccagtaa 1988 V.9: 1867 atcaaaatacaactcaaacctagctactccaatagcaattaaagcagtgccacccagtaa 1926 C1V.1: 1989 aag~ctaaagaaactcaagcgacagaataat 2048 V.9: 1927 aaggttccttaaacatggacagaacatccgagatgtcagcaacaaagaaaactgaaattc 1986 V-1: 2049 cagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctggaccgat 210.8 V.9: 1987 cagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctggaccgat 2046 00 V.1: 2109 tttaacattcacattgccctgcctctgtccccctttaaacgttga cccattttaaagaca 2168 V.9: 2047 tttaacattcacattgccctgcctctgtccccctttaaacgttgacccattttaaagaca 2106 V.:2169 aacatgaacattaacatcataatatgctttttatgaagtttcaataaggtttaaccttag 2228, V.9: 2107 aaagaataactaagtttagattatagtacta 2166 V.1: 22-29 tcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttcattgatac 228.8 V.9: 2167 tcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttcattgatac 2226 V.1: 2289 taatcccgtatacgagttagatcagatgaaga 2348 V.9: 2227 taaattacccagcttaatcaacagaatggtttaagtagtaccaggaagtaggacaagtaa 2286 V.1: 2349 ttcaattagtttcatagtagccctacttgcg 2408 V.9: 21287 ttcaattagtttcatagtagccctacttgcg 234*6 V.2: 2409 gaaatcgcgcgttctccacttctcgactcaa 2468 V.9: 2347 gaaatcgcgcgttctccacttctcgactcaa 2406 V.1: 2469 cttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaacaggaacacc 2528 V.9: 2407 cttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaacaggaacacc 2466 V.1: 2529 agttaaactaattaggaaacagagggagatttccaggcctgggtaactatatactgtgac 2588 V.9: 2467 agtacatagacggggttcagcggacaaatta 2526 V.1: 2589 cattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtcagggtgtg 2648 V.9: 2527 cattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtcagggtgtg 2586 V.1: 2649 Yacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatgttcacgc 2708 183 I1I11I1II1I11I11I1II1I 1I11I1II1I 1 I I t 1II1II 11I11I11II1II 1II 1 I II1II1II11I11I11II1II1 V.9: 2587 gacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatgttcacgc 2646 V.1: 2709 V.9: 2647 V.1: 2769 V.9: 2707 cttctattcccattggcgctaactcttaaggtcactctggtcgcttgtgaccccgtaac 2768 cttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgaccccgtaac 2706 cctgatgtacccctctaaaaggtgaggggc 2798 cctgatgtacccctctaaaaggtgaggggc 2736 Table LiII. Peptide sequences of protein coded by 193PIEIB v.9 (SEQ ID NO: 96) MEIQJSMDIMK IREYFQKYGY SPRVKKNSVH EQEAINSDPE LSNCENFQKT VASSCISGKS PRSPQLSDFG LERYIVSQVL PNPPQAVNNY KEEPVIVTPP TPKCALK-MDD FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAI FATPSPIIQQ LEKSDAEYT1N SPLVPTFCTP GLKIPSTKIS IALVSTNYPL EVEDRTSLaVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDIL ATPIAIKAVP PSKRFLKHGQ I'IRDVSNKEN

DVKDDLSDPP

TKQSLVKVLK

DTESRLNDNV

SKTNSSSNDL

QLLSKYNSNL

120 180 240 300 *330 Table LIV. Amino acid sequence alignment of 193P1 ElB v.1 (SEQ ID NO: 97) and 193PI ElB v.9 (SEQ ID NO: 98) Score 665 bits (1 716),: Expect O.0ldentilies =330/330 Positives= 330/330 (100%) V.1: 83 MEKNSM4DIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP. 142

MEKSMDIMKIRYFQKYGYSPRVCKKSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP

V.9: 1 MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP V.1: 143 VASSCISGKSPRSPQLSDFGLERYIVSQVLsPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202 VASSCI SGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 61 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120 V. 1: 203 TPKCAL KDDFBCVTPKLEI4FGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262 TPKCALKMDDFECVTPKLEHFGI SEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV V. 9: 121 TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKN'ARNNKSEEAIDTESRLNDNV 180 V.1: 263 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKN~SIALVSTNYPLSKTNSSSNDL 322 FATPSPI IQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNS IALVSTNYPLSKTNSSSNDL V.9: 181 FATPSPIIQQLEKSDA.EYTNSPLVPTFCTPGLKIPSTKHTSIALVSThYPLSKTNSSSNDL 240 323.EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 38? EVEDRTSLVLNSDTCFENLTDPSS PTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL V.9: 241 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLaRTPTPPEVTKIPEDILQLLSKYNSNL 300 V.1: 383 ATPIAIKAVPPSKRFLiKHGQNIRDVSNKEN 412

ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN

V.9: 301 ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 330 Table LV. Nucleotide sequence of transcript variant 193PiElB v.10 (SEQ ID NO: 99) 1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac ca-caggacag 61 121 181 241 301 361 421 481 541 caattagact Cattttgt~t tttcagttcc cgactcagga atctatgcgg tgcgcaccgc aatggttctc cgaaccgcgg ggagcgcagt tttaagtatt tgttttaat~a aggaccaggt ttaggaccat ggggaacttg ggcgtggccg cgcctcgatc ccgccggtgc caggaacatg ggggggttta aagactggtc aaagatggtc ttcttggtga gaagtggcgg cgctcctgct tccaggcggc ttcccgatcc gtgcctgccg gagctctaga c aaaggc tca agctccgtga cattgagatg ccgcctttat cc cgggt cat ggatcatgtt actgacggcc cgctgctcaa tattcgatat ttttcacaca tccataaaat gtcgagctqg ggcctcgaag gtagggcatg ctgcttgg gggaatgcgg gactctgcgt gcagactact agctacagtt ccaagggtga tccgcaatga gCCtCCCt;CC ctcagccagt cgcaacgaca ccgcgccaga Ct;CCgCggCC 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2 221 2281' 2341 2401 2461 2521 2581 2641.

gccagcagac gagacttcgg ccgagattca gagtccttc~c tctctggcca gaaagcgact actctaaagg attgatttca ataagagagt gagcaagaag gatgtgaaag ccacgtagtc ccaaaccctc accaaacaat tttgagtgtg aatgaagatt gatacagaat ttggaaaaaa ggtttgaaaa tcaaaaacaa aattcagaca gagaatctgc cagaaattcc tggaccgatt ttaaagacaa taaccttagt cattgatact gacaagtaat ctggccagag gtcccacacc aggaacacca tactgtgacc ca99gtgtgg tgttcacgcc ccccgtaacc gccgtggcgt ctctcgcgag aactagtggc ccgctgtgct gcacgctgga ttgaagatta atgatgttaa taaaggcaac at ttccagaa ccattaactc atgatctqtc cacaactttc cacaggcagt cactagtaaa taactcctaa acacaat9gg cc aggc tcaa gtgatgccga ttccatctac atagttcatc catgctttga tcagaacacc agtggatcta ttaacattca acatgaacat cttgttgaca aaattaccca ttcaaaaata agacattgct ttttaCttgC gttaaactaa attggcggtt aCttCggtgC ttctattccc ctgatgtacc aagcgcaccc agaggactgc ggga9gctgt cagcatggac ctgcgagacg tccaatgaga tattcttctt aaaagtacta 9tatqgatat tgacccagag tgatcctcct agattttgga gaacaactat agtactaaaa attagaacac acttaaaaat tgataatgtt atataccaac aaagaacagc aaatgatttg gaatttaaca tacacctcca tccaacacag Cattgccctg taacatcata tgtagcccag gcttaatcaa taaaggtgtt gccagccagc tcagtgctgt ttaggaaaca gagaccggtc tctztccaagt attgcgc g cctctaaaag gtctcgcggg gcctgcgcag gagctgagcg cc tatccgga gcccggctgc attttatatg gataaagcaa atggaaaaaa agtccacgt9 ttgtctaatt gttgcaagca cttgagcggt aaggaagagc actccaaaat t ttggtatct gcgaggaata tttgccactc tctcct t tgg atagctttgg gaagttgaag gatccctctt gaagtaacta aaactgaaca CCtctgtCCC atatgctttt tcattcactc cagaatggtt tgctactcag tctcctcc ctgaagatgc gagggagatt ttcaaccagt tttcacctgg aactcttaag gtgaggggc gtCtCCgggg agccgaggac gt999gtCtg gcttctgcgg agcgagcgct accttcattc gattggaaaa at tcaatgga tcaagaaaaa gtgaaaattt gttgtatttc acatcgtatc ccgtaattgt 9tgcactaaa ctgaatatac ataaaagtga ccagccccat tacctacatt tatccacaaa atcgtacttc cacctacgat aaattccaga aaatgagatg cctttaaacg tatgaagttt tttaaggact taagtagtac atgaggccgc catcatctcc ag ttgc tgtt tccaggcctg 9gaaccccga g9ggg9gagc gtcactctgg gcctcggcga gcgtccggcg cgtacgcctg gaagctgcgg ggacggagag agaagttcag tcaagaaggc tattatgaaa ttcagtacac tcagaagact tgggaagtct ccaagttcta aaccccacct aatggatgat tatgtgttta ggaggccata catccagcag CtgtactcCt ttacccatta gttggttttaL ttcttcttat agatattctc aaagccgagc ttgacccatt caataaggtt.

