AU2008200363A1 - Nucleic acids and corresponding proteins entitled 101P3A11 or PHOR-1 useful in treatment and detection of cancer - Google Patents

Description

AUSTRALIA

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

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Nucleic acids and corresponding proteins entitled 1 O1P3A 11 or PHOR-1 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 C NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 101P3AI1 or PHOR-1 USEFUL IN TREATMENT AND DETECTION OF CANCER 00 This is a divisional of AU 2002309873, the entire contents of which are incorporated herein by reference.

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CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority from U.S. Application Serial Number 00 10/017, 066, filed December 14, 2001, which is a continuation of; pending U.S.

Application Serial Number 10/001, 469, filed October 31, 2001, which claimed priority of; pending U.S. Provisional Application Serial Number 60/291,118, filed May 2001; the present application is also related to U.S. Application Serial Number 09/680,728, filed October 5, 2000, which claims benefit of priority from U.S.

Provisional Application Serial Number 60/157,902, filed October 5, 1999; each of the applications referenced in this paragraph are hereby incorporated in their entireties as if fully set forth herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

FIELD OF THE INVENTION The invention described herein relates to a gene and its encoded protein, termed 101P3A11 or PHOR-1, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 101P3A11.

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.

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SWorldwide, several cancers stand out as the leading killers. In particular, C carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the Sprimary 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 Cc cancers, common experience has shown that their lives are dramatically altered. Many

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Cc cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations 00 following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North N 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 0 the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated 0 with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the 00 serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the Sgeneration of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen C(i dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate Scancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 1445-51), STEAP (Hubert, e al., 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).

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

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma 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 carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

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

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 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 00 00 cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for Streatment 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. Colorcctal cancers are the third most common cancers in men 00 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 M 2000, accounting for about 11 of all U.S. cancer deaths.

SAt 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 00 0 permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed Sfor 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 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men per year) while rates for women were still significantly increasing per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women.

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

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

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

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black.

These decreases were probably the result of earlier detection and improved treatment.

00 Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer O 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 Shave shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar 0 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.

N Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast Stissue 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 00 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 children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

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

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

G Protein-Coupled Receptors G protein-coupled receptors (GPCR) share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane. The transmembrane helices are joined by strands of amino acids having a larger loop between the fourth and fifth transmembrane helix on the extracellular side of the membrane. Another larger loop, composed primarily of hydrophilic amino acids, joins transmembrane helices five and six on the intracellular side of the membrane. The carboxy terminus of the receptor lies intraccllularly with the amino terminus in the extracellular space. It is thought that the loop joining helices five and six, as well as the carboxy terminus, interact with the G protein. There is evidence that in certain GPCRs the first intracellular loop is also important for G-protein interactions. Currently, Gq, Gs, Gi, and Go are G proteins that have been identified.

00 Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different states or conformations: an "inactive" state and an "active" state. A receptor in an inactive state is unable to link Sto the intracellular transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway and produces a biological response.

A receptor may be stabilized in an active state by an endogenous ligand or an exogenous agonist ligand. 00 Recent discoveries, including but not exclusively limited to, modifications to the amino acid sequence of the receptor, provide alternative mechanisms other than ligands to stabilize the active state conformation. These Sapproaches effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the N receptor. Stabilization by such ligand-independent approaches is termed "constitutive receptor activation." A receptor for which the endogenous ligand is unknown or not identified is referred to as an "orphan receptor." Concerning traditional compound screening, in general, the use of an orphan receptor for screening Spurposes to identify compounds that modulate a biological response associated with such receptor has not been 0 possible. This is because the traditional "dogma" regarding screening of compounds mandates that the ligand for the receptor be known, whereby compounds that competitively bind with the receptor, by interfering or blocking the binding of the natural ligand with the receptor, are selected. By definition, then, this approach has no applicability with respect to orphan receptors. Thus, by adhering to this dogmatic approach to the discovery of therapeutics, the art, in essence, has taught and has been taught to forsake the use of orphan receptors unless and until the natural ligand for the receptor is discovered. The pursuit of an endogenous ligand for an orphan receptor can take several years and cost millions of dollars.

Furthermore, and given that there are an estimated 2,000 GPCRs in the human genome, the majority of which being orphan receptors, the traditional dogma castigates a creative approach to the discovery of therapeutics to these receptors. Numerous orphan G protein-coupled receptors are constitutively active in their endogenous state. Mouse olfactory receptor MOR 18-1 (>gi118479284, Figure 65). The endogenous ligand for 101P3A l is unknown.

SUMMARY OF THE INVENTION The present invention relates to a gene, designated 101P3A1 that has now been found to be overexpressed in the cancer(s) listed in Table I. Northern blot expression analysis of 101P3A I 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 101P3A11 are provided. The tissue-related profile of 101P3Al I in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 101P3A 1 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary to all or part of the 101P3A1 1 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 101P3A1 l-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, 317 or 318; or more than 317 or 318 contiguous amino acids of a 101P3AI I-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 101P3Al I genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 101P3A I1 genes, mRNAs, or to 101P3AI1- 00 o encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 101P3A 11. Recombinant DNA molecules containing 101P3A 11 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 101P3A11 gene products 00 0_ are also provided. The invention further provides antibodies that bind to 101P3A 1 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other Smammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies kN labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the O entire nucleic acid sequence of Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 (N is not prepared, either of which can be in respective human unit dose forms. In certain embodiments, the 00 entire nucleic acid sequence of Figure 2 is encoded and/or the entire amino acid sequence of Figure 2 is prepared, either of which can be in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 101P3A1 I polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 101P3AI 1. A typical embodiment of this invention provides methods for monitoring 101P3A 11 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 101P3A1 I such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 101P3AI I as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 101P3A11 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 101 P3A 11. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 101P3A1 I protein. Nonlimiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 101P3A 11 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety

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00 6A C1 that is immunologically reactive with 101P3AI 1 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 101P3A 1I. Non-limiting

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examples of such molecules include, but are not limited to, those complementary to a nucleotide 00 sequence essential for production of 101P3A 1 antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 101P3A 11 production) or a ribozyme effective to lyse 101P3A 1 mRNA.

N The present invention also provides a method to identify an agent that decreases the activity of a protein having the amino acid sequence set forth in positions 2-318 of SEQ ID NO:28, which method 0 comprises 00 providing a first sample of cells and a second sample of cells, wherein the cells of each sample Nc produce said protein; contacting the first sample with a candidate compound; measuring the activity of said protein in the first sample with the candidate compound; measuring the activity of said protein in the second sample, wherein the second sample has not been contacted with said candidate compound; comparing the measured activity of said protein in said first and second samples; whereby a decrease in the activity of said protein in said first sample as compared to said second sample identifies said compound as an agent that decreases activity of said protein; wherein said activity comprises cAMP accumulation mediated by said protein The present invention also provides a method to deliver a cytotoxic agent or a diagnostic agent to a cell, wherein said cell produces a protein having the amino acid sequence of positions 2-318 of SEQ ID NO:28, which method comprises exposing said cell to a conjugate of the cytotoxic agent or diagnostic agent coupled to an antibody or fragment thereof that binds said protein.

The present invention also provides a method to identify a peptide useful as a vaccine to elicit an immune response to a protein comprising positions 2-318 of the amino acid sequence of SEQ ID NO:28, which method comprises identifying an HLA supertype for which binding of epitopes of said vaccine is desired; selecting from the peptides listed in one of Tables VI to LI any epitopes of SEQ ID NO:28 disclosed to bind alleles of said identified supertype; experimentally assessing the ability of said peptides to bind to at least one allele of said HLA supertype and identifying as a peptide useful as a vaccine a peptide that binds with an IC 50 equal to, or less than, 500 nanomolar to said HLA supertype allele.

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Note: To determine the starting position of any peptide set forth in Tables V-XVIII and XXII to SIL (collectively HLA Peptide Tables) respective to its parental protein, variant 1, variant 2, etc., i reference is 00

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00 00 O made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table LII. Generally, a unique Search Peptide is used to obtain HLA peptides of a partiular for a Sparticular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table SLII. Accordingly if a Search Peptide begins at position one must add the value "X 1" to each position in 00 Tables V-XVIII and XXII to 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 is parental molecule, one must add 150 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

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n BRIEF DESCRIPTION OF THE FIGURES: Figure 1. The 101P3Al1 SSH sequence.

0 Figures 2A-2C. The cDNA and amino acid sequence of 101P3AI1 variants 1-3. The start methionine is underlined. The open reading frame for variants I and 3 extends from nucleic acid 133 to 1086 including the ci stop codon, The codon for the initial M in each variant can be omitted as the shorter peptide can have a more favorable Kozak sequence).

Figure 3A-C. Amino acid sequences of 10P3AI I variants 1-3.

Figure 4. Alignment of 101P3AI (Sbjct) with mouse olfactory receptor S25 (Query.) The transmembrane regions of 101P3A I and mouse olfactory receptor S25 (ORS25)predicted using the TMHMM algorithm are highlighted in gray. The amino acids of ORS25 predicted (Floriano, et al, 2000, Proc. Natl.

Acad. Sci., USA, 97:10712-10716) to be involved in binding of the ligand hexanol and/or involved in the formation of the ligand binding pocket are'italicized and bolded in the Figure, and are: Leu 131, Val 134, Val 135, Gly 138, Thrl39, Ser 193, Ser 197, Phe 225, Ala 230, lie 231, Gly 234, Thr 284, Phe 287, Gin 300, Lys 302.

Figure 5. Hydrophilicity amino acid profile of 101P3A 11 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp Woods 1981. Proc. Natl. Acad. Sci. U.S.A.

78:3824-3828) accessed on the Protscale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 6. Hydropathicity amino acid profile of 101P3A I 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 (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 7. Percent accessible residues amino acid profile of 101P3A1 I determined by computer algorithm sequence analysis using the method of Janin (Janin 1979 Nature 277:491-492) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 8. Average flexibility amino acid profile of 101P3A1 I 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 (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

Figure 9. Beta-turn amino acid profile of 101P3A I1 determined by computer algorithm sequence analysis using the method ofDeleage and Roux (Dclcage, Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

00 0 Figure 10A. Expression of 101P3A 1I by RT-PCR. First strand cDNA was prepared from vital pool 1 S(VP liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool, prostate Scancer pool, kidney cancer pool colon cancer pool, breast cancer pool, and cancer metastasis pool.

Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers 00 to 101P3Al 1, was performed at 30 cycles of amplification. Expression of 101P3AI I was observed in prostate xenograft pool, prostate cancer pool, kidney cancer pool, colon cancer pool, breast cancer pool, and cancer metastasis pool, but not in VP and VP2.

Figure 10B. Expression of 101P3AI 1 in human cancers demonstrated by dot blot analysis of tumor RNA M and normal RNA matched samples using patient-derived amplified cDNAs. Up-regulation ofPHOR-1 0 Sexpression was found in 3 of 3 prostate cancer patients, 6 of 14 kidney cancer patients, 2 of 8 uterine cancer patients, 3 CN of 8 stomach cancer patients and 7 of 7 rectal cancer patients.

0 Figure 11. Expression of 101P3A11 in human patient cancer specimens. RNA was extracted from a Spool of three prostate cancer tumors, kidney cancer tumors, colon cancer tumors, breast cancer tumors, and a cancer metastasis pool derived from cancer patients, as well as from normal prostate normal bladder (NB), normal kidney (NK) and normal colon Northern blots with 10 pg of total RNA/lane were probed with a 101P3A 11 fragment. Size standards in kilobases (kb) are indicated on the side. The results showed expression of 101P3A11 in prostate cancer tumors, kidney cancer tumors, colon cancer tumors, breast cancer tumors, cancer metastasis pool, bladder cancer pool, and in the normal prostate but not in the other normal tissues. A picture of the ethidium-bromide staining of the RNA gel is also presented.

Figure 12A. Expression of 101P3A11 in prostate cancer patient specimens. RNA was extracted from prostate tumors and their normal adjacent tissues (Nat) derived from prostate cancer patients. Northern blots with 10 pg of total RNA/lane were probed with 101P3Al 1 sequences. Results show upregulated expression of 101P3Al1 in 8 of 10 tumor specimens.

Figure 12B. Photomicrograph showing 101P3AI 1 expression in prostatic intraepithelial neoplasia (PIN) by in situ hybridization with an anti-sense 101P3A11 riboprobe.

Figure 12C. Photomicrograph showing 101P3A11 expression in prostate cancer tissue by in situ hybridization with an anti-sense 101P3AI nboprobe.

Figure 12D. Photomicrograph showing 101P3Al expression in prostate cancer by in situ hybridization with an anti-sense 101P3A11 riboprobe. Note up-regulation of expression relative to normal prostate, FIG. 12E.

Figure 12E. Photomicrograph showing 101P3A11 expression in normal prostate by in situ hybridization with an anti-sense 101P3AI riboprobe.

Figure 13. Expression of 101P3A 1 in colon cancer patient specimens. RNA was extracted from colon tumors and their normal adjacent tissues (Nat) derived from colon cancer patients. Northern blots with 10 Pg of total RNA/lane were probed with 101P3A1 I sequences. Size standards in kilobases (kb) are indicated on the side. Results showed expression of 101P3AI 1 in colon tumors but not in normal tissues. Expression was also seen in the colon cancer cell line T84. A picture of the ethidium-bromide staining of the RNA gel is also presented.

Figure 14. Expression or 101P3AI I in kidney cancer pallient slpcimens. RNA was extracled Iromn kidney tumors and their normal adjacent tissues (Nat)-derived from kidney cancer patients. Northern blots with 10 pg of total RNA/lane were probed with 101P3AI 1 sequences. Size standards in kilobases (kb) are 00 O indicated on the side. The results showed expression of 101P3A11 in five of six kidney tumor specimens. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) indicates that these tissues are not fully normal and that 101P3A 11 is expressed in early Sstage tumors. A picture of the ethidium-bromide staining of the RNA gel is also presented.

00 Figures 15A-15C. Androgen regulation of 101P3AI 1 in tissue culture cells. LAPC-9 cells were grown in charcoal-stripped medium and stimulated with the synthetic androgen mibolerone, for either 14 or 24 hours.

Northern blots with 10 lg of total RNA/lane were probed with 101P3A1 1 sequences (Figure 15A). A picture of the ethidium-bromide staining of the RNA gel is also presented (Figure 15C). Results showed expression of 101P3A11 was not regulated by androgen. The experimental samples were confirmed by testing for the 0 expression of the androgen-regulated prostate cancer gene PSA (Figure 15B). This experiment showed that, as 00 expected, PSA levels go down in presence of charcoal-stripped serum, and expression is induced at 14 and 24 hours in presence of mibolerone.

C, Figure 16. Androgen regulation of 101P3AI 1 in vivo. Male mice were injected with LAPC-9AD tumor cells. When tumors reached a palpable size (0.3-0.5cm in diameter), mice were castrated and tumors harvested at different time points following the castration. RNA was isolated from the xenograft tissues. Northern blots with jig of total RNA/lane were probed with 101P3Al1 sequences. Size standards in kilobases (kb) are indicated on the side. A picture of the ethidium-bromide staining of the RNA gel is also presented. The results showed that expression of 101P3A1 I is not androgen regulated.