attagtgttt; caggaagtag ccctgacctt tttcaggacc tgcaaacaac ggtaactata actctgctgt taacccccta tcgcttgtga Table LVII. Nuclootide sequence alignment of I93PIEiB1 v.1 (SEQ ID NO: 100) and 193P1E18 v.10 (SEQ ID NO: 101) Score= 3698-bi Is (1923), Expect= 0.0 Identities= 1923/1923 (100%) Strand= Plus/ Plus V.1 I tatcatctgtgactgaggaaatccctatcttcctatcagactaatgaaaccacaggacag V. 10:' 1 tatcatctgtgactgaggaaatccctatcttcctatcagactaatgaaaccacaggacag V.1 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 V.1 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 V-1 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 V.1 241 cgactcaggattaggaccatttcttggtyacattgagatggtcgagctgqtccgcaatga 300 00 00 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V.1 :301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360 V.1 :361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V. 1 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 V. 10: 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 V.1 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V. 10: 4:81 cgaaccgcggccgccggtgct.tcccgatccactgacggccgggaatgcggccgcgccaga 540 V.1 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 V.1 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 661. gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V.1 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.1 :781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.1 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.1l 901 gaaagcgactttgaagattatccaatgagaattttatatgaccttcattcagaagttcag 960 901 gaaagcgactttgaagattatccaatgagaattttatatgaccttcattcagaagttcag 960 V.1: 961 actctaaaggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaaggc 1020 V.10:961 actctaaaggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaaggc 1020 00 00 V.1: 1021 V. 10: 1022.

V.1: 1081 V- 0: 1081 V.1: 1141 V. 10: 1141 V.1: 1201 V. 10: 1201 V.1: 1261 V. 10: 1261 V.1: 1321 V. 10: 1321 V.1: 1381 V. 10: 1381 V.1: 1441 V. 10: 1441 1501 V. 10;1501 V.1: 1561 V. 10: 1561 V.1: 1621 V. 1*0 :1621 V. 1: 1681 1681 V.1: 1741 V. 10: 1741 attgatttcataaaggcaacaaaagtactaatggaaaaaaattcaatggatattatgaaa attgat ttcataaaggcaacaaaagtactaatggaaaaaaattcaatggatattatgaaa ataagagagtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacac ataagagagtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacac gagcaagaagccattaactctgacccagagttgtctaattgtgaaaattttcagaagact qagcaagaagccattaactctgacccagagttgtctaattgtgaaaattttcagaagact gatgtgaaagatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtct gatgtgaaagatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtct ccacgtagtccacaactttcagattttggacttgagcggtacatcgtatcccaagttcta ccacgtagtccacaactttcagattttggacttgagcggtacatcgtatcccaagttcta ccaaaccctccacaggcagtgaacaactataaggaagagcccgtaattgtaaccccacct ccaaaccctccacaggcagtgaacaactataaggaagagcccytaattgtaaccccacct accaaacaatcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgat accaaacaatcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgat tttgagtgtgtaactcctaaattagaacactttggtatctc~9aatatactatgtgttta tttgagtgtgtaactcctaaattagaacactttggtatctctgaatatactatgtgttta aatgaagat tacacaatgggacttaaaaatgcgaggaataataaaagtgaggaggccata aa tgaagattacacaatgyyacttaaaaatgcgaggaataataaaagtgaggaggccata gatacagaatccaggctcaatgataatgtttttgccactcccagccccatcatccagcag gatacagaatccaggctcaatgataatgtttttgccactcccagccccatcatccagcag ttggaaaaaagtgatgccgaatataccaactctcctttggtacctacattctgtactcct ttggaaaaaagtgatgccgaatataccaactctcctttggtacctacattctgtactcct ggtttgaaaattccatctacaaagaacagcatagctttggtatccacaaattacccatta ggtttgaaaattccatctacaaagaacagcatagctttggtatccacaaattacccatta tcaaaaacaaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggtttta tcaaaaacaaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggtttta I S7 1080 1080 1140 1140 1200 1200 1260 1260 1320: 1320 1380 1380 1440 1440 1500 1500 15.60 1560 1620.

1620 1680 1680 1740 1740 1800 1800 00 00 V.1: 1801 aattcagacacatgctttgagaatttaacagatccctcttcacctacgatttcttcttat 1860 V. 10: 1801 aattcagacacatgctttgagaatttaacagatccctcttcacctacgatttcttcttat 1860 V.1: 1861 gagaatctgctcagaacacctacacctccagaagtaactaaaattccagaagatattctc 1920 V. 10:1861 gagaatctgctcagaacacctacacctccagaagtaactaaaattccagaagatattctc 1920 V.1: 1921 cag 1923 111 V.10:1921 cag 1923 Score 1456 bits (757). Expect 0.Oldentities 757/757 (100%) Strand Plus Plus V. 1: 2042 gaaattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctg V. 10 :1923 gaaattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctg V. 1: 2102 V. 10 :1983 V. 1: 2162 V. 10: 2043 V. 1: 2222 V. 10 :2103 V. 1: 2282 2163 V. 1: '2342 V. 10:2223 V. 1: 2402 V. 10: 2283 V. 1: 2462 V. 10: :2343 V.1: 2522 V. 10: 2403 gaccgattttaacattcacattgccctgcctctgtccccctttaaacgttgacccatttt gaccgattttaacattcacattgccctgcctctgtccccctttaaacgttgacccatttt aaagacaaacatgaacattaacatcataatatgctttttatgaagtttcaataaggttta aaagacaaacatgaacat taacatcataatatgctttttatgaagtttcaataaggttta accttagtcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttca acctta gtcttgttgacatgtaycccagtcattcactctttaaggactattagtytttca ttgatactaaattacccagct taatcaacagaa tggtttaagtagtaccaggaagtagga ttgatactaaattacccagcttaatcaacagaatggtt taagtagtaccaggaagtagga caagtaatttcaaaaatataaaggtgtttgctac tcagatgaggccgcccctgaccttct caagtaatttcaaaaatataaaggtgtttgc tactcagatgaggccgcccctgaccttct ggccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgt ggccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgt cccacaccttttacttgc tcagtgctgtctgaagatgcagttgctgtttgcaaacaacag cccacaccttttacttgc tcagtgctgtctgaagatgcagttgctgtttgcaaacaacag gaacaccagttaaactaattaggaaacagagggagatttccaggcctgggtaactatata gaacaccagttaaactaattaggaaacagagggagatttccaggcctgggtaactatata 2101 1982 2161 2042 2221 2102 2281 2162 2341 2222 2401 2282 2461 2342 2521 2402 2581 2462 V.1: 2582 V. 10:2463 ctgtgacca.*ttggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtca 2641 ctgtgaccattggcggttgagaccggtcttzcaaccagtggaaccccgaactctgctgtca 2522 V.1: 2642 gggtgtggacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatg 2701 :2523 gggtgtggacttcgytgctcttccaagttttcacctgggggggggagctaaccccctatg 2582 V.1: 2702 ttcacgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgacc 2761 V.10:2583 ttcacgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgacc 2642 V.1: 2762 ccgtaaccctgatgtacccctctaaaaggtgagyggc 2798 V.10:2643 ccgtaaccctgatgtacccctctaaaaggtgaggggc 2679 Table LVII. Peptide sequences of protein coded by 193PIElB v.10 (SEQ ID NO: 102) MDPIRSFCGK LRSLASTLDC ETARLQRALD GEESDFEDYP MRILYDLHSE VQTLKDDVNI LLDKARLENQ EGIDFIKATK VLMEK1NSMDI MKIRRYFQKY QYSPRVKKNS VHEQEAINSD PELSNCENFQ KTDVKDDLSD PPVASSCISG KSPRSPQLSD FGLERYIVSQ VLPNPPQAVN N~YKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL EHFGISEYTM CLUEDYTMGL KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY TNSPLVPTFC TPGLKIPSTK NSIALVSTNY PLSKTNSSSN DLEVEDRTSL VLNSDTCFEN LTDPSSPTIS SYENLLRTPT PPEVTKIPED ILQKFQWIYP TQKLNKMR 120- 180 240 300 360- Table LVIII. Amino acid sequence alignment of 193PIEIB v.1 (SEQ ID NO: 103) and 193P1E1B v.10 (SEQ ID NO: 104) Score 749 bits (1935), Expect =0.Oldentities 373/373 Positives =373/373 (100%) V. 1:1

I

M4DPIRS FCGKLRSLASTLDCET.URLQRLDGEESDFEDYPMRILYDLHSEVQThDVI MDPIRSFCGKRSLASTLDCETARLQPALDGEESDFEDYPMR ILYEJLHSEVQTLKDDV1NI MDPIRS FCGKLRSLASTLDCETARLQRALDGEESDPEDYPMRILYDLHSEVQTLMDI V. 1 61 LLDKARLENQEGIDFIATKVMEIWSMD IMKIREYFQKYGYSPRVKKMSVHEQEAINSD 120 LLDKAPLE4QEGIDFIKATKLMEMSDIMKIREYFQKYGYS PRVKKI4SVHEQEAI1NSD 61 LLIALNEIFKT7MMMIMIEFKGSRKMVEEIS 120 V.1 121 PELSNCENFQKTDVKDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVJTPNPPQAVN 180 PELSNCENFQKTDVKDDLSDPPVASSCISGKS

PRSPQLSDFGLERYIVSQVLPNPPQAVN

121 PELSNCENFQKTDVfcLDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVJ-TPNPPQAN 180 V.1 181 NYKEEPVIVTPPTKQSLVKVLKTPKCALIMDDFECVTPKLEHFGISEYTMCLNP-YTMGL 240.

NYKEEPVIVTPPTKQSLVKLKTPKCALKDFECVTPKEHFGISEYTMCLNEYTMGL

181 NYKEEPVIVTPPTKQSLVKVIKTPKCLKMDDFECVTPKEHFGISEYTMCLNEDYTMGL 240 V.1 :241 241 KNARNNKSEEAIDTESRLNDNVFATPSPI IQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK 300 KNARNNKSEEAIDTESRLNDNVFATPSPI

IQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK

KNJARNNKSEEAIDTESRLNflNVFATPSPI IQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK 300 V.1 301 NSALSNPSTSSDEERSVNDCELDSPISELRP 360 NSILVSTNYPLSkTNSSSNDLEVEDRTSLVLNSDTCFENLT6'PSSPTI

SSYENLLRTPT

10: 301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT 360* V.1 361 PPEVTKIPEDILQ 373

PPEVTKIPEDILQ

361 PPEVTKIPEDILQ 373 Table LIX. Nucleotide sequence of transcript variant 193PIElB v.11 (SEQ 10 NO: 105) 1 61 121 181 241 301 361 421 481 54 1: 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 tatcatctgt caattagact catgtttgtt tttcagttcc cgactcagga atctatgcgg tgcgcaccgc aatggttctc cgaaccgcgg ggagcgcagt gccagcagac gagact tcg ccgagattca gagtcc ttcc tctctggcca gaaagcggat tgattt-cata aagagagtat gcaagaagcc tgtgaaagat acgtagtcca aaaccctcca caaacaatca tgagtgtgta tgaagattac tacagaatcc ggaaaaaagt tt tgaaaatt aaaaacaaat ttcagacaca gaa tctgctc gaaattccag gaccgatttt aaagac aaac accttagtct ttgatactaa caagtaattt gyccagagag cccacacctt 9aacaccagt ctgtgaccat gggtgtggac ttcacgcctt ccgtaaccct gactgaggaa tttaagtatt tttttaata aggaccaggt ttaggaccat ggggaacttg ggcgtggccg CgCCtCgatC ccgccggtgc caggaacatg gccgtggcgt ctctcgcgag aactagtggc ccgctgtgct gcacgctgga 9atgttaata aaggcaacaa ttccagaagt attaactctg gatctgtctg caactttcag caggcagtga ctagtaaaag actcctaaat acaatgggac aggctcaatg gatgccgaat ccatctacaa agt tcat caa tgctttgaga agaacaccta tggatctatc aacattcaca atgaacatta tgttgacatg attacccagc caaaaatata acattgctgc ttacttgctc taaactaatt tggcggttga ttcggtgctc ctattcccat gatgtacccc atccctatct gggggttta aagactggtc aaagatggtc ttcttggtga gaagtggcgg cgctCc-tgCt tccaggcggc ttcccgatcc gtgcctgccg aagcgc accc agaggactgc gggaggctgt cagcatggac ctgcgagacg ttcttcttga aagtactaat atggatatag acccagagtt atcctcctgt attttggact acaactataa tactaaaaac tagaacactt ttaaaaatgc ataatgtttt dtaccaactc agaacagcat atgatttgga atttaacaga cacctccaga caacacagaa ttgCCCtgCC acatcataat tagcccagtc ttaatcaaca aaggtgtttg cagc cag t~c agtgctgtct aggaaacaga gaccggtctt tt'ccaagttt tggcgctgaa tctaaaaggt tc ctatc aga gagctctaga caaaggctca agctccgtga cattgagatg ccgcctttat cccgggtcat ggatcatgtt actgacggcc cgctgctcaa gtCtCgCggg gcctgcgcag gagctgagcg CCtatccgga gcccggctgc taaagcaaga ggaaaaaaat tccacgtgtc gtctaattgt tgcaagcagt tgagcggtac ggaagagccc tccaaaatgt tggtatctct gaggaataat tgccactccc tcctttggta agctttggta agttgaagat tccctcttca agtaactaaa actgaacaaa tctgtCCCCC atgcttttta attcactctt gaatggttta ctactcagat tgccttccca gaagatgcag gggagatttc c aaccagtgg tcacctgggg ctcttaaggt gaggggc ctaatgaaac tattcgatat ttttcacaca tccataaaat gtcgagctgg ggcctcgaag 9tagggcatg ctycttggC9 gggaatgcgg gactctgcgt gtCtCCgggg agccgaggac gtggggtctg gcttctgcgg agcgagcgct ttggaaaatc tcaatggata aagaaaaatt gaaaattttc tgtatttctg atcgtatccc gtaattgtaa gaatatacta aaaagtgagg agccccatca cctacattct tccacaaatt .cgtacttcgt cctacgattt attccagaag atgagatgaa tttaaacgtt tgaagtttca taaggactat agtagtacca gaggccgccc tcatctcctt *ttgctgtttg caggcctggg aaccccgaac gggggagcta c acaggacag gcagactact agctacagtt ccaagggtga tccgcaatga gCCtCCCtCC ctcagccagt cycaacgaca ccgcgccaga CtccgcgCC gcctcggcga gcgtccggcg cgtacgcctg gaagc tgcgg ggacggagag aagaaggcat t tatgaaaat.

cagtacacga agaagactga ggaagtctcc aagttctacc ccccacctac tggatgattt tgtgtttaaa aggocataga t ccagcagt t gtactcctgg acccattatc tggttttaaa cttcttatga atattctcca agccgag'ctg gacccatttt ataaggttta tagtytttca ggaagtagga CtgaCCttCt tcaggaccgt caaacaacag taactatata tctgctgtca accccctatg 9cttgtgacc Table LX. Nucleotide sequence alignment of 193PI EiB v.1 (SEQ ID NO: 106) and 193PI ElB v.11 (SEQ ID NO: 107) Score 1744 bits (907), Expect 0.Oldentities 907/907 (100%) Strand Plus Plus V. 1 I tatcatctgtgactgaggaaatccctatcttcctatcagacaatgaaaccacaggacag V.11: 1 tatcatctgtgactgaggaaatccctatcttcctatcagactaatgaaaccacaggadag V.1 1 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 V. 11: 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 V.1 1 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 00 V.11: 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 V.1 181 tttcagttccaggaccaggtaaagatggtcagctccgtqatccataaaatccaagggtga 240 V.11: 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 V. 1 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V. 11: 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V'.1 :301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccetcc 360 00V.11:. 301 atttcgggatgaggcgcgctagctgagcccc 360 V.1 :361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V.11: 361.tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V.1 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480' V.1.1: 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 V1:481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V.11: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V.1 :541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 V.11: 541 ygagcgcagtcaygaacatggtgcctgccgcgctgctcaagactctgcgtctccycggcc 600 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 V.11: 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660.