Figure 17. Expression and detection of 101P3A11( 59-202)-psecFc fusion protein. The 101P3A11(159-202)-psecFc vector was constructed. The recombinant expression vector DNA was transfected into either 293T cells or Cos-7 cells. Cells as well as culture supernatants (media) were harvested 24 hours later.

The cells were lysed, and run on SDS-PAGE gel along with the media samples. The gel was transferred to nitrocellulose, stained with HRP-labeled anti-human IgG and developed using the ECL chemiluminescence detection kit Results showed expression of the 101P3A 1 (159-202)-psecFc fusion protein in the lysates of both 293T and Cos-7 cells. The 101P3A1 l(159-202)-psecFc fusion protein was also secreted and detected in the culture supematants of both cell types.

Figure 18. Expression of 101P3A 1 in 300.19 cells following retroviral-mediated gene delivery.

300.19 cells were transduced with the pSRa retroviral vector encoding the 101P3A11 gene. Following selection with neomycin, the cells were expanded and RNA was extracted. A Northern blot with 10 pg of total RNA/lane was probed with the 101P3A11 sequence. Size standards in kilobases (kb) are indicated on the side. Results showed expression of the 101P3A11 transcript driven from the retroviral LTR. LAPC-4AD and LAPC-9AD showed expression of the endogenous 101P3AI I transcript. The figure shows results of a short exposure of the autoradiogram.

Figures 19A-19C. Secondary structure and transmembrane prediction for 101P3A 11. Figure 19A: The secondary structure of 101P3A 1 protein was predicted using the HNN Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgi-bin/npsaautomat.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 random coils from the primary protein sequnce. The percent of the protein in a given secondary structure is also given. Figure 19B is a schematic representation of the probability of existence of transmembrane regions and orientation of 101P3AI 1 based on the TMpred algorithm of Hofmann and Stoffel 0 which utilizes TMBASE Hofmann, W. Stoffel. TMBASE A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). Figure 19C is a schematic representation of the probability ,of the existence of transmembrane regions and the extracellular and intracellular orientation of 101 P3AI I based c on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L.L. Sonnhammer, Gunnar von Heijne, 00 and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc.

of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F.

Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998). The TMpred and TMHMM M algorithms are accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/). The results of rn the transmembrane prediction programs presented in Figure 19B and 19C depict 101P3A 11 as containing 7 transmembrane domains consistent with that of a G-protein coupled receptor.

0 Figure 20. Expression of 101P3A11 in NIH-3T3 Tumors. Mice were injected subcutaneously with Scontrol 3T3-neo or NIH3T3 cells expressing 101P3A 11. Tumors were allowed to grow, the mice were then sacrificed and tumors harvested. RNA was isolated from LAPC-4AD and LAPC-4AI xenografts, 3T3-neo and 3T3-101P3A1 I cells grown in culture were used as controls. RNA isolated from six different tumors derived from 3T3-101P3A 11 cells (Tumor were compared by Northern blotting. Northern blots with 10 pg of total RNA/lane were probed with 101P3A11 sequence. A picture of the ethidium-bromide staining of the RNA gel is also presented. Results showed expression of 101P3A 1 in all 3T3-101P3A11 tumors as well as in 3T3/101P3A11 cells used to derive the tumors, but not in the negative control cells 3T3/neo cells.

Figure 21. 101P3AI 1 Induces Tumor Formation of 3T3 Cells. Injection of 106 3T3-neo, 3T3-Ras or 3T3-101P3A1 I cells (106 of the indicated cells mixed with Matrigel) subcutaneously into 6 male SCID mice (right flank) revealed that 6/6 3T3-vl 2Ras-injected mice formed tumors, 6/6 3T3-101P3AI injected mice formed tumors, and 0/6 3T3-neo-injected mice formed tumors. Each data point represents the mean tumor volume (n 6) in each group.

Figure 22. PTX reduces the in vivo growth of 3T3-101P3AI 1 Tumors. Pertussis toxin was found to inhibit the sub-cutaneous growth of3T3-101P3Al I tumors in SCID mice in a dose dependent manner.

Figure 23. Alignment of 101P3A 1-PHOR-I (Phor) with the rat GPCR RAIC (gi13420759). Identities 179/299 Positives 231/299 Gaps 1/299 Figure 24. Alignment of 101P3A lI-PHOR-I (Phor) with the human prostate specific GPCR.(gi113540539). Identities 179/299 Positives 233/299 Gaps 1/299 Figure 25. Alignment of O11P3A 1-PHOR.- (Phor) with human olfactory receptor 51112, (gi|14423836). Identities 163/304 Positives 214/304 Gaps 1/304 Figure 26. 101P3A11 Modulated Tyrosine Phosphorylation inNIH-3T3 Cells. 10P3A1 I mediated the de-phosphorylation of proteins at 200, 120-140, 85-90 and 55 kDa. 101P3A1 I induced the phosphorylation of proteins at 80 and 29 kDa in NIH-3T3 cells.

Figure 27. ERK Phosphorylation by PCR ligands in 101P3AI 1 Expressing Cells. FBS, lipophosphatidic acid, gastrin releasing pcptide, leukotriene and platelet activating factor induced the phosphorylation of ERK in 101P3AI 1 expressing cells.

Figure 28. Inhibition of 101P3Al1-Mediated ERK Activation by PD98059. ERK phosphorylation was inhibited by a MEK specific(PD98059) but not a p38 specific (SB203580) inhibitor in PC3-101P3Ai I cells.

Figure 29. Enhanced ERK Phosphorylation in Sodium Orthovanadate Treated PC3-101P3AI 1 Cells.

Sodium orthovanadate induced increased ERK phosphorylation in PC3-101P3A1 I cells relative to PC3-neo cells.

00 SFigure 30. Inhibition of 101P3AI 1-Mediated ERK Phosphorylation by AG1517. The EGFR inhibitor, C AG1517, inhibits EGF-mediated ERK phosphorylation in control and 10P3A 1-expressing PC3 cells. AG1517 partially inhibits 101P3AI 1 mediated ERK phosphorylation in PC3 cells.

SFigures 31A-31B. Activation of p38 in PC3-101P3AI 1 Cells. Expression of 101P3Al 1 mediates p38 00 phosphorylation in cells treated with 10% FBS as shown by blotting with antibodies to phospho-p38 (Figure 31A) compared to p38 (Figure 31B).

Figure 32. 101P3A1 I Induced Accumulation of cAMP in PC3 Cells. Expression of 101P3AI I increased the accumulation of cAMP in cells treated with 0. 1% and 10% FBS. FBS-induced cAMP accumulation in 101P3A11 cells was inhibited by pertussis toxin.

Figure 33. Pertussis Toxin Inhibits 101P3A11 Mediated ERK Phosphorylation. Pertussis toxin 00 inhibited FBS- mediated ERK phosphorylation in 101P3AI 1 expressing cells.

Figure 34. Pertussis Toxin Inhibited ERK Phosphorylation in PC3-101P3A1 I Cells. Pertussis toxin CN inhibited FBS- mediated ERK phosphorylation in 101P3A11 expressing cells. The inhibitory activity of pertussis toxin on ERK phosphorylation was more dramatic in FBS-trcatcd than EGF or GRP-trcatcd P'C3-1011'3Al 1 cells Figure 35. Inhibition of 101P3AI 1-mediated signaling by Suranim, a G protein inhibitor. Control NIH 3T3 and 3T3-101P3Al 1 cells were grown in the presence of absence of G protein inhibitors Surinam and NF449.

Proliferation was analyzed by Alamar blue after 72 hours. Suranim and NF449 inhibited the proliferation of 101P3A11 expressing but not control cells.

Figures 36A-36B. 101P3A11 Mediated ERK Phosphorylation By Conditioned Media. Figure 36A: blotting with anti-phospho ERK antibodies; Figure 36B: blotting with anti-ERK antibodies. Supernatants from PC3, PC3-101P3A11, PrEC and LAPC42 cells induce ERK phosphorylation in PC3 101P3AI I but not PC3 cells.

Supemantants from 3T3 and 293T cells had little specific effect on ERK phosphorylation.

Figure 37. 101P3A11 Enhances the Proliferation of 3T3 Cells. Control NIH 3T3 and 3T3-101P3A 1I cells were grown in the presence of absence 0.5 or 10% FBS. Proliferation was analyzed by Alamar blue after 48 hours. Expression of 101P3A11 induced a 6 fold increase in the proliferation of 3T3 cells grown in 0.5% FBS.

Figure 38. Inhibition of 101P3A 11 Mediated ERK Phosphorylation by 101P3A11 Specific Antibodies.

Expression of 101P3AI 1 induced ERK phosphorylation in 293T cells. Anti-101P3Al I pAb inhibited ERK Phosphorylation in 293T-101P3A 1I cells.

Figure 39. Anti-101P3Al 1 Ab Mediated cAMP Accumulation in PC3-101P3A 1 Cells. Control PC3 cells and cells expressing 101P3AI 1 were treated with anti-101P3Al 1 pAb for 2 min and evaluated for intracellular cAMP content. The assay was performed in duplicate.

Figures 40A-40F. Photomicrographs showing immunohistochemical analysis using anti-101P3Al 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded prostate cancer tissues (Figure 40A); anti-101P3Al I (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded prostate cancer cell line, LNCaP (Figure 40B); anti-101P3A 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded prostate cancer tissues (Figure 40C); anti-101P3A 11 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded normal prostate (Figure 40D); anti-101P3A 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded prostate cancer tissues (Figure 40E); and anti-101P3Al I (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded normal prostate (Figure 00 Figures 41A-41F. Photomicrographs showing immunohistochemical analysis using anti-101P3Al (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded prostate cancer l tissues (Figure 41A); anti-101P3Al 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded bladder cancer tissues (Figure 41B); anti-101P3Al 1 (peptide 1; amino acids 1-14) rabbit 00 polyclonal antibody on formalin fixed and paraffin embedded kidney cancer tissues (Figure 41C); anti-101P3Al 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded colon cancer tissues (Figure 41D); anti-101P3Al 1 (peptide 1; amino acids 1-14) rabbit polyclonal antibody on formalin fixed and paraffin embedded lung cancer tissues (Figure 41E); and anti-101P3A 1I (peptide 1; amino acids 1-14) rabbit polyclonal Santibody on formalin fixed and paraffin embedded breast cancer tissues (Figurc 41F).

SFigure 42 shows that 101P3A1 I induces orthotopic growth of tumors. 5x 10 cells were injected C' orthotopically into SCID mice, 7 mice per group; tumor weight was evaluated 24-25 days post cell injection.

00 SFigure 43 shows that 101P3A1 1 induces colony formation in a soft agar assay.

SFigure 44 Schematic of 101P3A11 Gene Variants Figure 45 Schematic of 101P3A 11 Proteins Variants Figure 46 Exon Map Figure 47: Recognition of PHOR-1 protein by PHOR-1 mAbs Figure 48: Recognition of PHOR-l protein in transfected 293T cells by sera from GST-PHOR-1 immunized mice.

Figure 49: Data showing that four hybridomas reactive to MBP-PHOR-I exhibited strong specific reactivity to PHOR-1 protein expressed in cells. This was demonstrated by Western analysis of 293T cells transfected with the epitope tagged PHOR-1 cDNA Figure 50: Mouse polyclonal antibodies raised to amino acids 1-23 detect PHOR- expressed in 293T cells Figure 51: Inhibition of ERK Phosphorylation by GPCR Inhibitors Figure 52: Inhibition of PC3 Proliferation by GPCR Inhibitors Figure 53: Inhibition of PC3-AGS-3 Proliferation by PTX Figure 54: AGS-3 Enhances Proliferation of 3T3 and PC3 Cells Figure 55: AGS-3 Induces in Vivo Tumor Formation of 3T3 Cells Figure 56: Inhibition of 3T3-AGS-3 Tumor Formation by PTX Figure 57: AGS-3 Induces the Orthotopic Growth of 3T3 Tumors Figure 58: AGS-3 Enhances Orthotopic Growth of PC3 Cells Figure 59: Partial Inhibition of 3T3-AGS-3 Tumor Formation by Suramin Figure 60: AGS-3 Induces Intratibial Tumor Growth of 3T3Cells Figure 61: AGS-3 Enhances Intratibial Tumor Growth of PC3 Cells Figure 62: Inhibition of AGS-3 Mediated ERK Phosphorylation by AGS-3 Specific Antibodies Figure 63: AGS-3 Enhances Cell Cycle Entry of 3T3 and PC3 Cells Figure 64: Anti-AGS3 Staining of MDCK Cells Figure 65: Nucleic Acid Alignments Figure 66 Expression and detection of 101P3A I.GFP fusion protein. The pcDNA3.1/101P3AI 1.GFP vector was constructed. 293T cells were transfected with either the pcDNA3.1/101P3Al 1.GFP recombinant expression vector pcDNA3.I/GFP vector or control pcDNA3.1 vector Cells were harvested 24 12 0 hours later and analyzed by microscopy for detection of green fluorescence. Results show expression of the SIOIP3AI I.GFP fusion protein is localized mostly at the cell membrane, whereas expression of the free GFP is d throughout the cells. The control vector did not show any fluorescence. We conclude that the IO1P3A1 I.GFP fusion protein is expressed from the pCDNA3.1/101P3AI I.GFP construct, and that the fusion protein is localized 0 at the cell membrane.

Figure 67 The cDNA and amino acid sequence of the open reading frames of codon optimized sl01P3A11 v.1 and sl01P3AlI v.3 C Figure 68 Expression and detection of codon optimized sl01P3AI I.GFP fusion protein. The N pcDNA3.I/sl01P3Al .GFP vector for codon optimized 101P3A11 was constructed. 293T cells were transfected 8 with either pcDNA3.1 vector control (light line), or one of the three different pcDNA3.1/sl01P3Al I.GFP vector CN clones, G2, 2G3, or 3H5 (dark line). Cells were harvested 24 hours later and either analyzed directly for green 00 0 fluorescence or stained viably using polyclonal anti-101P3Al I antibody and analyzed by flow cytometry.

Results show strong expression of the codon optimized PHOR- I.GFP fusion protein at the cell surface.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections Definitions II.) 101P3A11 Polynucleotides II.A.) Uses of 101P3A11 Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities II.A.2.) Antisense Embodiments Primers and Primer Pairs II.A.4.) Isolation of 101P3All-Encoding Nucleic Acid Molecules Recombinant Nucleic Acid Molecules and Host-Vector Systems 101P3A11-related Proteins IILA.) Motif-bearing Protein Embodiments III.B.) Expression of 101P3All-related Proteins III.C.) Modifications of 101P3All-related Proteins Uses of 101P3A11-related Proteins IV.) 101P3A11 Antibodies 101P3A11 Cellular Immune Responses VI.) 101P3A11 Transgenic Animals VII.) Methods for the Detection of 101P3A11 VIII.) Methods for Monitoring the Status of 101P3All-related Genes and Their Products IX.) Identification of Molecules That Interact With 101P3A11 Therapeutic Methods and Compositions Anti-Cancer Vaccines 101P3A11 as a Target for Antibody-Based Therapy 101P3A11 as a Target for Cellular Immune Responses X.C.I. Minigene Vaccines 00 SX.C.2. Combinations of CTL Peptides with Helper Peptides X.C.3. Combinations of CTL Peptides with T Cell Priming Agents X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides C. Adoptive Immunotherapy 00 Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 101P3A1I.

XII.) Inhibition of 101P3All Protein Function \0 XII.A.) Inhibition of 101P3AI1 With Intracellular Antibodies SXII.B.) Inhibition of 101P3AI1 with Recombinant Proteins XII.C.) Inhibition of 101P3A11 Transcription or Translation SXII.D.) General Considerations for Therapeutic Strategies XIII.) KITS/Articles of Manufacture C XIV.) Evaluation of GPCRs and Modulators Thereof XV.) Screening of Candidate Compounds XVI.) GPCR Fusion Proteins Definitions: Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and 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 advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage Cl C2 disease under the Whitmore-Jewett system, and stage T3 T4 and N+ disease under the TNM (tumor, node, metastasis) system.

In general, surgery is not 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 101P3A 11 (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 0O sites that are not present in the native sequence 101P3Al 1. 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 Scarbohydrate moieties present.

The term "analog" refers to a molecule which is structurally similar or shares similar or corresponding 00 attributes with another molecule a 101P3A1 l-related protein). For example an analog of a 101P3A 11 protein can be specifically bound by an antibody orT cell that specifically binds to 101P3A11.

The term "antibody" is used in the broadest sense. Therefore an "antibody" can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-101P3Al I Santibodies 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.

00 An "antibody fragment" is defined as at least a portion of the variable region of the immunoglobulin Smolecule that binds to its target, the antigen-binding region. In one embodiment it specifically covers single anti-101P3Al 1 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti- 101P3A 1 antibody compositions with polyepitopic specificity.

The term "codon optimized sequences" refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences." The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, 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, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as Ate", I13', Y 9 0 Re" 6 SmIS3, Bi 2 P" and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

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

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

The terms "hybridize", "hybridizing", "hybridizes" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in formamidc/6XSSC/0.1% SDS/100 pg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.IXSSC/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 00 in atccordance with the invention preferably do not contain materials normally associated with the peptides in their O in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from Scontaminant polynucleotides that correspond or are complementary to genes other than the 101P3AI 1 genes or that S encode polypeptides other than 101P3A1 I gene product or fragments thereof. A skilled artisan can readily employ OO nucleic acid isolation procedures to obtain an isolated 101P3A 11 polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical or chemical methods are employed to remove the 101P3AI 1 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard Spurification methods to obtain an isolated 101P3AI I protein. Alternatively, an isolated protein can be prepared by C chemical means.

The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, 00 cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of Sthe invention, the mammal is a human.

C The terms "metastatic prostate cancer" and "metastatic disease" mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone.

Prostate cancer bone metastases are often osteoblastic rather than osteolytic resulting in net bone formation).

Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, Le, 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 101P3AI -related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function protein-protein interaction, protein-DNA interaction, etc) or modification that is phosphorylated, glycosylated or amidated), or localization secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, "motif' refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino 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 pattenm 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, and/or composition that is physiologically compatible with humans or other mammals.

00 0 The term "polynucleotide" means a polymeric form ofnucleotides of at least 10 bases or base pairs in Slength, either ribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, 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 00 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 ¢C specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".

C An HLA "primary anchor residue" is an amino acid at a specific position along a peptide sequence which 00 is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, Susually 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. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

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 101P3A1 1, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 101P3AI I 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, 101P3AI 1 protein; arc not found in naturally occuITing metabolic pathways; and/or are more soluble in aqucous thlan nonaqueous solutions "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration.

In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of 00 hybridization reactions, see Ausubel el al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

S"Stringent conditions" or "high stringency conditions", as defined herein, are identified by, but not t limited to, those that: employ low ionic strength and high temperature for washing, for example 0.015 M 00 sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 0 C; 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, imM sodium citrate at 42 or employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), c mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm SDNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42 with washes at 42 0 C in 0.2 x SSC (sodium C chloride/sodium, citrate) and 50% formamide at 55 OC, followed by a high-stringency wash consisting of 0.1 x 0 SSC containing EDTA at 55 "Moderately stringent conditions" are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37"C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in I x SSC at about 37-50 0 C. 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.

As used herein "to treat" or "therapeutic" and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.

A "transgenic animal" a mouse or rat) is an animal having cells that contain a transgene, which transgcne was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A "transgene" is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response "vaccine" is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, a minigene that encodes a polyepitopic peptide. The "one or more peptides" can include any whole unit integer from 1-150 or more, at least 2, 3, 4,5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29,30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, dendritic cells.

0 The term "variant" refers to a molecule that exhibits a variation from a described type or norm, such as a 0 protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein the 101P3AI 1 protein shown in Figure 2 or Figure 3. An analog is an example of a variant protein.

t Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.

00 The "101P3AI 1-related proteins" of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art.

SFusion proteins that combine parts of different 101P3Al 1 proteins or fragments thereof, as well as fusion proteins of a S101P3A 1I protein and a heterologous polypeptide are also included. Such 101P3Al 1 proteins are collectively referred to as the 101P3A1 1-related proteins, the proteins of the invention, or 101P3Al I. The term "101P3AI 1- Cl related protein" refers to a polypeptide fragment ora 101P3A 11 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19,20,21, 22,23, 24, 25, 30,35, 40,45, 50, 55, 60, 65, 70, or 317 or 318 or more amino acids.

"Active ingredient" in the context of a "Pharmaceutical Composition" shall mean a component of a Pharmaceutical Composition that provides the primary pharmaceutical benefit, as opposed to an "inactive ingredient" which would generally be recognized as providing no pharmaceutical benefit.

"Agonists" shall mean moieties that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes. In the context of the disclosed invention, a Pharmaceutical Candidate comprising a 101P3A 11 Agonist can be utilized for affecting metabolism. "Partial agonists" shall mean moieties that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists.

"Antagonist" shall mean moieties that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists. Antagonists do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.

"Candidate compound," in the context of the disclosed invention, shall mean a small molecule that is amenable to a screening technique.

"Composition" shall having a meaning in accordacne with standard use. For example, a composition can mean a material comprising at least two compounds, components or substituents; for example, and not limitation, a Pharmaceutical Composition comprising at least one Active Ingredient and at least one other component.

"Compound efficacy" shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity.

"Constitutive receptor activation" shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof.

"Contact" or "contacting" shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.

"Endogenous" shall mean a material that a mammal naturally produces. Endogenous in reference to, for example and not limitation, the term "receptor" shall mean that which is naturally produced by a mammal (for example, and not limitation, a human), yeast, bacterium or a virus. In contrast, the term "non-endogenous" in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) yeast, bacterium or a virus. For example, and not limitation, a receptor which is not constitutively active in its 19 00 C=I endogenous form, but when manipulated becomes constitutively active, is most preferably referred to Sherein as a "non-endogenous, constitutively activated receptor." Both terms can be utilized to describe Sboth "in vivo" and "in vitro" systems. For example, and not a limitation, in a screening approach, the 00 00 endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in Svivo system is viable.

"G protein coupled receptor fusion protein" and "GPCR fusion protein," in the context of the invention disclosed herein, each mean a non-endogenous protein comprising an endogenous, 00 constitutively activated orphan GPCR fused to at least one G protein, most preferably, the alpha (a) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. For example, and not limitation, in an endogenous state, the G protein "Gsa" is the predominate G protein that couples with 101P3AI I such that a GPCR Fusion Protein based upon 101P3AI I would be a non-endogenous protein comprising 101P3AI 1 fused to Gscc. The G protein can be fused directly to the c-terminus of the endogenous, constitutively active orphan GPCR or there may be spacers between the two.

"Inhibit" or "inhibiting", in relationship to the term "response" shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.

"Inverse agonists" shall mean moieties that bind the endogenous form of the receptor, and which inhibit the baseline intracellular response initiated by the active endogenous form of the receptor below the normal base level of activity that is observed in the absence of the endogenous ligand, agonists or partial agonists, or decrease GTP binding to membranes. Preferably, the baseline intracellular response is decreased in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist. Biologically, "101P3AI 1 inverse agonist" shall mean moieties that can be assessed in vivo by factors other than just determination that the moiety has interacted with 101P3A I1.

"Ligand" shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.

"Pharmaceutical composition" shall mean a composition comprising at one Active Ingredient and at least one ingredient that is not an Active Ingredient (for example and not limitation, a filler, dye, or a mechanism for slow release), whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal or in cells thereof such as in vitro without limitation, the mammal is a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

00 (I "Small molecule", in the context of the invention disclosed herein, is a non- protein based Smoiety; for example, and not limitation, NF449is a small molecule within the context of this invention.

SIn a preferred embodiment, the endogenous ligand for a receptor is not a "small molecule." 00 SThroughout 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 IND elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been C, included in the present specification is solely for the purpose of providing a context for the present 00 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.

II.) 101P3A11 Polynucleotides One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 101P3AI I gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 101P3AIl-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, 00 polynucleotides or oligonucleotides complementary to a 101P3AI 1 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotidcs that hybridize to a 101P3A1 I gene, mRNA, or to a 101P3A11 encoding C, polynucleotide (collectively, "I01P3A1 I polynucleotides"). In all instances when referred to in this section, T Scan also be U in Figure 2.

SEmbodiments of a 101P3A I polynucleotide include: a 101P3A I polynucleotide having the sequence 0 shown in Figure 2, the nucleotide sequence of 101P3A 11 as shown in Figure 2 wherein T is U; at least contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous Snucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, ND embodiments of 101P3Al 1 nucleotides comprise, without limitation: 00 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 130 through nucleotide residue number 1086, optionally including the last, stop codon, wherein T can also be U; (III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2A, from nucleotide residue number 133 through nucleotide residue number 1086, optionally including the last, stop codon, wherein T can also be U; (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B, from nucleotide residue number 130 through nucleotide residue number 348, optionally including the last, stop codon, wherein T can also be U; a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B, from nucleotide residue number 133 through nucleotide residue number 348, optionally including the stop codon, wherein T can also be U; (VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2C, from nucleotide residue number 130 through nucleotide residue number 1086, optionally including the last, stop codon, wherein T can also be U; (VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2C, from nucleotide residue number 133 through nucleotide residue number 1086, optionally including the last, stop codon, wherein T can also be U; (VIII) a polynucleotide that encodes a 101P3A I1-related protein that is at least 90% homologous to an entire amino acid sequence shown in Figure 2A-C; 00 (IX) a polynucleotide that encodes a 101P3AI I-related protein that is at least 90% identical to an C" entire amino acid sequence shown in Figure 2A-C; a polynucleotide that encodes at least one peptide set forth in Tables V-XVIII and XXII to IL, 00 optionally with a proviso that the polynucleotide is not a contiguous sequence from a nucleic acid sequence of Figure 2; S(XI) a polynucleotide that encodes at least two peptides seleected from the peptides set forth in STables V-XVIII and XXII to IL, optionally with a proviso that the polynucleotide is not a contiguous 0sequence from a nucleic acid sequence of Figure 2; 00 (XII) a polynucleotide that encodes at least two peptides selected from the peptides set forth in Tables SV-XVIII and XXII to IL, optionally with a proviso that the polynucleotide is not a contiguous sequence C(N from a nucleic acid sequence of Figure 2; (XIII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure (XIV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; (XV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; (XVI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XVII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9; (XVII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3B in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure (XIX) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3B in any whole number increment up to 317 or 318 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; 00 O (XX) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure S3B in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; ct 0 (XXI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3B in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; IND (XXII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3B in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9; 00 S(XXIII) a polynucleotide that encodes monoloncal antibody or binding region thereof secreted by a C- hybridoma entitled X18(1)4 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; (XXIV) a polynucleotide that encodes monoloncal antibody or binding region thereof secreted by a hybridoma entitled X 8(1)10 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; (XXV) a polynucleotide that encodes monoloncal antibody or binding region thereof secreted by a hybridoma entitled XI 8(1)23 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; (XXVI) a polynucleotide that encodes monoloncal antibody or binding region thereof secreted by a hybridoma entitled X18(4)7deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; (XXVII)a polynucleotide that is fully complementary to a polynucleotide of any one of(I)-(XXVI); and, (XXVIII) a peptide that is encoded by any of (I)-(XXVI); (XXIX) a peptide that occurs at least twice in Tables V-XVIII and XXII to IL collectively, or an oligonucleotide that encodes such HLA peptide; (XXX) a peptide that occurs at least once in Tables V-XVIII and at least once in tables XXII to IL, or an oligonucleotide that encodes such HLA peptide; (XXXI) a peptide which comprises peptide regions, or an oligonucleotide encoding the peptide, 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 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure ii) a 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 00 Svalue equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity 0, profile of Figure 6; iii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number c- increment up to the full length of that protein in Figure 3, that includes an amino acid position having a 00 value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number Sincrement up to the full length of that protein in Figure 3, that includes an amino acid position having a Svalue 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 0Flexibility profile of Figure 8; 00 v) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number 0increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9; (XXXII)a polynucleotide of any of(I)-(XXVII) or peptide of(XXVII)-(XXXI) together with a pharmaceutical excipient and/or in a human unit dose form.

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

Typical embodiments of the invention disclosed herein include 101P3A11 polynucleotides that encode specific portions of 101P3A 11 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, 70, 80, 85, 90., 95, 100, 125, 150, 175, 200, 225, 250, 375, 300 or317 or 318 contiguous amino acids of 101P3AI 1.

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 101P3A 11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 101P3AI 1 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 101P3Al I protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 101P3A 1 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 101P3A1 1 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 101P3AI 1 protein shown in Figure 2 or Figure 3, or polynucleotides encoding about amino acid 60 to about amino acid 70 or amino acid 317 or 318 of the 101P3Al 1 protein shown in Figure 2 or Figure 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 1 through the carboxyl terminal amino acid of the IOIP3AI I protein arc embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 101P3A1 I 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 00 amino acid 20, (or 30, or 40 or 50 etc.) of the 101P3AI I 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 101P3AI I sequence as shown in Figure 2.

Additional illustrative embodiments of the invention disclosed herein include 101P3A 11 polynucleotide 00 fragments encoding one or more of the biological motifs contained within a 101P3A11 protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 101P3A1 I protein "or variant" set forth in Tables V-XVIII and XXII to IL. In another embodiment, typical polynucleotide fragments of the invention Sencode one or more of the regions of 101P3AI 1 protein or variant that exhibit homology to a known molecule. In N another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 101P3A1 1 Sprotein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

00 Note that to determine the starting position of any peptide set forth in Tables V-XVIII and Tables XXII 0 to IL (collectively HLA Peptide Tables) respective to its parental protein, variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table LVII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table LLII.

Accordingly if a Search Peptide begins at position one must add the value "X minus 1" to each position in Tables V-XVIII and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule.

For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 1, 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

II.A.) Uses of 101P3All Polynucleotides Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 101P3A 11 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 101P3A11." For example, because the 101P3AI 1 gene maps to this chromosome, polynucleotides that encode different regions of the 101P3AI 1 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 (sec c.g. Krajinovic et Mutat. Res. 382(3-4): 81-83 (1998); Johansson et Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)).