V.1 :661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V.11: 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V.1 721 ccaataatggcgagttggtacgggttctcct 780 V.11: 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtac 90 ctg 780 V.1 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.11: 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V-1 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.11: 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 00 V.1 :901 gaaagcg 907 ~fl V.11: 901 gaaagcg 907 Score 1836 bits (955), Expect 0.Oldentities =9551955 (100%) Strand Plus Plus V.1: 969 ggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaaggcattgattt 1028 V.11:907 ggatgatgttaatattcttcttgataaagcaagatyaaaatcaagaaggcattgattt 966 1029 cataaaggcaacaaaagtactaatygaaaaaaattcaatggatattatgaaaataagaga 108-8 00 V.1:6 cataaaggcaacaaaagtactaatggaaaaaaattcaatggatattatgaaaataagaga 1026 V.1: 1089 gtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacacgagcaaga 1148 V.11:1027 qtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacacgagcaaga 1006 1: 1149.: agccattaactctgacccagagttgtctaattgtgaaaattttcagaagactgatgtgaa 1208 V.11:1087 agccattaactctgacccagagttgtctaattgtgaaaattttcagaagactgatgtgaa 1146 V.1: 1209 agatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtctccacgtag 1268 V.11:1147 agatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtctccacgtag 1206 V.1: 1269 tccacaactttcagattttggacttgagcggtacatcgtatcccaagttctaccaaaccc 1328 V.11:1207 tccacaacttztcagattttggacttgagcggtacatcgtatcccaagttctaccaaaccc 1266 1329 tccacaggcagtgaacaactataaggaagagcccgtaattgtaaccccacctaccaaaca 1388 V.11:1267 tccacaggcagtgaacaactataaggaagagcccgtaattgtaaccccacctaccaaaca 1326 V.1: 1389 atcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgattttgagtg 1448 V.11:1327 atcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgattttgagtg 1386 V.1: 1449 tgtaactcctaaattagaacactttggtatctctgaatatactatgtgtttaaatgaaga 1508 V.11:1387 tgtaactcctaaattagaacactttggtatctctgaatatactatgtgtttaaatgaaga 1446 V.1: 1509 ttacacaatgggacttaaaaatgcgaggaataataaaagtgaggaggccatagatacaga 1568 V.11:1447 ttacacaatgggacttaaaaatgcgaggaataataaaagtgaggaggccatagatacaga 1506.

V.1: 1569 atccaggctcaatgataatgtttttgccactcccagccccatcatccagcagttggaaaa 1628 V.11:1507 atccaggctcaatgataatgtttttgccactcccagccccatcatccagcagttggaaaa 1566 V.1: 1629 V. 11: 1567 V.1: 1689 V. 11: 1627 V. 1: 1749 V. 11: 1687 V.1: 1809 V. 11: 1747 V.1: 1869 V. 11: 1807 aagtgatgccgaatataccaactctcctt tggtacctacattctgtactcctggtttgaa aagtgatgccgaatataccaactctcctttggtacctacattctgtactcctggtttgaa aattccatctacaaagaacagcatagctttggtatccacaaattacccattatcaaaaac aattccatctacaaagaacagcatagctt tggtatccacaaattacccattatcaaaaac aaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggt tttaaattcaga aaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggttttaaattcaga cacatgctt tgagaatttaacagatccctcttcacctacgatttcttcttatgagaatct cacatgctttgagaatttaacagatccctcttcacctacgatttcttcttatgagaatct gctcagaacacctacacctccagaagtaactaaaattccagaagatattctccag 1923 gctcagaacacctacacctccagaagtaactaaaattccagaagatattctccag 18GI Score= 1456 bits (757), Expect 0.Oldentities 757/757 (100%) Strand= Plus! Plus V.1: 2042 gaaattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctg 11 -1861 gaaattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctg V.1: 2102 gaccgattttaacattcacattgccctgcctctgtccccctttaaacgttgacccattt V.11: 1921 9accgattttaacattcacattgccctgcctctgtccccctttaaacgttgacccatttt V.1: 2162 aaagacaaacatgaacattaacatcataatatgctttttatgaagtttcaataaggttta V.11: 1981 aaagacaaacataacattaacatcataaatgctttttatgaagtttcaataaggttta V.1:2222.. accttagtcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttca V.11:2041 accttagtcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttca V. 1: 2282 .ttgatactaaattacccagcttaatcaacagaatggttaagtagtaccaggaagtagga V.11:2101 ttgatactaaattacccagcttaatcaacagaatggtttaagtataccaggaagtagga V.1: 2342 caagtaatttcaaaaatataaaggtgtttgctactcagatgaggccgcccctgaccttct V.11:2361 caagtaatttcaaaaatataaaggtgtttgctactcagatgaggccgcccctgaccttct V-1: 2402 ggccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgt V.11:2221 ggccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgt 1688 1626 1748 1686 1808 1746 1868 1*806 2101.

1920.

2161.

1980 2221 2040 22-81 2100 2341 2160 2401 2220 246.L 2280 V.I: 2462 V. 11: 2291 V.I: 2522 V. 11:-2341 V.1: 2582 V.11:2401 V.1: 2642 V. 11: 2461 V.I: 2702 V.11:2521 V.1: 2762 V.11:2581 cccacaccttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaacag 2521 cccacaccttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaacag 2340 gaacaccagttaaactaattaggaaacagagggagatttccaggcctgggtaactatata 2581 gaacaccagttaaactaattaggaaacagagggagatttccaggcctgggtaactatata 2400 ctgtgAccattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtca 2641 ctgtgaccattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtca 2460 gggtgtggacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatg 2701 gggtgtggacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatg 2526 ttcacgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgacc 2761.

ttcacgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtg* cc, 2580 ccgtaaccctgatgtacccctctaaaaggtgaggggc 27918 ccgtaaccctgatgtacccctctaaaaggtgaggggc 2617 Table LXI. Peptide sequences of protein coded by 193PIEIB v.11 (SEQ ID NO: 108)

MEKNSMDIMK

VASSCI SGKS

TPKCALKMDD

FATPSP IIQQ

EVEDRTSLTL

KLNKMR

IREYFQKYGY

PRS PQLSDFG

FECVTPKLEH

LEKSDAEYTN

MSDTCFENLT

S PRVKKNSVH

LERYIVSQVL

FGISEYTMCL

S PIVPTFCTP

DPSSPTISSY

EQEAINSDPE

PNPPQAVNNY

NEDYTMGLKHN

GLKI PSTKNS

ENLLRTPTPP

LSNCENFQKT

KEEPVIVTPP

ARNN'KSEEAI

IALVSTNYPL

EVTKI PEDIL

DVKDDLSDPP

TKQSLVKVLK

DTESRLNDNV

SKTNSSSNDL

QKFQWIYPTQ

Table LXII. Amino acid sequence alignment of i93PIEIB M. (SEQ ID NO: 109) and I93PIE1B v.11 (SEQID NO: 110) Score 589 bits (1518), Expect e-l671dentities =291/291 Positives 291/291 (100%) V.1I 93 V. 11: 1 V.1 143 V. 11: 61 V. 1 203 V. 11: 121 263 V.11: 181 V-1 323 V. 11: 241 MEKNSMDIMKIREYPQKYGYSPRVKKNSVHIEQE-AINSDPELSNCENFQKTDVKDDLSDPP 142

MEKSDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP

MEKlSMDIMKIREYFQKYGYSPRVKKRSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP VASSCISGKSPRS PQLSDF'GLERYIVSQV PNPPQAVbJNYKEEPVIVTPPTKQSLVKVLK 202 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEBPVIVTPPTKQSLVK.mK VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120 TPKCALKMDDFECVTPKLEHFGISEYTMC[NEDYTMGLKNARNNKSEEAIDTESRLNDNV 262