Thus, polynucleotides encoding specific regions of the 101P3A11 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 101P3AI 1 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 101P3AI l was shown to be highly expressed in bladder and other cancers, 101P3A 11 polynucleotides are used in methods assessing the status of 101P3A11 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 101P3A11 proteins are used to assess the 0 presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 101P3AI gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding 00 specific regions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an altemative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules 0 capable of inhibiting the RNA or protein expression of 101P3AI 1. For example, antisense molecules can be RNAs 00 or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as Sphosphorothioate 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 101P3A 11 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, 101P3A11. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988).

The 101P3A11 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (0oligo) 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-1,2-benzodithiol-3one-l,1-dioxide, which is a sulfur transfer reagent. See, lycr, R. P. et al., J. Org. Chcm. 55:4693-4698 (1990); and Iyer, R. P. el al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 101P3AI I antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, Partridge et al., 1996, Antisense Nucleic Acid Drug Development 6: 169-175).

The 101P3A11 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons ofa 101P3AI 1 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 101P3A 11 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 101P3A11 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 101P3A I mRNA. Optionally, 101P3A11 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 101P3A11. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 101P3AI 1 expression, see, L. A. Couture D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs Further specific embodiments of this 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 26 00 O 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, g bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 101P3AI 1 polynucleotide in a sample and as a means for detecting a cell 00 expressing a 101P3A I protein.

Examples of such probes include polypeptides comprising all or part of the human 101P3A1 I cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 101P3AI 1 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can n be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 101P3AI 1 mRNA.

The 101P3AI polynucleotides of the invention are useful for a variety of purposes, including but not 0C limited to their use as probes and primers for the amplification and/or detection of the 101P3A 1 gene(s), 0 mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other C cancers; as coding sequences capable of directing the expression of 101P3AI 1 polypeptides; as tools for modulating or inhibiting the expression of the 101P3AI I genc(s) and/or translation of the 101P3A1 I transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 101P3A1 I or 101P3AI1 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 101P3All-Encodlng Nucleic Acid Molecules The 101P3AI 1 cDNA sequences described herein enable the isolation of other polynucleotides encoding 101P3AI 1 gene product(s), as well as the isolation of polynucleotides encoding 101P3A1 I gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 101P3AI I gene product as well as polynucleotides that encode analogs of 101P3Al 1-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 101P3Al I gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems Lambda ZAP Express, Stratagene). Phage clones containing 101P3A11 gene cDNAs can be identified by probing with a labeled 101P3A 11 cDNA or a fragment thereof. For example, in one embodiment, a 101P3AI I 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 101P3A I gene. A 101P3A11 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 101P3AI 1 DNA probes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems The invention also provides recombinant DNA or RNA molecules containing a 101P3A11 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

00 0 The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 101P3A11 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host Scell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a Smammalian cell or an insect cell a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of 00 suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins COS, CHO, 293, 293T cells). More particularly, a \0 polynucleotide comprising the coding sequence of 101P3AI or a fragment, analog or homolog thereof can be used Sto generate 101P3AI 1 proteins or fragments thereof using any number of host-vector systems routinely used and Swidely known in the art.

OO A wide range of host-vector systems suitable for the expression of 101P3A 1 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra).

C, Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogcn) and the retroviral vector pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 101P3A1 I can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-l, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 101P3A 1 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 101P3A11 and 101P3A11 mutations or analogs.

Recombinant human 101P3A11 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 101P3A l-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 101P3A11 or fragment, analog or homolog thereof, a 101P3A1 l-related protein is expressed in the 293T cells, and the recombinant 101P3AI 1 protein is isolated using standard purification methods affinity purification using anti-101P3Al I antibodies). In another embodiment, a 101P3AI I coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 101P3A11 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 101P3A11 coding sequence can be used for the generation of a secreted form of recombinant 101P3AI 1 protein.

As discussed herein, redundancy in the genetic code permits variation in 10 IP3A11 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 www.dna.affrc.go.jp/~nakamura/codon.html.

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

The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation 28 00 O consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 cN (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively g at the 5' proximal AUG codon is abrogated only under rare conditions (see, Kozak PNAS 92(7): 2662-2666, C- (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

00 III.) 101P3All-related Proteins Another aspect of the present invention provides 101P3AI -related proteins. Specific embodiments of IN 101P3Al 1 proteins comprise a polypeptide having all or part of the amino acid sequence of human 101P3A I as shown in Figure 2 or Figure 3. Alternatively, embodiments of 101P3A 11 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 101P3A11 shown in Figure 2 or Figure 3.

00 Embodiments of a 101P3AI 1 polynucleotide include: a 101P3A 11 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 101P3A1 I as shown in Figure 2 wherein T is U; at least CKN contiguous nucleotides 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 101P3A 11 nucleotides comprise, without limitation: an protein comprising, consisting essentially of, or consisting of a sequence as shown in Figure 2; (II) a 101P3Al l-related protein that is at least 90% homologous to an entire amino acid sequence shown in Figure 2A-C; (III) a 101P3Al 1-related protein that is at least 90% identical to an entire amino acid sequence shown in Figure 2A-C; (IV) a protein that comprises at least one peptide set forth in Tables V-XVIII or Tables XXII to IL, 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 V-XVIII, collectively, which peptide is also set forth in Tables XXII to IL, 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 V- XVIII and XXII to IL, 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 V- XVIII and XXII to IL, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2; (VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables V- XVIII; and at least one peptide set forth in Tables XXII to IL, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2; 0 (IX) a polypeptide comprising at least 5 amino acids of a protein of Figure 3A or 3C in any whole 0 number increment up to 317 or 318 that includes an amino acid position having a value greater than in the Hydrophilicity profile of Figure a polypeptide comprising at least 5 amino acids of a protein of Figure 3A or 3C in any whole 00 number increment up to 317 or 318 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; S(XI) a polypeptide comprising at least 5 amino acids of a protein of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than in the Percent Accessible Residues profile of Figure 7; 00 O (XII) a polypeptide comprising at least 5 amino acids of a protein of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than in the Average Flexibility profile of Figure 8; (XIII) a polypeptide comprising at least 5 amino acids of a protein of Figure 3A or 3C in any whole number increment up to 317 or 318 that includes an amino acid position having a value greater than in the Beta-turn profile of Figure 9; (XIV) a polypeptide comprising at least 5 amino acids of a protein of Figure 3B in any whole number increment up to 72 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure (XV) a polypeptide comprising at least 5 amino acids of a protein of Figure 3 in any whole number increment up to 72 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; (XVI) a polypeptide comprising at least 5 amino acids of a protein of Figure 3 in any whole number increment up to 72 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; (XVII) a polypeptide comprising at least 5 amino acids of a protein of Figure 3 in any whole number increment up to 72 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; (XVIII) a polypeptide comprising at least 5 amino acids of a protein of Figure 3 in any whole number increment up to 72 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9; (XIX) a monoloncal antibody or binding region thereof secreted by a hybridoma entitled XI 8(1)4 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; 00 00 (XX) a monoloncal antibody or binding region thereof secreted by a hybridoma entitled X18(1)10 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA F 20110-2209 USA) as Accession No. on 15 May 2002; (XXI) a monoloncal antibody or binding region thereof secreted by a hybridoma entitled X18(1)23 deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209 USA) as Accession No. on 15 May 2002; I\ (XXII) a monoloncal antibody or binding region thereof secreted by a hybridoma entitled X 18(4)7deposited with American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, SVA 20110-2209 USA) as Accession No. on 15 May 2002; 00 0 (XXIII) a peptide that occurs at least twice in Tables V-XVIII and XXII to IL, collectively; CN1 (XXIV) a peptide that occurs at least once in Tables V-XVIII, and at least once in tables XXII to IL; (XXV) 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 ii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of Figure 8; or, v) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9; (XXVI) a peptide of together with a pharmaceutical excipient and/or in a human unit dose form.

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

00 Typical embodiments of the invention disclosed herein include 101P3A 1 polynucleotides that encode Sspecific portions of 101P3A11 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, 70, or 317 or 318 contiguous amino acids of 101P3A1 1.

In general, naturally occurring allelic variants of human 101P3A I protein share a high degree of structural identity and homology 90% or more homology). Typically, allelic variants of a 101P3A I protein contain Sconservative amino acid substitutions within the 101P3AI 1 sequences described herein or contain a substitution of an I amino acid from a corresponding position in a homologue of 101P3A11. One class of 101P3AI 1 allelic variants are Sproteins that share a high degree of homology with at least a small region of a particular 101P3A11 amino acid ci sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution,

OO

0 truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology Seach have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions. Such changes include substituting any of isoleucine valine and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa; glutamine for asparagine and vice versa; and serine for threonine and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional 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" 2' d ED. Lubert Stryer ed (Stanford University); Henikoff et a., 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 101P3A11 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 101P3A11 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl.

Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 101P3A1 variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the 00 0 variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently Sfound in both buried and exposed positions (Creighton, The Proteins, Freeman Co., Chothia, J.

SMol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino C acid can be used.

00 As defined herein, 101P3AI 1 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 101P3A 1I 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 101 P3A 1I m variant also specifically binds to a 101P3A I protein having an amino acid sequence set forth in Figure 3. A N polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 101P3A I protein. Those c skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a 0 grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, Nair et al, J. Immunol 2000 165(12): 6949-6955; Hebbes et Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 101P3Al1-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 101P3AI 1 protein variants or analogs comprise one or more of the 101P3A 11 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 101P3A 11 fragments (nucleic or amino acid) that have altered functional immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of Figure 2 or Figure 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 101P3A 1 protein shown in Figure 2 or Figure 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 101P3A 11 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 101P3A1 1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 101P3Al 1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 101P3AI I protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 101P3A1 I protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 101P3AI I protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 101P3A11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 101P3A11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 101P3AI I protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 101P3AI 1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 101P3AI 1 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 101P3Al 1 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 101P3A11 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

00 0 101P3A I1-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 g molecules that encode a 101P3A 11-related protein. In one embodiment, nucleic acid molecules provide a means to Sgenerate defined fragments of a 101P3A11 protein (or variants, homologs or analogs thereof).

00 III.A.) Motif-bearing Protein Embodiments Additional illustrative embodiments of the invention disclosed herein include 101P3AI 1 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 101P3A 11 polypeptide sequence set forth in Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be M evaluated for the presence of such motifs by a number of publicly available Internet sites (see, URL C addresses: pfam.wustl.edul; searchlauncher.bcm.tmc.edu/seq-seach/struc-predict.html; psort.ims.u-tokyo.ac.jp/; 0 www.cbs.dtu.dk/; www.ebi.ac.uk/interpro/scan.html; www.expasy.ch/tools/scnpsitl.html; Epimatrix T M and Epimer

T

Brown University, www.brown.edu/ResearchfB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, (K bimas.dcrt.nih.gov/.).

Motif bearing subsequences of all 101P3AI 1 variant proteins are set forth and identified in Tables V- XVIII and XXII TO IL.

Table XIX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table XIX list motif name abbreviation, percent identity found amongst the different member of the motif family, motif name or description and most common function; location information is included if the motif is relevant for location.

Polypeptides comprising one or more of the 101P3AI 1 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 101P3AI 1 motifs discussed above are associated with growth dysregulation and because 101P3A11 is overexpressed in certain cancers (See, Table Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g.

Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochcm. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et J. Natl. Cancer Inst.

Monogr. 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XVIII and XXII TO IL. CTL epitopes can be determined using specific algorithms to identify peptides within a 101P3A 1 protein that are capable of optimally binding to specified HLA alleles Table IV; Epimatrix T and Epimer

T

Brown University, URL www.brown.edu/ResearchfTB.HIV_Lab/epimatrix/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.

0 Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For 00 example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV.

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 Sof interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics r' 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 00 S58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480- ^C 90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker e 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 al., 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.

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

101P3A11-related proteins are embodied in many forms, preferably in isolated form. A purified 101P3AI 1 protein molecule will be substantially free of other proteins or molecules that impair the binding of 101P3A I1 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 101P3A 1-related proteins include purified 101P3AI l-related proteins and functional, soluble 101P3A11-related proteins. In one embodiment, a functional, soluble 101P3A 1 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 101P3A I1 proteins comprising biologically active fragments of a 101P3AI 1 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 101P3A 11 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 101P3A 11 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.

101P3Al I-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 on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti- 00 0 101P3AI I antibodies, orT cells or in identifying cellular factors that bind to 101P3A 1. Forexample, hydrophilicity 0 profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and g 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 Doolittle, 1982, J. Mol. Biol.

00 157:105-132. Percent Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran Ponnuswamy 1988, Int.

J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, Roux 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a 101P3A 11 protein S that are capable of optimally binding to specified HLA alleles by using the SYFPEITHI site at World Wide Web 0 URL syfpeithi.bmi-heidelberg.com/; the listings in Table Epimatrix T and Epimer

T

Brown University, 0 URL (www.brown.edu/ResearchB-HIVLabepimatri/epimatrix.htm); and BIMAS, URL bimas.dcrt.nih.gov/).

Illustrating this, peptide epitopes from 101P3A 1 that are presented in the context of human MHC Class I molecules, HLA-AI, A2, A3, All, A24, B7 and B35 were predicted (see, Tables V-XVIII, XXII TO IL). Specifically, the complete amino acid sequence of the 101P3A11 protein and relevant portions of other variants, for HLA Class I predictions 9 flanking redisues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side ofa point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; and the site SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/ was used.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 10 or 1 l-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 101P3AI 1 predicted binding peptides are shown in Tables V-XVIII and XXII TO IL herein. In Tables V-XVIII and XXII TO IL, selected candidates, 9-mers, 10-mers, and 15-mers for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37*C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

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

It is to be appreciated that every epitope predicted by the BIMAS site, Epimer T M and Epimatrix T sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, 36 00 00 bimas.dcrt.nih.gov/) are to be "applied" to a 101P3A11 protein in accordance with the invention. As used in this context "applied" means that a 101P3AI 1 protein is evaluated, visually or by computer-based patterns Sfinding methods, as appreciated by those of skill in the relevant art. Every subsequence ofa 101P3Al 1 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 00 residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 101P3All-related Proteins SIn an embodiment described in the examples that follow, 101P3A 11 can be conveniently expressed in g cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 101P3A1 I with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or "i TagS, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be 0 used to facilitate the production of a secreted 101P3A1 1 protein in transfected cells. The secreted HIS-tagged S10P3AI I in the culture media can be purified, using a nickel column using standard techniques.

III.C.) Modifications of 101P3All-related Proteins Modifications of 101P3A11-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 101P3A1 I polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 101P3A1 I protein. Another type of covalent modification of a 101P3A1 I polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 101P3A1 1 comprises linking a 101P3AI 1 polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 101P3A1 l-related proteins of the present invention can also be modified to form a chimeric molecule comprising 101P3A11 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 101P3AI I sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of 101P3A11. A chimeric molecule can comprise a fusion of a 101P3AI 1-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of al01P3Al 1 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 101P3AII related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 101P3A 11 polypcptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, U.S. Patent No.

5,428,130 issued June 27, 1995.

00 III.D.) Uses of 101P3All-related Proteins The proteins of the invention have a number of different specific uses. As 101P3A 1 is highly expressed Sin prostate and other cancers, 101P3AI -related proteins are used in methods that assess the status of 101P3AI I C gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, 00 polypeptides from specific regions of a 101P3AI1 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 101P3AI -related proteins comprising the amino acid M residues of one or more of the biological motifs contained within a 101P3A11 polypeptide sequence in order to Sevaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 101P3AI -related proteins that contain the amino acid residues of one or more of the C biological motifs in a 101P3A1 I protein are used to screen for factors that interact with that region of 101P3A 1.

0 101P3A11 protein fragments/subsequences are particularly useful in generating and characterizing domainspecific antibodies antibodies recognizing an extracellular or intracellular epitope of a 101P3A1 I protein), for identifying agents or cellular factors that bind to 101P3AI 1 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 101P3Al I 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 101P3Al I gene product Antibodies raised against a 101P3A 1 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 101P3A1 I protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 101P3Al1-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 101P3A I proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 101P3A I -expressing cells in radioscintigraphic imaging methods). 101P3A11 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 101P3A1l Antibodies Another aspect of the invention provides antibodies that bind to 101P3AI 1-related proteins. Preferred antibodies specifically bind to a 101P3A11-related protein and do not bind (or bind weakly) to peptides or proteins that are not 101P3A 1-related proteins. For example, antibodies that bind 101P3A11 can bind 101P3Al 1-related proteins such as the homologs or analogs thereof.

101P3A 1 antibodies of the invention are particularly useful in cancer (see, Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 101P3AI 1 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 101P3A11 is involved, such as advanced or metastatic prostate cancers.

00 The invention also provides various immunological assays useful for the detection and quantification of S101P3A 11 and mutant 101P3A Il-related proteins. Such assays can comprise one or more 101P3A 1I antibodies Scapable of recognizing and binding a 101P3A 11-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 00 radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays 0(ELIFA), and the like.

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

SIn addition, immunological imaging methods capable of detecting prostate cancer and other cancers O expressing 101P3AI are also provided by the invention, including but not limited to radioscintigraphic imaging Smethods using labeled 101P3AI 1 antibodies. Such assays are clinically useful in the detection, monitoring, and 00 prognosis of 101P3A 11 expressing cancers such as prostate cancer.

101P3AI 1 antibodies are also used in methods for purifying a 101P3AI 1-related protein and for isolating 101P3A11 homologues and related molecules. For example, a method of purifying a 101P3A1 1-related protein comprises incubating a 101P3A 1 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 101P3A1 -related protein under conditions that permit the 101P3AI 1 antibody to bind to the 101P3AI 1related protein; washing the solid matrix to eliminate impurities; and eluting the 101P3AI l-related protein from the coupled antibody. Other uses of 101P3A1 I antibodies in accordance with the invention include generating antiidiotypic antibodies that mimic a 101P3A11 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 101P3Al 1-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 101P3A1 I can also be used, such as a 101P3A I GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 101P3A11-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 101P3AI 1-related protein or 101P3A 11 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 101P3AI 1 protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 101P3AI 1 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 101P3A1 I amino acid sequence are used to identify hydrophilic regions in the 101P3AI structure.

Regions of a 101P3A11 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method ofHopp, T.P. and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, 1982, J. Mol. Biol. 157:105-132. Percent Accessible Residues profiles can be generated using the method ofJanin 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method ofBhaskaran Ponnuswamy 1988, Int. J. Pept.

Protein Res. 32:242-255. Beta-turn profiles can be generated using the method ofDeleage, Roux 1987, 00 0 Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the C"i scope of the present invention. Methods for the generation of 101P3A 11 antibodies are further illustrated by way of f the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well c- known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a 00 carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 101P3A11 immunogen is often conducted by injection over a suitable O time period and with use of a suitable adjuvant, as is understood in the art During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

S101P3A1 monoclonal antibodies can be produced by various means well known in the art. For example, 00 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 CN known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 101P3A1 1-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 101P3AI 1 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 101P3A1 I 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 at, 1993, Proc. Natl. Acad. Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 101P3A1 1 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 101P3A I monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits et al., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin.

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

Reactivity of 101P3A11 antibodies with a 101P3A 1 -related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 101P3AI -related proteins, 101P3A1 l-expressing cells or extracts thereof. A 101P3A11 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or 0 more 101P3AI 1 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art Wolffet Cancer Res. 53: 2560-2565).

SThus, the present invention relates to polyclonal and monoclonal antibodies raised in response to either 101P3A 1, or biologically active fragments thereof. The polyclonal and/or monoclonal antibodies of the present 00 invention, especially 101P3AI I neutralizing antibodies (antibodies that block 101P3A 1 function and/or block its binding with ligands), will also be useful as therapeutics to modulate 101P3AI 1 expression and/or activity. In addition, the polyclonal and/or monoclonal antibodies of the present invention are useful as diagnostics for rn qualitative and/or quantitative detection of 101P3A 1.1 expression in a variety of immunoassays, Western blotting; immunohistochemistry and immunoprecipitation (of samples/biopsies of material such as tissue, serum, blood, urine or semen); and, (ii) as noted above, inhibition of 101P3Al I function using 101P3AI neutralizing 00 antibodies for the treatment of diseases associated with 101P3A1 I overexpression such as cancers of tissues listed Sin Table I. Antibodies that antagonize the effect of 101P3A1 I (for example, inhibition of 101P3Al l's ability to protect certain cells from apoptosis, cell death or inhibition of 101P3Al l's ability to bind to its ligand) can be administered directly by methods known in the art (see, Antibodies in Human Diagnosis and Therapy by Raven Press, New York (1977)). Monoclonal antibodies are especially preferred for the treatment of tumors associated with an abnormal 101P3Al 1 expression. For example, a 101P3AI 1 monoclonal antibody which slows the progression of a cancer associated with an increase in 101P3AI 1 expression within cancer cells, and in turn can cause tumor shrinkage over time, will be especially useful.

A useful paradigm is the early success ofHerceptinTM, a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor 2 (HER2). The antibody is produced in CHO cells and the final product is available as a lyophilized powder.

HER2 has been shown to be overexpressed in 25-30% of primary breast cancers. In turn, administration of Herceptin T has been shown to inhibit the proliferation of tumor cells which ovcrexprcss HER2. A prospective 101P3AI monoclonal antibody can be "humanized" by methods well known in the art, such as XcnomouscTM technology (Abgenix), or antibody phage display, in order to reduce any unwanted immunological effects of human administration of the antibody. Alternatively, the 101P3A 11-based antibody can be a chimera, most likely a mouse/human or rat/human chimera. In addition, any such therapeutic 101P3Al I-based antibody can be administered alone or within a regime that includes other cancer therapies, such as known chemotherapeutic agents, which can act in concert to reduce tumor growth associated with increased 101P3AI 1 expression.

Another example of the use of monoclonal antibodies to treat various cancers is Rituxan

T

a recombinant DNAbased mouse/human chimeric monoclonal antibody which has been shown to be effective in treating patients with low grade B-cell non-Hodgkin's lymphoma (NHL), a cancer of the immune system. Rituxan targets and destroys white blood cells (B cells) involved in the disease, resulting insignificant tumor shrinkage with less severe sideeffects than most cancer treatments. Additional monoclonal antibodies currently under development include an monoclonal antibody to treat patients with low-grade lymphomas, (ii) a combination anti-EGFr antibody with doxorubicin in patients with hormone refractory prostate cancer as well as a combination anti- EGFr antibody with cisplatin in patients with head and neck and lung cancer. A 101P3A11 anti-idiotype antibody can also be administered so as to stimulate a host immune response to tumors overexpressing 101P3A11.

Therefore, it is evident that 101P3AI I antibodies, especially 101P3AI 1 monoclonal antibodies, are potentially useful tools, along or in combination with other cancer therapies, for direct therapeutic intervention of cancers characterized by an increase in 101P3A 11 expression.

0 Skilled artisans understand that equivalent molecules known in the art which mimic the inhibitory Sactivity of an antibody capable of inhibiting 101P3A1 I function are aspects of the presently disclosed methods which employ an antibody capable of inhibiting 101P3A 1I function. Examples of such molecules include antic 101P3AI 1 peptide mimetics which inhibit the growth of at least a comparable or like manner to an antibody 00 capable of inhibiting 101P3A1 function.

Polvclonal Antibodies The antibodies of the invention also comprise polyclonal antibodies. Methods of preparing polyclonal Santibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one r<f 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. The immunizing 00agent can include a 101P3A11 polypeptide or a fusion protein thereof. It can be useful to conjugate the Simmunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such Simmunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which can be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The immunization protocol can be selected by one skilled in the art without undue experimentation. The mammal can then be bled, and the serum assayed for antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus.

Monoclonal Antibodies The antibodies of the invention can, alternatively, be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).

In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or arc capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include a 101P3AI 1 polypeptide or a fusion protein thereof.

Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].

Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRTdeficient cells. Alternatively, SLAM technology can be employedfor screening as appreciated by one of skill in the art.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Vir.

00 O An example of such a murine myeloma cell line is P3X63AgU.1. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.

SImmunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

00 The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the 101P3A1 1. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in in vitro binding 0 assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques M and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

00 After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution Sprocedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic C, Press, (1986)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see, U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)) or by covalcntly joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. Optionally, chimeric antibodies can be constructed which include at least one variable or hypervariable domain of an anti-101P3Al I antibody selected from the antibodies disclosed herein.

Optionally, the antibody capable of inhibiting 101P3AI 1 function of the present invention will bind to the same epitope(s) as any of the antibodies disclosed herein. This can be determined by conducting various assays, such as described herein. For instance, to determine whether a monoclonal antibody has the same specificity as the antibodies referred to herein, one can compare its activity in blocking assays or inhibition assays or functional assays.

The antibodies of the invention include "cross-linked" antibodies. The term "cross-linked" as used herein refers to binding of at least two IgG molecules together to form one (or single) molecule. The 101P3AI 1 antibodies can be cross-linked using various linker molecules and optionally the antibodies are cross-linked using 00 an anti- IgG molecule, complement, chemical modification or molecular engineering. It is appreciated by those Sskilled in the art that complement has a relatively high affinity to antibody molecules once the antibodies bind to Scell surface membrane. Accordingly, complement can be used as a cross-linking molecule to link two or more antibodies bound to cell surface membrane. Among the various murine Ig isotypes, IgM, IgG2a and IgG2b are 00 known to fix complement.

The antibodies of the invention can optionally comprise dimeric antibodies, as well as multivalent forms of antibodies. Those skilled in the art can construct such dimers or multivalent forms by techniques known in the art and using the anti-101P3All antibodies herein.

SThe antibodies of the invention can also comprise monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of C immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the 00 Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted Swith another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

Single chain Fv fragments can also be produced, such as described in Iliades et al, FEBS Letters, 409:437-441 (1997). Coupling of such single chain fragments using various linkers is described in Kortt et al., Protein Engineering, 10:423-433 (1997).

In addition to the antibodies described herein, it is contemplated that chimeric or hybrid antibodies are prepared using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.

Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

The 101P3A 11 antibodies of the invention further comprise humanized antibodies or human antibodies.

Humanized forms of non- human murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the 00 O human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, Svariable domains, in which all or substantially all of the CDR regions correspond to those of a non-human 00 immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et 0 al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- 00 human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" 0 variable domain. Humanization can be essentially performed following the method of Winter and co- workers NC (Jones et al., Nature, 321:522-525 (1986); Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al., Science 239;1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies Pat. No.

4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Sources of such import residues or import variable domains (or CDRs) include antibodies that specifically bind 101P3A11.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296-2308 (1993); Chothia and Lesk, J. Mol.

Biol., 196:901- 917 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992); Presta et al., J. Immunol., 151:2623- 2632 (1993)).

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679 published 3 March 1994).

00 Human monoclonal antibodies can be made via an adaptation of the hybridoma method first described by Kohler and Milstein by using human B lymphocytes as the fusion partner. Human B lymphocytes producing an F1 antibody of interest can, for example, be isolated from a human individual, after obtaining informed consent. For instance, the individual can be producing antibodies against an autoantigen as occurs with certain disorders such 00 as systemic lupus erythematosus (Shoenfeld et al. J. Clin. Invest., 70:205 (1982)), immune-mediated thrombocytopenic purpura (ITP) (Nugent et al. Blood, 70(1):16-22 (1987)), or cancer. Alternatively, or additionally, lymphocytes can be immunized in vitro. For instance, one can expose isolated human peripheral Sblood lymphocytes in vitro to a lysomotrophic agent L- leucine-0-methyl ester, L-glutamic acid dimethly Sester or L-leucyl-L- leucine-O-methyl ester) Pat. No. 5,567,610, Borrebaeck et and/or T-cell depleted human peripheral blood lymphocytes can be treated in vitro with adjuvants such as 8-mercaptoguanosine and C cytokines Pat. No. 5,229,275, Goroffet al.).

00 SThe B lymphocytes recovered from the subject or immunized in vitro, are then generally immortalized in order to generate a human monoclonal antibody. Techniques for immortalizing the B lymphocyte include, but ate not limited to: fusion of the human B lymphocyte with human, murine myelomas or mouse-human heteromyeloma cells; viral transformation with an Epstein-Barr virus; see Nugent et al., supra, for example); fusion with a lymphoblastoid cell line; or fusion with lymphoma cells.

Lymphocytes can be fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). The hybridoma cells thus prepared ate seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Suitable human myeloma and mouse-human heteromyeloma cell lines have been described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Human antibodies can also be generated using a non-human host, such as a mouse, which is capable of producing human antibodies. As noted above, transgenic mice are now available that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody 00 production. Transfer of the human germ-line immunoglobulin gene array in such germ- line mutant mice resulted C in the production of human antibodies upon antigen challenge. See, Jakobovits et al., Proc. Natl. Acad. Sci.

SUSA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); U.S. Pat. No. 5,591,669; U.S. Pat. No. 5,589,369; and U.S. Pat. No. 5,545,807. Human antibodies can 00 also be prepared using SCID-hu mice (Duchosal et al Nature 355:258-262 (1992)).

In another embodiment, the human antibody can be selected from a human antibody phage display library. The preparation of libraries of antibodies or fragments thereof is well known in the art and any of the known methods can be used to construct a family of transformation vectors which can be introduced into host C cells. Libraries of antibody light and heavy chains in phage (Huse et al., Science, 246:1275 (1989)) or of fusion proteins in phage or phagemid can be prepared according to known procedures. See, for example, Vaughan et al, Nature Biotechnology 14:309-314 (1996); Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991); 00 O Marks et al., J. Mol. Biol., 222:581-597 (1991); Hoogenboom and Winter, J. Mol Biol., 227:381- 388 (1992); Barbas et al., Proc. Natl. Acad. Sci., USA, 89: 4457-4461 (1992); Griffiths et al., EMBO Journal, 13:3245-3260 (1994); de Kruif et al., J. Mol. Biol., 248:97-105 (1995); WO 98/05344; WO 98/15833; WO 97/47314; WO 97/44491; WO 97/35196; WO 95/34648; U.S. Pat. No. 5,712,089; U.S. Pat. No. 5,702,892; U.S. Pat. No.