TPCLKDEVPLHGSYMLEYMLNRNSE-DERNN

TPKCALKMDDFECVTPKLHFGISEYTMCLNEDYTMGLNARNKSEEAJDTESPLgDN 180' FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKJSIALVSTNYPLSKTN~SSSNDL 322 FATPS P1IQQLEKSDAEYTNS PLVPTFCTPGLKIPSTKNS IAIJVSTNYPLSKTNSSSNDL FATPS PTIQQLEKSDAEYTNS PLVPTFCTPGLKI PSTKNS IALVSTNYPLSKTNS SSt'DL 240 EVEDRTSLVLNSDTCFEN'LTDPSSPTISSYENLLRTPTPPEVTrKIPEDILQ 373 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKI PEDILQ EVEDRTSLVLNSDTCFENTLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ 291 Table L-XIII. Nucleotide sequence of transcript variant 193PIElB v.12 (SEQ ID NO: 111) 00 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 tatcatctgt caattagact catgtttgtt tttcagttcc cgactcagga atctatgcgg tgcgcaccgc aatggttctc cgaaccgcg ggagcgcagt -gccagcagac gagacttcg ccgagattca gagtccttcc tctctggcca gaaagccttt ccacccagta aactgaaatt gctggaccga ttttaaagac tttaacctta ttcattgata aggacaagta ttCtggccag ccgtcccaca acaggaacac tatactgtya 9tcagggtgt tatgttcacg gaccccgtaa gactgaggaa tttaagtatt tgttttaata aggaccaggt ttaggaccat ggggaacttg ggcgtggCCg cgcctcgatc ccgccggtgc caggaacatg gccgtggcgt ctctcgcgag aactagtggc ccgctgtgct gcacgc tgga tatcaaaata aaaggttcct ccagtggatc ttttaacatt aaacatgaac gtcttgttga ctaaattacc atttcaaaaa agagacattg ccttttactt cagttaaact cca ttyycgy ggacttcggt CCttCtattC ccctgatgta atccctatct 9ggggttta aagactgg tc aaagatggtc ttcttggtga gaagtggcgg cgctcctgct tccaggcggc ttcccgatcc gtgcctgccg aagcgcaccc agaggactgc 999aggctgt cagcatggac ctgcgagacg caactcaaac taaacatgga tatccaacac cacattgccc attaacatca catg tagc c cagcttaatc tataaaggtg ctgccagcca gCtcagtgct aat taggaaa ttgagaccgg gctcttccaa ccat tggcgc cccctctaaa tcctatcaga gayctctaga caaaggctca agctccgtga cattgagatg ccycctttat cccgggtcat ggatcatgtt .actgacyggc cgctgctcaa gtctcgcggg gCCtgcgcag gagctgagcg cctatccgga gccyggotgc ctagctactc cagaacatcc agaaactgaa tgcctctgtc taatatgctt agtcattcac aacagaatgg tttgctactc gctctgcctt gtctgaagat cagaggyaga tcttcaacca gttttcacct tqaactctta aggtgagggg c taa tga aa a tattcgatat ttttcacaca tccataaaat gtcgagctgg ggcctcgaag gtaggcatg ctgcttggcg gggaatgcgg gactgcgt gtctccgggg agccgaggac g tggggtctg 9CttctgCgg agcgagogo t caatagcaat gagatgtcag caaaatgaga cccctttaaa tttatgaagt tctttaagga tttaagtagt agatgaggcc cccatcatct gcagttgctg tttccaggcc gtggaacccc 9gg9gg9gga aggtcactct cacaggacag gcagactact agctacagtt ccaagggtga tccgcaatga gcctccctCC ct cago cay t cgcaacgaca ccgcgocaga o tCCgCggCC gcctcggcga gcgtccggcg cgtacgcctg gaagc tgcgg ggacggagag taaagcagtg caacaaagaa tgaaagccga cgttgaccca t *tcaataag ctattagtgt, accaggaagt.

gococctgacc cctttcagga tttgcaaaca tg9gtaacta gaactctgct gctaaccccc ggtcgcttg t Table LXIV. Nucleotide sequence alignment of 193P1EIB v.1 (SEQ ID NO: 112) and I93PlEIB v.12 (SEQ ID NO: 113) Score 1742 bits (906), Expect O.0idenlities 900/06 (100%) Strand Plus t' Plus V. 1 1 tatcatctgtgactgaggaaatccctatct'cctatcagactaatgaaaccaaggacag V.1:1 tatcatctgtgactgaggaaatccctatcttcctatcagactaatgaaacaaggacag V.1 :61 ataattagatgggtaggttgttcaagaatc 120 V.12: 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgagactact 120 V.1 :121 cagtgtgttaaaatgccagccttcccacaat 180 V.12: 121 cagtgtgttaaaatgccagccttcccacaat 180 V.1 181 V. 12: 181 V.1 241 V.12: 241 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga.240 ttcgtcgacgtagtgcaccggtctaacaggg 240 cgactcaggattaggaccatttcttggtgacattgaatggtcgagctggtcgcaaga 300 cgactcaggattaggaccatttcttgggacattgagatggtcgagctggtcgaatga 300 00 V.1 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360 V.12. 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctcc ctcc 360 V.1 :361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V. 12: 361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V.1 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 V.12: 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 00 V.1 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V.12: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V.1 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 *V.12: 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc-600 V. 1 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 V. 12: 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V. 12: 663. gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V.1 :721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.12: 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.1 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.12: 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcggaagctgcgg 840' V. 1 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V. 12: 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.1 :901 gaaagc 906 V.12: 901 gaaagc 906 Score =1683 bits (875), Expect .Oldentities =875/875 (100%) Strand Plus /Plus V.1: 1924 cttttatcaaaatacaactcaaacctagctactccaatagcaattaaagcagtgccaccc 1983 V.12:907 cttttatcaaaatacaactcaaacctagctactccaatagcaattaaagcagtccaccc 966 V.1: 1984 agtaaaaggttccttaaacatggacagaacatccqagatgtcagcaacaaagaaaactga 2043 V.12:967 agtaaaaggttccttaaacatggacagaacatccgagatgtcagcaacaaagaaaactga 1026 V. 1: 2044 aattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctgga 2103 V.12:1027 aattccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctgga 1086 V.1: 2104 ccgattttaacattcacattgccctgcctctgtccccctttaaacgttgacccattttaa 2163 V. 12 :1087 ccattaataatgctctttccttaagtacatta1146 V.1: 2164 agcactactactaattgttttagtcaagtta 2223 V.12:1147 agacaaacatgaacattaacatcataatatgctttttatgaagtttcaataaggtttaac 1206 V. 1i 2224 ctattgtaagaccgcttatttagcatggtct 2283 V- 12:1207 cttagtcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttcatt 12.66 V.1: 2284 gatactaaattacccagcttaatcaacagaatggtttaagtagtaccaggaagtaggaca 2343 V.12:1267 gatactaaattacccagcttaatcaacagaatggtttaagtagtaccaggaagtaggaca 1326; V. 1: 2344 agtaatttcaaaaatataaaggttttgctactcagatgaggccgcccctgacctctgg 2403 V.12:1327 agtaatttcaaaaatataaaggtgtttgctaccagatgaggccgcccctgaccttctgg 1386.