5,427,908; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,270,170; WO 92/06176; WO 99/06587; U.S. Pat. No. 5,514,548; WO 97/08320; and U.S. Pat. No. 5,702,892. The antigen of interest is panned against the phage library using procedures known in the field for selecting phage- antibodies which bind to the target antigen.

The 101P3A11 antibodies, as described herein, will optionally possess one or more desired biological activities or properties. Such antibodies can include but are not limited to chimeric, humanized, human, and affinity matured antibodies. As described above, the antibodies can be constructed or engineered using various techniques to achieve these desired activities or properties. In one embodiment, the 101P3A 1 antibody will have a 101P3AI 1 binding affinity of at least 10 s M, preferably at least in the range of 10 M to 10 7 M, mote preferably, at least in the range of 10 M to 101 2 M and even more preferably, at least in the range of 10" M to 1 2 M. The binding affinity of the antibody can be determined without undue experimentation by testing the antibody in accordance with techniques known in the art, including Scatchard analysis (Munson and Pollard, Anal. Biochem., 107:220 (1980)). For example, a 101P3AI 1 antibody can be assayed for binding affinity to 101P3AI 1, including constructs or fragments thereof.

In another embodiment, the antibody interacts in such a way to create a steric conformation which prevents binding of an antibody capable of inhibiting or enhancing 101P3A 11 function. The epitope binding property of the antibody of the present invention can be determined using techniques known in the art.

Other Modifications Other modifications of the 101P3AI 1 antibodies are contemplated herein. The antibodies of the present invention can be modified by conjugating the antibody to a cytotoxic agent (like a toxin molecule) or a prodrugactivating enzyme which converts a prodrug a peptidyl chemotherapeutic agent, see W081/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278. This technology is also referred to as "Antibody Dependent Enzyme Mediated Prodrug Therapy" (ADEPT).

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting 47 00 0 phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5- fluorocytosine into the anti-cancer drug, fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such t as cathepsins B and that are useful for converting peptide-containing prodrugs into free drugs; caspases such 00 as caspase-3; D-alanylcarboxypeptidases, useful for converting prodrugs (hat contain D- amino acid substituents; carbohydrate-cleaving enzymes such as beta- galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; beta-lactamase useful for converting drugs derivatized with beta-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs N derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs.

Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the KN prodrugs of the invention into free active drugs (see, Massey, Nature 328: 457-458 (1987)). Antibodyabzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.

The enzymes can be covalently bound to the antibodies by techniques well known in the art such as the use ofheterobifunctional crosslinking reagents. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, Neuberger et al., Nature, 312: 604-608 (1984).

Further modifications to the polypeptides of the invention are contemplated. For example, the antibodies can be linked to one of a variety ofnonproteinaceous polymers, polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, Ed., (1980). To increase the serum half life of the antibody, one can incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described, in U.S. Pat. No. 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule IgG 1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

Formulations The antibody capable of inhibiting 101P3AI 1 function are preferably administered in a carrier. The molecules can be administered in a single carrier, or alternatively, can be included in separate carriers. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the carrier to render the formulation isotonic. Examples of the carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7.4 to about 7.8. It will be apparent to those persons skilled in the art that certain carriers are preferred depending upon, for instance, the route of administration and concentration of agent being administered. The carrier can be in the form of a lyophilized formulation or aqueous solution.

Acceptable carriers, excipients, or stabilizers are preferably nontoxic to cells and/or recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; 00 antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and mcresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, 00 or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or Ssorbitol; salt-forming counter-ions such as sodium; and/or non-ionic surfactants such as TWEEN®, N PLURONICS® or polyethylene glycol (PEG).

SThe formulation can also contain more than one active compound as necessary for the particular Sindication being treated, preferably those with complementary activities, and preferably that do not adversely

OO

Saffect each other. Alternatively, or in addition, the composition can comprise a cytotoxic agent, cytokine or Sgrowth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The antibody capable of inhibiting 101P3A 11 function can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Oslo, A.

Ed. (1980).

The formulations to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides Pat. No.

3,773,919), copolymers of L-glutamic acid and ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

Modes of Administration An antibody(s) capable of inhibiting 101P3A 11 function can be administered in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Optionally, administration can be performed through mini-pump infusion using various commercially available devices.

Effective dosages and schedules for administering an antibody capable of inhibiting 101P3A function can be determined empirically, and making such determinations is within the skill in the art. Effective dosage or amount of an antibody capable of inhibiting 101P3A 11 function used alone may range from about 1 tg/kg to about 100 mg/kg of body weight or more per day. Interspecies scaling of dosages can be performed in a manner O known in the art, as disclosed in Mordenti et al., Pharmaceut. Res., 8:1351 (1991). Those skilled in the art Swill understand that the dosage of an antibody capable of inhibiting 101P3A 11 function that must be administered F: will vary depending on, for example, the mammal which will receive the an antibody capable of inhibiting 101P3A11 function, the route of administration, and other drugs or therapies being administered to the mammal.

00 Depending on the type of cells and/or severity of the disease, about 1 pg/kg to 15 mg/kg 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 pg/kg to M 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or Slonger, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens can be useful.

SIt is contemplated that yet additional therapies can be employed in the methods. The one or more other 0 therapies can include but are not limited to, other chemotherapies (or chemotherapeutic agents) and/or radiation 0 therapy, immunoadjuvants, growth inhibitory agents, cytokines, and other non-Her-2 antibody-based therapies.

Examples include interleukins IL-I, IL-2, IL-3, IL-6), leukemia inhibitory factor, interferons, erythropoietin, thrombopoietin, and anti-VEGF antibody. Other agents known to inhibit the growth of mammalian cells -can also be employed, and such agents include TNF-, CD30 ligand, 4-1BB ligand, and Apo-l ligand.

Additional chemotherapies contemplated by the invention include chemical substances or drugs which are known in the art and are commercially available, such as Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside Cyclophosphamide, Leucovorin, Thiotcpa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carrainomycin, Amimopterin, Dactinomycin, Mitomycins, Esperamicins (see U. S. Pat. No. 4,675,187), Melphalan and other related nitrogen mustards. Also included are agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onaptistone.

Preparation and dosing schedules for such chemotherapy can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams Wins, Baltimore, Md.

(1992). The chemotherapeutic agent can precede, or follow administration with the antibody capable of inhibiting 101P3A11 function, or can be given simultaneously therewith.

The chemotherapy is preferably administered in a carrier, such as those described above. The mode of administration of the chemotherapy can be the same as employed for an antibody capable of modulating, such as inhibiting or enhancing, 101P3A 11 function, or it can be administered via a different mode.

Radiation therapy can be administered according to protocols commonly employed in the art and known to the skilled artisan. Such therapy can include cesium, iridium, iodine, or cobalt radiation. The radiation therapy can be whole body irradiation, or can be directed locally to a specific site or tissue in or on the body. Typically, radiation therapy is administered in pulses over a period of time from about I to about 2 weeks. The radiation therapy can, however, be administered over longer periods of time. Optionally, the radiation therapy can be administered as a single dose or as multiple, sequential doses.

An antibody capable of inhibiting 101P3A1 I function (and one or more other therapies) can be administered concurrently or sequentially. Following administration of an antibody capable of inhibiting 101P3AI function, treated cells in vitro can be analyzed. Where there has been in vivo treatment, a treated 00 g mammal can be monitored in various ways well known to the skilled practitioner. For instance, tumor mass can Sbe observed physically, by biopsy or by standard x-ray imaging techniques.

SV.) 101P3A11 Cellular Immune Responses 00 The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the D invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, 00 Annu. Rev. Immunol. 7:601, 1989; Germain, R. Annu. Rev. Immunol. 11:403, 1993). Through the study of O single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed C1 peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been idcntificd and arc set forth in Table IV (see also, Southwood, et al., J. Immunol. 160:3363, 1998; Rammensec, et al., Inmunogenetics 41:178, 1995; Rammenscc et al., SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V.

Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F.

and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.

155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, et 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, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. J. Mol. Biol. 219:277, 1991.) Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

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

Various strategies can be utilized to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal individuals (see, Wentworth, P. A. et 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 00 peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen Ci presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during F1 this time and are detected using, a lymphokine- or 5 Cr-release assay involving peptide sensitized target Scells.

0 2) Immunization of HLA transgenic mice (see, Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For Sexample, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA D transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the Spresence of test peptide for approximately one week. Peptide-specific T cells are detected using, a 5 1 Cr- C~ release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

0 3) Demonstration of recall T cell responses from immune individuals who have been either effectively 0 vaccinated and/or from chronically ill patients (see, Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et a., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells. At the end of the culture period, T cell activity is detected using assays including 5 1 Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 101P3A11 Transgenic Animals Nucleic acids that encode a 101P3AI I-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 101P3A1 1 can be used to clone genomic DNA that encodes 101P3A11. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 101P3A11. 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 101P3A11 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 101P3AI 1 can be used to examine the effect of increased expression of DNA that encodes 101P3AI 1. 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 101P3AI 1 can be used to construct a 101P3A1 I "knock out" animal that has a defective or altered gene encoding 101P3A1 I as a result of homologous recombination between the endogenous gene encoding 101P3AI 1 and altered genomic DNA encoding 101P3A 11 introduced into an embryonic cell of the animal. For example, cDNA that encodes 101P3A1 I can be used to clone genomic DNA 00 encoding 101P3A 1 in accordance with established techniques. A portion of the genomic DNA encoding 101P3A I can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be g used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous 00 recombination vectors). The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras (see, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A SPractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be Simplanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock 0 out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by Sstandard techniques and used to breed animals in which all cells of the animal contain the homologously c 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 101P3A1 I polypeptide.

VII.) Methods for the Detection of 101P3A11 Another aspect of the present invention relates to methods for detecting 101P3A11 polynucleotides and 101P3AI l-related proteins, as well as methods for identifying a cell that expresses 101P3A11. The expression profile of 101P3A11 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 101P3A11 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 101P3AI 1 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 Northern 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 101P3A 11 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like.

Detectable 101P3A1 I polynucleotides include, for example, a 101P3A11 gene or fragment thereof, 101P3A11 mRNA, alternative splice variant 101P3AI 1 mRNAs, and recombinant DNA or RNA molecules that contain a 101P3A1 I polynucleotide. A number of methods for amplifying and/or detecting the presence of 101P3A1 I 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 101P3AI I 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 101P3A11 polynucleotides as sense and antisense primers to amplify 101P3A I cDNAs therein; and detecting the presence of the amplified 101P3A1 1 cDNA. Optionally, the sequence of the amplified 101P3AI 1 cDNA can be determined.

In another embodiment, a method of detecting a 101P3A 11 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 101P3A11 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 101P3AI 1 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 101P3A11 nucleotide sequence (see, Figure 2) and used for this purpose.

00 The invention also provides assays for detecting the presence of a 101P3A 11 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 101P3A1 I-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of 00 detecting the presence ofa 101P3A 1l-related protein in a biological sample comprises first contacting the sample with a 101P3A 11 antibody, a 101P3A11-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 101P3AI 1 antibody; and then detecting the binding of 101P3AI 1-related protein in Sthe sample.

C Methods for identifying a cell that expresses 101P3AI I are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 101P3A I gene comprises detecting the presence of I^101P3A 1 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for 00 Sexample, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled S101P3A11 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 101P3AI 1, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 101P3A11 gene comprises detecting the presence of 101P3AI -related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 101P3A l-related proteins and cells that express 101P3A1 -related proteins.

101P3A11 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 101P3A11 gene expression. For example, 101P3AI 1 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 101P3AI expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 101P3A 11 expression by RT-PCR, nucleic acid hybridization or antibody binding.

V1I.) .Methods for Monitoring the Status of 101P3A11-related Genes and Their Products Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 101P3A I 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 101P3A 11 in a biological sample of interest can be compared, for example, to the status of 101P3AI 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 101P3AI in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, Grever et al., J. Comp.

Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 101P3A11 status in a sample.

The term "status" in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or OC state of a gene and its products. These include, but are not limited to the location of expressed gene products 0 (including the location of 101P3A I expressing cells) as well as the level, and biological activity of expressed gene products (such as 101P3AI I mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 101P3A11 comprises a change in the location of 101P3A 11 and/or 101P3A11 expressing cells and/or an increase 00 in 101P3A 1 mRNA and/or protein expression.

101P3AI 1 status in a sample can be analyzed by a number of means well known in the ar, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected Ssamples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 101P3A 11 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular SBiology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 101P3AI 1 in a biological sample is evaluated by various methods utilized by skilled artisans 00 including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 101P3AI 1 0 gene), Northern analysis and/or PCR analysis of 101P3A 11 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 101P3A11 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 101P3A1 I proteins and/or associations of 101P3A 11 proteins with polypeptide binding partners). Detectable 101P3AI 1 polynucleotides include, for example, a 101P3AI I gene or fragment thereof, 101P3AI 1 mRNA, alternative splice variants, 101P3AI I mRNAs, and recombinant DNA or RNA molecules containing a 10 IP3A 11 polynucleotide.

The expression profile of 101P3A I1 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 101P3A1 I provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 101P3A1 I status and diagnosing cancers that express 101P3A 1, such as cancers of the tissues listed in Table I. For example, because 101P3Al 1 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 101P3A11 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 101P3AI 1 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 101P3AI I 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 101P3AI I 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 101P3AI 1 in a biological sample can be examined by a number of wellknown procedures in the art. For example, the status of 101P3A 11 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 101P3AI 1 expressing cells those that express 101P3A11 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 101P3A1 I-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 101P3AI I in a biological sample are often associated with dysregulated cellular growth. Specifically, one 00 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 0 (see, Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 Aug 154(2 Pt 1):474.8).

In one aspect, the invention provides methods for monitoring 101P3A1 I gene products by determining the status of 101P3A I gene products expressed by cells from an individual suspected of having a disease N associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so Sdetermined to the status of 101P3AI 1 gene products in a corresponding normal sample. The presence of aberrant 101P3All gene products in the test sample relative to the normal sample provides an indication of the presence of 00 dysregulated cell growth within the cells of the individual.

O In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 101P3A1 1 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 101P3Al 1 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 101P3A I1 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 101P3A11 mRNA or express it at lower levels.

In a related embodiment, 101P3A11 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 101P3A1 I protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 101P3A I1 expressed in a corresponding normal sample. In one embodiment, the presence of 101P3A 1 protein is evaluated, for example, using immunohistochemical methods. 101P3A11 antibodies or binding partners capable of detecting 101P3AI protein expression are used in a variety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 101P3AI 1 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid 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 mutation in the sequence of 101P3AI 1 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 101P3AI 1 indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 101P3A1 I gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, U.S. Patent Nos. 5,382,510 issued 7 September 1999, and 5,952,170 issued 17 January 1995).

Additionally, one can examine the methylation status of a 101P3A 11 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in 00 immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter Shypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not Sexpressed 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 el al., Am. J. Pathol. 155(6): 1985- 00 1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral (N 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 CI hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain 0 methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of 101P3AI 1. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.

Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 101P3AI 1 expression. The presence of RT-PCR amplifiable 101P3AI 1 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In tle prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer, In one embodiment, a method for predicting susceptibility to cancer comprises detecting 101P3A11 mRNA or 101P3A1 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 101P3A1 I mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 101P3AI 1 in prostate or other tissue is examined, with the presence of 101P3AI 1 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 101P3AI 1 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 0 perturbations in-101P3Al I gene products in the sample is an indication of cancer susceptibility (or the emergence or 0 existence of a tumor).

r1 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 101P3A 11 mRNA or 101P3A 11 protein 0 expressed by tumor cells, comparing the level so determined to the level of 101P3A 11 mRNA or 101P3A 1 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 101P3AI 1 mRNA or 101P3AI 1 protein expression in the tumor sample relative to the normal f sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by Sdetermining the extent to which 101P3A 11 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 101P3A1 I nucleotide and amino N acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as 0 insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive Stumors.

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 101P3Al I mRNA or 101P3A11 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 101P3AI1 mRNA or 101P3A 1 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 101P3A 1 mRNA or 101P3A11 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 101P3AI 1 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 101P3AI 1 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 101P3A1 gene and 101P3AI 1 gene products (or perturbations in 101P3A11 gene and 101P3A 11 gene products)ind a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, Bocking et al., 1984, Anal. Quant. Cytol.

6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 101P3A 1 gene and 101P3AI 1 gene products (or perturbations in 101P3A1 I gene and 101P3AI 1 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between the expression of 101P3A I gene and 101P3A 11 gene products (or perturbations in 101P3A I1 gene and 101P3AI 1 gene products) and another factor associated with malignancy entails detecting the overexpression of 101P3A I mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and 00 observing a coincidence of 101P3AI1 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or SPSM expression). In a specific embodiment, the expression of 101 P3A 1 and PSA mRNA in prostate tissue is Sexamined, where the coincidence of 101P3A 11 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

00 Methods for detecting and quantifying the expression of 101P3A11 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 101P3AI I mRNA include in situ hybridization using labeled 0 101P3A1 I riboprobes, Northern blot and related techniques using 101P3A11 polynucleotide probes, RT-PCR analysis Susing primers specific for 101P3A11, and other amplification type detection methods, such as, for example, branched 0 DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 00 101P3A 11 mRNA expression. Any number of primers capable of amplifying 101P3A 1 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal c or monoclonal antibodies specifically reactive with the wild-type 101P3AI 1 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 101P3A1l The 101P3A 1 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 101P3A 11, as well as pathways activated by 101P3AI I via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the "two-hybrid assay"). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, U.S. Patent Nos. 5,955,280 issued 21 September 1999, 5,925,523 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, Marcotte, et al., Nature 402: 4 November 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 101P3AI 1 protein sequences. In such methods, peptides that bind to 101P3AI 1 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 101P3A I1 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 101P3A 11 protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.

Alternatively, cell lines that express 101P3A 11 are used to identify protein-protein interactions mediated by 101P3A11. Such interactions can be examined using immunoprecipitation techniques (see, Hamilton etal. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 101P3AI I protein can be immunoprecipitated from 101P3A 1-expressing cell lines using anti-10IP3Al I antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 101P3A11 and a His-tag (vectors mentioned above).

The immunoprecipitated complex can be examined for protein association by procedures such as Western 00 blotting, 5S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 101P3AI 1 can be identified through related embodiments c. of such screening assays. For example, small molecules can be identified that interfere with protein function, 00 including molecules that interfere with 101P3A l's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 101P3Al1-related ion channel, protein pump, or cell IN communication functions are identified and used to treat patients that have a cancer that expresses 101P3A 11 (see, g Hille, Ionic Channels of Excitable Membranes 2 Ed., Sinauer Assoc., Sunderland, MA, 1992).

SMoreover, ligands that regulate 101P3A11 function can be identified based on their ability to bind 101P3A1 I and OO activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 g 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 lusion protein of 101P3AI 1 and a DNA-binding protein arc used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators which activate or inhibit 101P3A 11.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a 101P3A11 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 101P3AI 1 amino acid sequence, allowing the population of molecules and the 101P3A1 I amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 101P3A 11 amino acid sequence, and then separating molecules that do not interact with the 101P3Al I 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 101P3A11 amino acid sequence. The identified molecule can be used to modulate a function performed by 101P3AI 1. In a preferred embodiment, the 101P3A 11 amino acid sequence is contacted with a library ofpeptides.

Therapeutic Methods and Compositions The identification of 101P3A1 I as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 101P3AI I functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a 101P3A 1 protein are useful for patients suffering from a cancer that expresses 101P3A 11. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 101P3AI 1 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 101P3Al 1 gene or translation of 101P3AI 1 mRNA.

Anti-Cancer Vaccines 00 The invention provides cancer vaccines comprising a 101P3AI 1-related protein or 101P3A I-related nucleic acid. In view of the expression of 101P3A 1, cancer vaccines prevent and/or treat 101P3A1 -expressing cancers with g minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate 00 cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et a., 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 101P3A1 l-related protein, or a 101P3AI 1encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 101 P3A 11 Simmunogen (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 C et al., Ann Med 1999 Feb 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32) 0 Briefly, such methods of generating an immune response humoral and/or cell-mediated) in a mammal, C comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope an epitope present in a 101P3AI I protein shown in Figure 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope generates antibodies that specifically recognize that epitope). In a preferred method, a 101P3A 11 immunogen contains a biological motif, see Tables V-XVIII and XXII TO IL, or a peptide of a size range from 101P3AI 1 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.

The entire 101P3AI 1 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g.,Vitiello, A. et al., J.

Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide)

("PLG")

microspheres (see, Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see Tam, J. Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, et AIDS Bio/Technology 4:790,1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. etal., 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. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. e 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. Hunt, L. and Webster, R. Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. ed., p. 423, 1996; Cease, K.

and Berzofsky, J. Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. etal., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

00 In patients with 101P3A 1-associated cancer, the vaccine compositions of the invention can also be used (N in 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.

SCellular Vaccines: 00 CTL epitopes can be determined using specific algorithms to identify peptides within 101P3A I protein that bind corresponding HLA alleles (see Table IV; Epimer" and Epimatrix T M Brown University (URL www.brown.edu/Research/TB-HIvLab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; INO SYFPEITHI at URL syfpeithi.bmi-heidelberg.com). In a preferred embodiment, a 101 P3A 11 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII and XXII TO IL or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I 00 motif7supermotif(e.g., Table IV Table IV or Table IV and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif(e.g., Table IV or Table IV As is appreciated in the art, the CK1 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 I molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, additional amino acids can be attached to the amino and/or carboxyl termini of a motifbearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or amino acids long, or longer than 25 amino acids.

Antibody-based Vaccines A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein a 101P3A 1 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 101P3Al1 in a host, by contacting the host with a sufficient amount of at least one 101P3A I B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter recontacting the host with the 101P3A 11 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 101P3A1 I-related protein or a man-made multiepitopic peptide comprising: administering 101P3A11 immunogen a 101P3AI 1 protein or a peptide fragment thereof, a 101P3AI fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, U.S. Patent No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see, Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et Immunity 1994 751- 761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 101P3A11 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 101P3A11 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 0 facilitator, such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 1 101P3AI 1, in order to generate a response to the target antigen.

Nucleic Acid Vaccines: 00 Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 101P3A11. Constructs comprising DNA encoding a 101P3AI 1-related protein/immunogen and Cappropriate 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 101P3A1 I protein/immunogen.

C Alternatively, a vaccine comprises a 101P3AI1 -related protein. Expression of the 101P3A 11-related protein 00 0 immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells 0 that bear a 10IP3A 11 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address www.genweb.com).

Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S.

Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivius, and sindbis virus (see, Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et at. J. Natl. Cancer Inst 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 101P3AI -related protein into the patient intramuscularly or intradermally) to induce an anti-tumor response.

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

Thus, gene delivery systems are used to deliver a 101P3A 1-related nucleic acid molecule. In one embodiment, the full-length human 101P3A11 cDNA is employed. In another embodiment, 101P3A 1 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 101P3AI I 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 0 prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer Spatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, l dendritic cells can be used to present 101P3A I1 peptides to T cells in the context of MHC class I or II molecules.

In one embodiment, autologous dendritic cells are pulsed with 101P3A I peptides capable of binding to MHC 00 class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 101P3A 11 protein. Yet another embodiment involves engineering the overexpression of a 101P3A 11 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-associated virus, DNA N transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 101P3A1 I can also be engineered to express immune C' modulators, such as GM-CSF, and used as immunizing agents.

OO

0 101P3A11 as a Target for Antibody-based Therapy 101P3A11 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 ofintrabodies). Because 101P3A11 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 101P3A ll-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 101P3A11 are useful to treat 101P3A1 I-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

101P3A I antibodies can be introduced into a patient such that the antibody binds to 101P3A11 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 101P3A 1, 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 101P3AI 1 sequence shown in Figure 2 or Figure 3.

In addition, skilled artisans understand that it is routine to conjugate antibodies to cytoloxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g.

101P3A 11), 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-101P3Al I antibody) that binds to a marker 101P3A11) 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 101P3A 1, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 101P3A 11 epitope, and, exposing the cell to the antibody-agent conjugate.

00 Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized Scancer, comprising a step of administering parenterally to said individual a pharmaceutical composition Scomprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-101P3A1 I antibodies can be done in accordance with various 00 approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, SCancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol.

S19:93-101), leukemia (Zhong et 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.

(I Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or 0 radioisotope, such as the conjugation of Y 9 or to anti-CD20 antibodies ZevalinTM, IDEC SPharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin T M (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 101P3A11 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin MylotargM, Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG 4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see US Patent 5,416,064).

Although 101P3A11 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et at. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Although 101P3Al 1 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 101P3A1 I expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 101P3A I1 imaging, or other techniques that reliably indicate the presence and degree of 101P3A1 I 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.

00 g Anti-101P3Al 1 monoclonal antibodies that treat prostate and other cancers include those that initiate a Spotent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-101P3Al 1 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent c- cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin 00 molecule for interaction with effector cell Fe receptor sites on complement proteins. In addition, anti-101P3Al I mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 101P3A 11.

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-101P3Al 1 mAb exerts an anti-tumor effect is evaluated using any Snumber of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and 00 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 C, chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure.

Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 101P3AI 1 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-101P3A 11 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-101P3Al 1 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-101P3Al I mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agcnt(s) conjugated to them.

Anti-101P3Al 1 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, intradermal, and the like. Treatment generally involves repeated administration of the anti-I01P3Al 1 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin T M mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-101P3A 1 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 101P3AI 1 expression in the patient, the extent of circulating shed 101P3A 11 antigen, the desired steady-state antibody concentration level, frequency of 00 O treatment and the influence ofchemotherapeutic or other agents used in combination with the treatment method C'1 of the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 101P3AI 1 in a given sample the levels of Scirculating 101P3A11 antigen and/or 101P3AI 1 expressing cells) in order to assist in the determination of the 0 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, Surine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate 0 cancer therapy).

SAnti-idiotypic anti-101P3Al I antibodies can also be used in anti-cancer therapy as a vaccine for Sinducing an immune response to cells expressing a 101P3All-related protein. In particular, the generation of 00 anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate antiidiotypic anti-101P3All antibodies that mimic an epitope on a 101P3AI -related protein (see, for example, C' Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

101P3Al as a Target for Cellular Immune Responses Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with 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 invention are well known in the art, and include, e.g., 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 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 cytosinephosphorothiolated-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 at least partially immune to later development of cells that express or overexpress 101P3A11 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

67 00 O 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 Spreferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in 00 accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, CA) molecule (described in U.S. Patent Number 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), M as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, with a minigene in accordance with the invention, or are pulsed with peptides.

Q The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, Seither DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization Swhereby loading of dendritic cells occurs in vivo.

Preferably, the following principles arc utilized when selecting an array of epitopcs 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 ICs of 500 nM or less, often 200 nM or less; and for Class II an ICso of 1000 nM or less.

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

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

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

00 If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the CN 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, ct the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in 00 the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional cpitopcs (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made Sjuxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation.

I Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitopc. Of particular concern is a junctional epitope that is a "dominant epitope." A dominant 0epitope may lead to such a zealous response that immune responses to other epitopes are diminished or 00 suppressed.

Where the sequences of multiple variants of the same target protein are present, potential CN 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.I. Minigene Vaccines A number of different approaches are available which allow simultaneous delivery of multiple epitopes.

Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention.

Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. J Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. etal., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 101P3A11, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 101P3A 1, (see Tables V-XVIII and XXII to IL), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The immunogenicity ofa 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 transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: generate a CTL response and that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class n epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum 00 targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these Slarger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus 00 and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the Sepitope 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 C1 cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli 0 origin of replication; and an E. coli selectable marker ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, the human cytomegalovirus (hCMV) promoter. See, U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity.

In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

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

In some embodiments, a bi-cistronic expression vector which allows production of both the minigeneencoded 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 LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE

T

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

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc.

0 (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms Susing gel electrophoresis or other methods.

SPurified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as 00 "naked DNA," is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available.

SCationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see. as described Sby WO 93/24640; Mannino Gould-Fogerite, BioTechniques 682 (1988); U.S. Pat No. 5,279,833; WO S91/06309; and Feigner, et al., Proc. Nat lAcad. Sci. USA 84:7413 (1987). In addition, peptides and compounds C" referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to p0urified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment oftransfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium- 51 (sCr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 5 'Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations.

Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent IM for DNA in PBS, intraperitoneal for lipidcomplexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, "Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vive induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

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

X.C.2. Combinations of CTL Peptides with Helper Peptides 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 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 Sspacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or 00 amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a 0 hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually Sthree to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper Speptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The 00 amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a (C majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 QYIKANSKFIGITE (SEQ ID NO: Plasmodiumfalciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNVVNS (SEQ ID NO: and Streptococcus 18kD protein at positions 116-131 GAVDSILGGVATYGAA (SEQ ID NO: Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes PADRE

T

Epimmune, Inc., San Diego, CA) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO: where is either cyclohexylalanine, phenylalanine, 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 ofT helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C3. Combinations of CTL Peptides with T Cell Priming Agents In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the e-and a- amino groups of a lysine residue and then linked, via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser- Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered cither 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 00 0 e- and a- amino groups of Lys, which is attached via linkage, Ser-Ser, to the amino terminus of the CK immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S- Sglycerylcysteinlyseryl- serine (PjCSS) can be used to prime virus specific CTL when covalently attached to an 00 appropriate peptide (see, Deres, el al., Nature 342:561, 1989). Peptides of the invention can be coupled to

P

3 CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P 3

CSS-

conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cellc mediated responses.

SX.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides OO An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's C1 blood. A pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Pharmacia- Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptidepulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vive with a cocktail of peptides, some of which stimulate CTL responses to 101P3AI1. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 101P3A 1.

X.D. Adoptive Immunotherapy Antigenic 101P3AI I-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 101P3A11. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 101P3A11. The peptides or 73 00 0O DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, Sas appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 101P3AI 1-associated 00 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) Sdelivered to the patient may vary according to the stage of the disease or the patient's health status. For example, Sin a patient with a tumor that expresses 101P3A1 a vaccine comprising 101P3AI 1-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

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

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg.

Dosage values for a human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient.