V. 1: 2404 ccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgtcc 2463 V.12:1387 ccagagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgtcc 1446 V.1: 2464 cacaccttttacttgctcagtgctgtctgaagatgcagttgctgtttcaaaaacagga 2523 V.12:1447 cacaccttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaaagga. 1506 V.1: 2524 accataatatgaaaagggttcgctgtatttc 2583 V.12:1507 accataatatgaaaagggttcgctgtatttc 1566 V.1: 2584 gtactgcgtaacgctaacggaccgattcgcg 2643 V.12 :1567 gtactgcgtaacgctaacggaccgattcgcg 1626 V.1: 2644 gttgctgtcctcattcacggggggtaccttt 2703 V-12 :1627 gttgctgtcctcattcacggggggtaccttt 1686 V.1: 2704 cagctttcctgccgatctagcccgtgtggcc 2763 V. 12 :1687 cacgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgacccc 1746 197 V.2: 2764 gtaaccctgatgtacccctctaaaaggtgaggggc 2798 V.122:1747 gtaaccctgatgtacccctctaaaaggtgaggggc 1781 Table LXV. Peptide sequences of protein coded by 193PI ElB v.1 2 (SEQ ID NO: 114) MDPIRSFCGK LRSLASTLDC ETARLQRA1JD GEESLLSKYN SNLATPIAIK AVPPSKRFLK HGQNIRDVSN KEN 73 ,Table LXVI. Amino acid sequence alignment of 193P1IB v.1 (SEQ ID NO: 115) and 193P1IBv.12 (SEQ ID NO: 116) Score 72.0 bits (175), Expect 2e-1 21dentities =35/39 Positives 35/39 (89%) V.1 I MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDY 39 MDPIRS FCGKLRSLASTLDCETARLQRA 4 DGEES Y V.12: 1 MDPIRSPCGKLRSLA.STLDCETARLQRALDGEESLLSKY 39 Score 80.9 bits (198), Expect =4e-15Identities 39/39 Positives -39/39 (1.00%) V.1 374 LLSKY'NSNLATPIAIKAVPPSKRFLK{GQI'IRDVSNKEN 412 LLSKCYNSNLATPIAIKaVPPSKRFLKHGQNIRDVSNKEN V-12: 35 LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 73 Table LXVII. Nucleotide sequence of transcript variant 193P31EIB v.13 (SEQ ID NO: 117) 1 tatcatctgt gactgaggaa 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 18G1 caattagact catgtttgtt tttcagttcc cgactcagga atctatgcgg tgcgcaccgc aatggttctc cgaaccgcgg ggagcgcagt gccagcagac gagacttcgg ccgagattca gagtccttcc tctctggeca gaaagcggtg ttatatgacc aaagcaagat gaaaaaaatt ccacgtgtca tctaattgtg gcaagcagtt gagcggtaca gaagagcccg ccaaaatgtg ggtatctctg aggaataata gccactccca cctttggtac gctttggtat gttgaagatc ccctcttcac tttaagtatt tgttttaata aggaccaggt ttaggaccat ggggaacttg ggcytggccg cgcctcgatc ccgccggtgc caggaacatg gccgtggcgt ctctcgcgag aactagtggc ccgctgtgct g ca cgc tgga cgtgaggcgg ttcattcaga tggaaaatca caatggatat agaaaaattc aaaattttca gtatttctgg tcgtatccca taattgtaac cactaaaaat aatatactat aaagtgagga gccccatcat ctacattctg ccacaaatta gtacttcgtt ctacgatttc atccctatct ggggggttta aagactggtc aaagatggtc ttcttggtga gaagtggcgg cgctcctgct tccaggcggc ttcccgatcc gtgcctgccg aagcgcaccc agaggac tgc gggaggctgt cagcatggac ctgcgagacg gcggccaggg agttcagact agaaggcatt tatgaaaata agtacacgag gaagactgat gaagtctcca agttctacca cccacctacc ggatgatttt gtgtttaaat ggccatagat ccagcagttg tactcctggt cccattatca ggttttaaat ttcttatgag tcc tatcaga gagctctaga caaaggctca agc tccgtga cattgagatg ccgcctttat cccgggtcat 9gatcatgtt ac tgacggcc cgctgctcaa gtctcgcggg gcctgcgcag gagc tgagcg cctatccgga gcccggc tgc cacgactttg ctaaaggatg gatttcataa agagagtatt caagaagcca gtgaaagatg cgt agtccac aaccctccac aaacaatcac gagtgtgtaa gaagattaca acagaatcca gaaaaaagtg ttgaaaattc aaaacaaata tcagacacat aatctgctca ctaatgaaac tattcgatat ttttcacaca tccataaaat gtcgagctgg ggcctcgaag gtagggcatg ctgcttggcg gggaatgcgg gactctgcgt gtctccgggg agccgaggac gtggggtctg gcttctgcgg agcgagcgct aagattatcc atgttaatat aggcaacaaa tccagaagta ttaactctga atctgtctga aactttcaga aggcagtgaa tag taaaagt ctcctaaatt caatgggact ggctcaatga atgccgaata catctacaaa gttcatcaaa gctttgagaa gaacacctac cacaggacag gcagactact agctacagtt ccaagggtga tccgcaatga gcctccctcc ctcagccagt cgcaacgaca ccgcgccaga c t ccgcgg cc gcctcggcga gcgtccggcg cgtacgcctg gaagctgcgg ggacggagag aatgagaatt tcttcttgat agtactaatg tggatatagt cccagagttg tcctcctgtt ttttggactt caactataag actaaaaact agaacacttt taaaaatgcg taatgttttt taccaactct gaacagcata tgatttggaa tttaacagat acctc cagaa 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 gtaactaaaa ttccagaaga tattctccag Cttttatcaa aatacaactc aaacCtaact actccaatag atccgagatg tgaacaaaat tgtccccctt gct ttttatg tcactcttta atggtttaag a ctcagatga ccttCccatc agatgcagtt, gagatttcca accagtggaa acctgggggg cttaaggtca 9g9gC caattaaagc tcagcaacaa gaga tgaaag taaacgttga aagtttcaat aggactatta tagtaccagg ggccgcccct atctcctttc gctgtttgca ggcctgggta Ccccgaactc gggagctaac ctctggtcgc agtgccaccc agaaaactga ccgagctgga cccattttaa aaggtttaac gtgtttcatt aagtaggaca gaccttctgg aggac:cgtcc aacaacagga actatatact tgctgtcagg cccctatgtt ttgtgacccc agtaaaaggt aattccagtg ccgattttaa agacaaacat Cttagtcttg gatactaaat agtaatt tca ccagagagac cacac~ctttt acaccagtta gtgaccattg gtgtggactt, cacgccttct gtaaccctga tccttaaaca tggacagaac gatctatcca acacagaaac cattcacatt gccctgcctc gaacattaac atcataatat ttgacatgta gcccagtcat tacccagctt aatcaacaga aaaatataaa ggtgtttgct attgctgcca gccagctctg acttgctcag tgctgtctga aactaattag gaaacagagg gcggttgaga ccggtcttca Cggtgctctt ccaagttttc attcccattg gcgctgaact tgtacccctc taaaaggtga Table L-XVIII. Nucleotide sequence alignment of 193PIElB M. (SEQ ID NO: 118) and 193PIEIB v.13 (SEQID NO: 119) Score =1744 bits (9 07), Expect =O.Oldentities =907/907 (100%) Strand= Plus /Plus V.I1 1 tactttatagatcttctcacgcataaccgaa V.-13- 1 tactttatagatcttttcacgcataaccgaa V.I1 61. cataattagatgggttgg tgttcaagaatat 120 V.13. 61 cataattagatgggtt gttgaatgttcgcat 120 181 ttcgtcgacgtagtgcaccggtctaacaggg 240 V. 13: 181 ttcgtcgacgtagtgcaccggtctaacaggg 240 V.1 241 V.13: 241 V.1I 301 V.13: 301 V.1 361 V.13: 361 cgaccagattagacattcttgtgaattagatgtcagcggtcgcatga300 11111111 Ilttlgll 111t1tc11g11gIcItIIIIIII gt 11111 tgg111 gc(a111 300 atttcgggatgaggcgcgctagctgagcccc 360 atttcgggatgaggcgcgctagctgagcccc 360 Ilglglallgligc111gccgIg11c11gI 111 cgIIt111111111111111111111111 420 tggaccgggcggtcgtccgtagagctccgcg 360 V1:421 aatggttctccgcctcgatctccggacattcctgcccaca 8 00 V. 13: 421 aatggttctccgcctcgatctccaggcggcggatcatgttctgcttggcgcgcaacgaca 480 V.1: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V. 13: 481 cgaaccgcggccgccggtgcttcccgatccactgacggccgggaatgcggccgcgccaga 540 V.1 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 V. 13: 541 ggpgcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 V-1 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 00 V.13: 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 V.1 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V. 13: 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720..

V.1 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.13. 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V. 1 781 gagtcctticcccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.13: 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V. 1: 841 tctctgccagcacgctggactgcgaacggcccggctcagcgagcgtggacggagag 900 V. 13: 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.1 :901-gaaagcg 907 I I 1111 V.13: 901 gaaagcg 907 Score =3640 bits (1893), Expect .Oldentities 1893/1893 (100O) Strand Plus /Plus V.1 906 cgactttgaagattatccaatgagaattttatatgaccttcattcagaagttcagactct 965 V. 13: 933 cgactttgaagattatccaatgagaattttatatgaccttcattcagaagttcagactct 992 V.1: 966 aaaggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaaggcattga 1025 V.13:993 aaaggatgatgttaatattcttcttgataaagcaagattggaaaatcaagaaggcattga 1052 V. 1: 1026 tttcataaaggcaacaaaagtactaatggaaaaaaattcaatggatattatgaaaataag 1085 V. 13 :1053 tttcataaaggcaacaaaagtactaatggaaaaaaattcaatggatattatgaaaataag 1112 V.1: 1086 agagtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacacgagca 1145 200 V. 13:1113 V.1: 1146 V. 13:1173 1206 V. 13:1233 V.1: 1266 V. 13 :1293 V.1: 1326 V.13:1353 V.1: 1386 V.13;1413 V. 1: 1446 V. 13: 1473 V.1; 1506 V-.13: 1533 V.1: 1566 V.13: :1593 V.1: 1626 V.13:1653 V. 1: 1686 V. 13:1713 V. 1: 1746 V. 13:1773 V.1: 1806 V.13 :1833 agagtatttccagaagtatggatatagtccacgtgtcaagaaaaattcagtacacgagca agaagccattaactctgacccagagt tgtctaattgtgaaaattttcagaagactgatgt agaagccattaactctgacccagagt tgtctaattgtgaaaattttcagaagactgatgt gaaagatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtctccacg gaaagatgatctgtctgatcctcctgttgcaagcagttgtatttctgggaagtctccacg tagtccacaactttcagattttggac ttgagcggtacatcgtatcccaagttctaccaaa tagtccacaactttcagattttgacttgagcggtacatcgtatcccaagtttac~a ccctccacaggcagtgaacaactataaggaagagcccgtaat tgtaaccccacctaccaa ccctccacaggca9 tgaacaactataaggaagagcccgtaattgtaaccccacctaccaa acaatcactagtaaaagtactaaaaactccaaaatgtgca ctaaaaatggatgattt tga acaatcactagtaaaagtactaaaaactccaaaatgtgcactaaaaatggatgattttga gtgtgtaactcctaaattagaacactttggtatctctgaatatactatgtgtttaaatga gtgtgtaactcctaaattagaacactttggtatctctgaatatac Latgtgtttaaatga agattacacaatgggacttaaaaatgcgagqaataataaaaqtgaggaggccatagatac agattacacaatgggacttaaaaatgcgaggaataataaaagtgaggaggccatagatac agaatccaggctcaatgataatgtttttgccactcccagccccatcatccagcagttgga agaatccaggctcaatgataatgtttttgccactcccagccccatcatccagcagttgga aaaaagtgatgccgaatataccaactctcctttggtacctacattctgtactcctggttt gaaaattccatctacaaagaacagcatagctttggtatccacaaat tacccattatcaaa till 1t1111titacft gaa11g1111111t11111t1111111111111111111111 aacaaatagttcatcaaatgatttggaagttgaagatcgtacttcgttggttttaaattc aacaaatagttcatcaaatga tttggaagttgaagatcgtacttcgttggttttaaattc agacacatgctttgagaatttaacagatccctcttcacctacgatttcttcttatagaa agacacatgctttgagaatttaacagatccctcttcacctacgatttcttcttatgagaa 1172 1205 1232 1265 1292 1325 1352 1385 1412 1445 1472 1505 1532 1565 1592 1625 1652 1685- 1712 1745 1772 1805 1832 1865 1892 V.1: 1866 V. 13:1893 V. 1: 1926 V. 13 :1953 V.1: 1986 V. 13:2013 2046 V.13 :2073 V. 1: 2106 V. 13:2133 V. 1: 2166 V. 13 :2193 V. 1: 2226 V. 13 :2253 V. 1: 2286 V. 13:2313 V.1: 234.6 V. 13:2373 V.1: 2406 V. 13!2433 1: 2466 V. 13: 2493 2526 V. 13:2553 V. 1: 2586 V. 13 :2613 tctgctcagaacacctacacctccagaagtaactaaaat tccagaagatattctccagct tctgctcagaacacctacacctccagaagtaactaaaat tccagaagatattctccagct tttatcaaaatacaactcaaacctagctactccaatagcaattaaagcagtgccacccag tttatcaaaatacaactcaaacctagctactccaatagcaattaaagcagtgccacccag taaaaggttccttaaacatggacagaacatccgagatgtcagcaacaaagaaaactgaaa taaaaggttccttaaacatggacagaacatccgagatgtcagcaacaaagaaaactgaaa ttccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctggacc ttccagtggatctatccaacacagaaactgaacaaaatgagatgaaagccgagctggacc gattttaacattcacattgccctgcctctgtccccctttaaacgt tgacccattttaaag gattttaacattcacattgccctgcctctgtccccctttaaacgttgacccattttaaag acaaacatgaacattaacatcataatatgctttttatgaagt ttcaataaggtttaacct acaaacatgaacattaacatcataatatgctttttatgaagtttcaataaggtttaacct tagtcttgttgacatgtagcccagtcattcactctttaaggactattagtgtttcattga tagtcttgttgacatgtagcccagtcattcactctttaaggacta ttagtgtttcattga tactaaattacccagcttaatcaacagaatggtttaagtagtaccaggaagtaggacaag tac taaattacccagcttaatcaacagaatggtt taagtagtaccaggaagtaggacaaq taatttcaaaaatataaaggtgtttgctactcagatagccgcccctgaccttctggcc taatttcaaaaa tataaaggtgtttgctactcagatgaggccgcccctgaccttctggcc agagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgtccca agagagacattgctgccagccagctctgccttcccatcatctcctttcaggaccgtccca caccttttacttgctcagtgctgtctgaagatgcagttgctgtt tgcaaacaacaggaac caccttttacttgctcagtgctgtctgaagatgcagttgctgtttgcaaacaacaggaac accagttaaactaattaggaaacagagggagatttccaggcctgggtaactatatactgt accagttaaactaattaggaaacagagagatttccaggcctgggtaactatatactgt gaccattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtcagggt gaccattggcggttgagaccggtcttcaaccagtggaaccccgaactctgctgtcagggt 202 192S 1952 1985 2012 2045 2072 2105 2132 2165 2192 2225 2252 2285 2312 2345 2372 2405 2432 2465 2492 2525 2552 2585 2612 2645 2672 00 V.1: 2646 gtygacttcggtgctcttccaagttttcacctgggggggggagctaaccccctatgttca 2705 V.13 :2673 gtggacttcggtgctcttccaagttttcacctggggggggagctaacccctatgttca 2732 V. 1: 2706 cgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgaccccgt 2765 V.13:2733 cgccttctattcccattggcgctgaactcttaaggtcactctggtcgcttgtgaccccgt 2792 V.1 :2766 aaccctgatgtacccctctaaaaggtgaggggc 2798 V.13:2793 aaccctgatgtacccctctaaaaggtgaggggc 2825 00 Score =1744 bits (907), Expect 0Oldentities 907/907 (100%) Strand Plus /Plus V.1 1 tatcatctgtgactgaggaaatccctatcttcctatcagactaatgaaaccacaggacag V.13: 1 tatcatctgtgactgagqaaatccctatcttcctatcagactaatgaaaccacaggacag V.1 1 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 V. 13: 61 caattagacttttaagtattggggggtttagagctctagatattcgatatgcagactact 120 V.1 121 cagtgtgttaaaatgccagccttcccacaat 180 V.13: 121 catgtttgtttgttttaataaagactggtccaaaggctcattttcacacaagctacagtt 180 v.1 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 V.13: 181 tttcagttccaggaccaggtaaagatggtcagctccgtgatccataaaatccaagggtga 240 V.1 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V.13: 241 cgactcaggattaggaccatttcttggtgacattgagatggtcgagctggtccgcaatga 300 V.1 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctgaaggcct~ccctcc 360 V.13: 301 atctatgcggggggaacttggaagtggcggccgcctttatggcctcgaaggcctccctcc 360.

V-1 :3G1 tggaccgggcggtcgtccgtagagctccgcg 420 V. 13: 361 tgcgcaccgcggcgtggccgcgctcctgctcccgggtcatgtagggcatgctcagccagt 420 V.1 :421 aatggttctccgcctcgatctccaggcggCggatcatgttctgcttggcgcgcaacgaca 480 V.13: 421 aatggttctccgcctcgatctccaggcggcggatcatottctgttggcgcgcaacgaca 480 V. 1 481 cgaaccgcggccgccggtgcttcccgatccactgacgccggaatgcggcccgccaga 540 V.13: 481 cgaaccgcggccgccggtgcttcccgatccactgacgccgggaatgcggccgcgccaga 540 00 V.1 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 V.13: 541 ggagcgcagtcaggaacatggtgcctgccgcgctgctcaagactctgcgtctccgcggcc 600 V.1 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcgqggtctccgggggcctcggcga 660 V.13: 601 gccagcagacgccgtggcgtaagcgcacccgtctcgcggggtctccgggggcctcggcga 660 V.1 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 V. 13: 661 gagacttcggctctcgcgagagaggactgcgcctgcgcagagccgaggacgcgtccggcg 720 00 V.1 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.13: 721 ccgagattcaaactagtggcgggaggctgtgagctgagcggtggggtctgcgtacgcctg 780 V.1: 781 gaytccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.13: 781 gagtccttccccgctgtgctcagcatggaccctatccggagcttctgcgggaagctgcgg 840 V.1 841 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.13: 842 tctctggccagcacgctggactgcgagacggcccggctgcagcgagcgctggacggagag 900 V.1 901 gaaagcg 907 V.13: 901 gaaagcg 907 Table LXIX.Pcptide sequences of protein coded by 193PI ElB v.13 (SEQ ID NO: 120) MRILYDLHSE VQTLKDDVNI LLDKARLENQ EGIDFIKATK VLMEKNSMDI MKIREYFQKY GYSPRVKKNS VHEQEAINSD PELSNCENFQ KTDVIKDDLSD PPVASSCISG KSPRSPQLSD 120 P GLERYIVSQ VLPNPPQAVN N-YKEEPVIVT PPTKQSLVKV LKTPKCALK<M DDFECVTPITJ 180 EHFGISEYTM CLNEDYTMGL KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY .240 TNSPLVPTFC TPGLKIPSTK NSIALVSTNY PLSKTNSSSN~ DLEVEDRTSL VLNSDTCFEN 300 LTDPSSPTIS SYENLLRTPT PPEVTKIPED ILQLILSKYNS NLATPIAIKA VPPSKRFLKH 360 GQN'IRDVSNK EN 372 Table LXX. Amino acid sequence alignment of 193PI ElB v.1 (SEQ ID NO: 121) and 193PIEl B v.1 3 (SEQ ID NO: 122) Score =745 bits (1923), Expect 0.Oldentities =3721372 Positives =372/372 (100%) V. 1 :41 MRILYDLiHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKINSMDIMKIREYFQKY 100

MRILYDLHSEVQTLKDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY

V. 13: 1 MR IJYDLHSEVQTILKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY V.1 :101 GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 160 GYS PRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDIJSDPPVASSCISGKSPRSPQLSD V. 13: 61 GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 120 V. 1 161 FGLERYIVSQVLPNPPQAVNNYKE4EPVIVTPPTKQSLVKVLKTPKCALKDDFECVT1PKL 220 FGLERYIVSQVLPNPPQAVNN~YKEEPVIVTPPTKQ9LVKVLKTPKCALKMVDDFECVTPKL V.13: 121 FGLERYIVSQVLPNPPQAVNN KEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL 180 204 00 00 V.1 221 V-13: 181 V.1 :281 V.13: 241 EHFGSEYMCLNDYTGLKARNNSEEIDTSRLUNVFTPSiIQQEKSAEY280 EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLiNJVFATPS P1IQQLEKSDAEY EHFGISEYTMCLNEDYTMGLI1(NSEEAIDESRLFATPSPIIQQLEKDAEY 240 TNPVTCPLISKSAVTYPSTSSDEERSVNDCE 340

TNPVTCPLISMILSUPSTSSDEERSVNDCE

TNPVTCPLISKSAVTYPSTSSDEERSVNDCE 300 V.1 341 LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLS YSNATPIAIKVPPSJRFLyj 400 LTDPSSPTISSYENLLRTPTPPEVTKI PEDILQLLSKYNSNLATPIAIKAVPPSyJRFLyJ{ V.13: 301 LTPSTSYNLTTPVKPDLLSYSLTIIAPSRL 360 V. 1 401 V. 13: 3 61 GQNIRDVSNKEN 412 GQMIRDVSrncE GQNIRDVSNKENI 372

Claims (28)

1. A recombinant expression vector comprising a polynucleotide that encodes the polypeptide sequence at least 90% identical to SEQ ID NO:3. O O (N o (N 00 0-

2. The recombinant expression vector of claim 1, wherein the selected from the group consisting of: a polynucleotide comprising the sequence of SEQ nucleotide residue numbers 805 through 2043; 10 a polynucleotide comprising the sequence of SEQ nucleotide residue numbers 805 through 2043; and a polynucleotide comprising the sequence of SEQ nucleotide residue numbers 805 through 2043. polynucleotide is ID NO:2, from ID NO:10, from ID NO:12, from

3. The recombinant expression vector comprising a polynucleotide of any one of claims 1 or 2, wherein the vector is a viral vector.

4. The recombinant expression vector of any one of claims 1, 2, or 3, wherein the viral vector is selected from the group consisting of vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbis virus. The recombinant expression vector of any one of claims 1, 2, or 3, wherein the viral vector is adeno-associated virus.

6. The recombinant expression vector of any one of claims 1, 2, or 3, wherein the viral vector is vaccinia virus.

7. The recombinant expression vector of any one of claims 1, 2, or 3, wherein the viral vector is fowlpox virus.

8. A host cell that contains an expression vector of any of the preceding claims.

9. A process for producing a protein comprising culturing the host cell of claim 7 under conditions sufficient for the production of the protein, wherein the amino acid sequence of the protein is selected from the group consisting of SEQ ID NO:3, 11, and

13. 00 207 10. The process of claim 9, further comprising recovering the protein so produced. 11. The process of claim 10, wherein the protein is recovered using C chromatography. S12. A composition comprising a pharmaceutically acceptable carrier and the viral vector of any one of claims 3 to 7. 00 13. The composition of claim 12, wherein the viral vector is fowlpox. i 14. An antibody or fragment thereof that immunospecifically binds to an epitope on a protein comprising the amino acid sequence of SEQ ID NO: 11, or 13. The antibody or fragment thereof of claim 14, which is monoclonal.

16. The antibody or fragment thereof of claim 15, wherein the monoclonal antibody is a recombinant protein.

17. The antibody or fragment thereof of claim 16, which is a single chain monoclonal antibody.

18. The antibody or fragment thereof as described in any one of claims 14 to 17, wherein the fragment is an Fab, F(ab') 2 Fv or Sfv fragment.

19. The antibody or fragment thereof of claim 14, which is a human antibody. The antibody or fragment thereof of any one of claims 14 to 19, which is labeled with a cytotoxic agent.

21. The antibody or fragment thereof of claim 20, wherein the cytotoxic agent is selected from the group consisting of radioactive isotopes, chemotherapeutic agents and toxins.

22. The antibody or fragment thereof of claim 21, wherein the radioactive isotope is selected from the group consisting of 21 At, 131I, 125, 90Y, 86Re, 88Re, 53 Sm, 212Bi, 32p and radioactive isotopes of Lu. 00 208 O

23. The antibody or fragment thereof of claim 21, wherein the chemotherapeutic Sagent is selected from the group consisting of taxol, actinomycin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, gelonin, and calicheamicin.

24. The antibody or fragment thereof of claim 21, wherein the toxin is selected from 0 the group consisting of diphtheria toxin, enomycin, phenomycin, Pseudomonas 0 exotoxin (PE) A, PE40, abrin, abrin A chain, mitogellin, modeccin A chain, and alpha- Ssarcin. 00

25. The antibody or fragment thereof of any one of claims 14 to 24, wherein the 1 antibody or fragment thereof further comprises a pharmaceutically acceptable carrier.

26. A hybridoma that produces an antibody of claim

27. A vector comprising a polynucleotide encoding a monoclonal antibody according to claims 16 or 17.

28. An in vitro method for detecting the presence of a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13 or a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2, 10, or 12, in a test sample comprising: contacting the sample with an antibody or polynucleotide, respectively, that specifically binds to the protein or polynucleotide, respectively; and detecting binding of protein or polynucleotide, respectively, in the sample thereto.

29. The method of claim 28, wherein the polynucleotide is an mRNA. The method of claim 28, wherein the polynucleotide is a cDNA produced from the sample by reverse transcription.

31. The method of any one of claims 28-30, wherein the determining step comprises comparing an amount of binding of the antibody or polynucleotide that specifically binds to the protein or the polynucleotide to the presence of the protein or polynucleotide in a corresponding normal sample. 00 209 S32. The method of claim 31, wherein the presence of elevated polynucleotide or Sprotein in the test sample relative to the normal tissue sample provides an indication of the presence of cancer.

33. The method of claim 32, wherein the cancer is selected from the group 0consisting of leukemia and cancer of the prostate, testis, kidney, brain, bone, skin, ovary, breast, pancreas, colon, and lung, and the test and normal tissue samples are selected from the group consisting of serum, blood or urine and tissues of the prostate, 00 testis, kidney, brain, bone, skin, ovary, breast, pancreas, colon, and lung. 00 i 34. An in vitro method of inhibiting growth of a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, comprising providing an effective amount of an antibody according to any one of claims 13 to 24 to the cell, whereby the growth of the cell is inhibited. An in vitro method of delivering a cytotoxic agent to a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, comprising providing an effective amount of an antibody according to any one of claims 20 to 24 to the cell.

36. Use of a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, for the preparation of a medicament to induce an immune response from a subject, whereby a T cell or B cell is activated.

37. The use of claim 36, wherein the activated B cell generates antibodies that specifically bind to the protein.

38. The use of claim 36, wherein the activated T cell is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the protein.

39. The use of claim 36, wherein the activated T cell is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a CTL or the antibody producing activity of a B cell. Use of an effective amount of antibody or antigen binding fragment thereof for the preparation of a medicament which delivers a cytotoxic agent to a cell expressing a protein comprising the amino acid sequence of SEQ ID NO:3, 11, or 13, wherein the 00 210 N, antibody or antigen binding fragment thereof comprises the antibody according to any (1 one of claims 20 to 24. C1 41. The use of claim 40, wherein the medicament which inhibits growth of a cell expressing the protein. 00