Boosting dosages of between about 1.0 pg to about 50,000 pg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 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, intratlecal, or local as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, intravenously, subcutaneously, intradermally, or intramuscularly.

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

00 SA variety of aqueous carriers may be used, water, buffered water, 0.8% saline, 0.3% glycine, C" 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 Slyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

00 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.

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

C1 A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, Remington's Pharmaceutical Sciences, 17"' Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial inununization can be from about I to about 50,000 pg, 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) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu.

For antibodies, a treatment generally involves repeated administration of the anti-101P3A 11 antibody preparation, via an acceptable route of administration such as intravenous injection typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 101P3A1 I mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 10 1P3AI 1 expression in the patient, the extent of circulating shed 101P3AI I antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient Non-limiting preferred human unit doses are, for example, 500/ g lmg, Img 50mg, 50mg 100mg, 100mg 200mg, 200mg 300mg, 400mg 500mg, 500mg 600mg, 600mg 700mg, 7 00mg 800mg, 800mg 900mg, 900mg Ig, or Img 700mg. In certain embodiments, the dose is in a range of mg/kg body weight, with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3,4, 5, 6, 7, 8, 9, body weight followed, in two, three or four weeks by weekly doses; 0.5 10mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225,250, 275, 300,325, 350, 375, 400mg m 2 of body area weekly; 1-6 0 0mg m 2 of body area weekly; 2 25-400mg 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.

00 SIn one embodiment, human unit dose forms ofpolynucleotides comprise a suitable dosage range or C, effective amount that provides any therapeutic effect. As appreciated by.one of ordinary skill in the art a g therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health 00 care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than M the lower limit, of about 60, 80, 100, 200, 300,400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 0Q mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, I to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, I to 100 mg/kg, S100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 C 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 ofT-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 10' cells to about 106 cells, about 106 cells to about 10' cells, about 10' to about 10" cells, or about 108 to about 5 x 1010 cells. A dose may also about 106 cells/m to about 1010 cells/m 2 or about 10' cells/m to about 10' cells/m 2 Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposone, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, Szoka, et al., Ann. Rev. Biophys. Bioeng.

9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

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

00 For solid compositions, conventional nontoxic solid carriers may be used which include, for example, CN pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable C- nontoxic composition is formed by incorporating any of the normally employed cxcipicnts, such as those carriers 00 previously listed, and generally 10-95% 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 O 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.

0 Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon 00 atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be C1 employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25- 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 101P3AI1.

As disclosed herein, 101P3A11 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled "Expression analysis of 101P3AI 1 in normal tissues, and patient specimens").

101P3AI 1 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 el al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J.

Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, Tulchinsky et Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto et al., Cancer Detect Prey 2000;24(1):1-12). Therefore, this disclosure of 101P3A 1I polynucleotides and polypeptides (as well as 101P3A 11 polynucleotide probes and anti-101P3Al I antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 101P3A 11 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays which employ, PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, Sharief et al., Biochem. Mol. Biol.

Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 101P3A11 polynucleotides described herein can be utilized in the same way to detect 101P3A1 I overexpression or the metastasis of prostate and other cancers 77 0 expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA Swhich 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. Pract 192(3):233-7 (1996)), the 101P3A 11 polypeptides described herein 00 can be utilized to generate antibodies for use in detecting 101P3A I 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 m the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a N biological sample for the presence of cells expressing 101P3A 1I polynucleotides and/or polypeptides can be used Sto provide evidence of metastasis. For example, when a biological sample from tissue that does not normally C",I contain 101P3A 1 -expressing cells (lymph node) is found to contain 101P3A 1 -expressing cells such as the

OO

0 101P3A1l expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, Srespectively, this finding is indicative of metastasis.

Alternatively 101P3A 11 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 101P3A11 or express 101P3A 11 at a different level are found to express 101P3AI or have an increased expression of 101P3AI 1 (see, the 101P3A1 I 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 101P3AI I) such as PSA, PSCA etc. (see, Alanen et al., 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, 101P3A 11 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478- 480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled "Expression analysis of 101P3A11 in normal tissues, and patient specimens," where a 101P3A11 polynucleotide fragment is used as a probe to show the expression of 101P3A 11 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, Sawai et al., Fetal Diagn. Ther.

1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M.

Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence a 101P3A11 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 specifically binds to that epitope are used in methods of monitoring PSA. 101P3A 1 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the Sart with a wide variety of systems such as fusion proteins being used by practitioners (see, Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel el al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses 00 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 101P3Ai I biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based C on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as Slong as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence a 101P3A 11 polypeptide shown in Figure 3).

As shown herein, the 101P3AI 1 polynucleotides and polypeptides (as well as the 101P3AI 1 0 polynucleotide probes and anti-101P3A 11 antibodies or T cells used to identify the presence of these molecules) Sexhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 101P3A11 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 101P3A11 polynucleotides and polypeptides (as well as the 101P3Al I polynucleotide probes and anti-101P3Al I antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 101P3A 11 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 101P3A11 gene maps (see the Example entitled "Chromosomal Mapping of 101P3AI below). Moreover, in addition to their use in diagnostic assays, the 101P3A 11-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, Takahama K Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).

Additionally, 101P3A1 I-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of I01P3A 1. 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 101P3A11 antigen. Antibodies or other molecules that react with 101P3AI I can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 101P3A11 Protein Function The invention includes various methods and compositions for inhibiting the binding of 101P3A11 to its binding partner or its association with other protein(s) as well as methods for inhibiting 101P3A1 I function.

XI.A.) Inhibition of 101P3A11 With Intracellular Antibodies In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 101P3A11 are introduced into 101P3A11 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-101P3Al I antibody is expressed intracellularly, binds to 101P3AI I protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known.

79 00 Such intracellular antibodies, also known as "intrabodies", are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology il has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).

Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem.

00 S 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 Slinker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as Sa single chain variable region fragment joined to the light chain constant region. Well-known intracellular Strafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies 0 targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C- O terminal ER retention signal, such as the KDEL (SEQ ID NO: amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 101P3A1 I in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals arc engineered into such 101P3A1 I intrabodics in order to achieve the desired targeting. Such 101P3AI I intrabodics arc designed to bind specifically to a particular 101P3AI 1 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 101P3AI 1 protein are used to prevent 101P3A11 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus preventing 101P3AI 1 from forming transcription complexes with other factors).

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

XI.B.) Inhibition of 101P3A11 with Recombinant Proteins In another approach, recombinant molecules bind to 101P3A 11 and thereby inhibit 101P3A11 function.

For example, these recombinant molecules prevent or inhibit 101P3A 11 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 101P3A1 I specific antibody molecule. In a particular embodiment, the 101P3Al I binding domain of a 101P3A1 I binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 101P3AI 1 ligand binding domains linked to the Fc portion of a human IgG, such as human IgGl. Such IgG portion can contain, for example, the CH2 and CH 3 domains and the hinge region, but not the CHI domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 101P3Al 1, whereby the dimeric fusion protein specifically binds to 101P3AI I and blocks 101P3A1 I interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

00 SXII.C.) Inhibition of 101P3AI 1 Transcription or Translation C The present invention also comprises various methods and compositions for inhibiting the transcription a of the 101P3AI gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 101P3A 1 mRNA into protein.

OO In one approach, a method of inhibiting the transcription of the 101P3A I gene comprises contacting the 101P3AI 1 gene with a 101P3AI antisense polynucleotide. In another approach, a method of inhibiting 101P3AI 1 mRNA translation comprises contacting a 101P3Al1 mRNA with an antisense polynucleotide. In IN another approach, a 101P3Al1 specific ribozyme is used to cleave a 101P3AI 1 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 101P3A11 gene, such as 101P3A11 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting 00 a 101P3AI 1 gene transcription factor are used to inhibit 101P3AI 1 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of C antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 101P3AI 1 by interfering with 101P3A I1 transcriptional activation are also useful to treat cancers expressing 101P3A11. Similarly, factors that interfere with 101P3A11 processing are useful to treat cancers that express 101P3A11. 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 101P3A11 antisense, ribozyme, polynucleotides encoding intrabodies and other 101P3AI 1 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 101P3A11 antisense polynucleotides, ribozymes, factors capable of interfering with 101P3A1 1 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 101P3A11 to a binding partner, etc.

In vivo, the effect of a 101P3A11 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Patent 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic mctastascs characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

0 In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions.

SIn one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence ofapoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

00 The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include Sany material that when combined with the therapeutic composition retains the anti-tumor function of the IN therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but Sare 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, Remington's Pharmaceutical Sciences 16' Edition, A.

00 Osal., Ed., 1980).

STherapeutic formulations can be solubilizcd 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 bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

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

XIII.) Kits/Articles of Manufacture For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled.

Such probe can be an antibody or polynucleotide specific for a Figure 2-related protein or a Figure 2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

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

A label can be present on the container to indicate that the composition is used for a specific therapy or nontherapeutic application, such as a diagnostic or laboratory application, and can also indicate directions for either in vive 0 or in vitro use, such as those described herein. Directions and or other information can also be included on an Sinsert(s) or label(s) which is included with or on the kit.

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

In another embodiment of the invention, an article(s) of manufacture containing compositions, such as 00 amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable Scontainers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a M variety of materials such as glass or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), in one embodiment the container holds a polynucleotide for use in examining the mRNA expression profile of a cell,. together with reagents used for this purpose.

00 SThe container can alternatively hold a composition which is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or-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 101P3AI 1 and modulating the function of 101P3A11.

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 neoplasia of a tissue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.

XIV.) Evaluation of GPCRs and Modulators Thereof The traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the.receptor. Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been recognized is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists, and inverse agonists of the receptor. For those diseases that result from an overly active receptor, what is desired in a therapeutic, drug is a compound which acts to diminish the active state of a receptor, not necessarily a drug which is an antagonist to the endogenous ligand. This is because a compound therapeutic, prophylactic, diagnostic, prognostic, or laboratory reactant) that reduces the activity of the active receptor state need not bind at the same site as the endogenous ligand. In accordance with the present disclosure, any search for relevant compounds should start by screening compounds against the ligand-independent active state. The search, then, is for an inverse agonist to the active state receptor.

0 Screening candidate compounds against orphan receptors, for example, including and not limited to, S101P3A1 I and 101P3A 1 Fusion Protein, allows for the direct identification of candidate compounds which act at the orphan cell surface receptor, without requiring any prior knowledge or use of the receptor's endogenous ligand. By determining areas within the body where such receptors are expressed and/or over-expressed, it is 00 possible to determine related disease/disorder states which are associated with the expression and/or over expression of these receptors; such an approach is disclosed herein.

Disease/Disorder Identification and/or Selection.

g Inverse agonists and agonists to 101P3A 11 can be identified by the methodologies disclosed herein.

IN Such inverse agonists and agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases related to this receptor. Indeed, an antagonist to such a receptor (even if the ligand were known) C, may be ineffective given that the receptor is activated even in the absence of ligand-receptor binding. Because of 0 the ability to directly identify inverse agonists and agonists to these receptors, thereby allowing for the Sdevelopment of pharmaceutical compositions, a search for diseases and disorders associated with these receptors is possible. For example, 101P3A I1 is expressed in cancers of the tissues set forth in Table I.

XV.) Screening of Candidate Compounds General GPCR Sscreening Assay techniques When a G protein receptor becomes constitutively active, it binds to a G protein (for example Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as.a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [35S]GTP7S, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35 S]GTP7S can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995 (Mol Pharmacol. 1995 Apr;47(4):848-54).

Generally, this preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.

Specific GPCR screening assay techniques Once candidate compounds arc identified using the "generic" G protein- coupled receptor assay an assay to select compounds that are agonists, partial agonists, or inverse agonists), farther screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.

Thus, by screening those candidate compounds, which have been identified using a "generic" assay in an agonist and/or antagonist competitive binding assay, farther refinement in the selection process is provided.

In the case of 101P3AI1 it has been determined that this receptor couples the G protein Gs.

Gs stimulates the enzyme adenylyl cyclase (Gi, on the other hand, inhibits this enzyme).

Adenylyl cyclase catalyzes the conversion of ATP to cANT; thus, assays that detect cANT can be utilized, for example and not limitation, cell-based cANT assay, to determine if a candidate compound is an inverse agonist to the receptor such a compound which contacts the receptor would decrease the levels of

I

00 cAMP relative to the uncontacted receptor). As a result, "cyclase-based assays" can be used to further screen Sthose compounds selected from an agonist and/or antagonist competitive binding assay.

SXVI.) GPCR Fusion Proteins 00 The use of an endogenous, constitutively activated orphan GPCRs, such as 101P3AI 1, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists, provides a unique challenge in that, by definition, the endogenous receptor is active even in the absence of an Sendogenous ligand bound thereto.

Thus, in order to differentiate between, the endogenous receptor in the presence of a candidate compound and the endogenous receptor in the absence of that compound, with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial agonist or 0have no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation.

A preferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that an endogenous orphan GPCR is constitutively activate, using the assay techniques set forth herein (as well as others known in the art), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the endogenous, constitutively active orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, an inverse agonist to the receptor, it is more likely that one will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.

A GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the endogenous GPCR. The GPCR Fusion Protein appears to be important for screening with an endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques. This is important in facilitating a significant "signal to noise" ratio. A significant ratio is preferred for the screening of candidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the endogenous GPCR sequence and the G protein sequence both be in- frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence) and that the "stop" codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Both approaches have been evaluated, and in terms of measurement of the activity of the GPCR, the results are substantially the same; however, there is a preference (based upon convenience) for use of a spacer in that some restriction sites that are not used will, upon expression, effectively, become a spacer. Most preferably, the G protein that couples to the endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein a universal G protein construct) be available for insertion 00 S of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.

Pharmaceutical Compositions Candidate compounds selected for further development as active ingredients can be formulated into pharmaceutical compositions using techniques well known to those in the art.

00 Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16t" Edition, 1980, Mack Publishing Co., (Oslo et al., eds.).

O

SEXAMPLES:

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

00 S^ Example 1: Expression analysis of 101P3AI In normal tissues and patient specimens Analysis of 101P3AI 1 by RT-PCR is shown in Figure 10A. 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, prostate cancer pool, kidney cancer pool, colon cancer pool, breast cancer pool, and cancer metastasis pool.

Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 101P3AI 1, was performed at 30 cycles of amplification. Expression of 101P3AI 1 was observed in prostate xenograft pool, prostate cancer pool, kidney cancer pool, colon cancer pool, breast cancer pool, and cancer metastasis pool, but not in VP1 and VP2. Dot blots using patient-derived amplified cDNAs (Clontech, CA) show upregulation of PHOR-1 in 3/3 prostate cancer patients, 6/14 kidney cancer patients, 2/8 uterine cancer, 1/1 cervical cancer, 3/8 stomach cancer, and in 7/7 rectal cancer patients (Figure 10B). Expression of 101P3A1 I was assayed in a panel of human patient cancer specimens (Figure 11). RNA was extracted from a pool of three prostate cancer tumors, kidney cancer tumors, colon cancer tumors, breast cancer tumors, and cancer metastasis pool derived from cancer patients, as well as from normal prostate normal bladder normal kidney (NK) and normal colon Northern blots with 10 pg of total RNA/lane were probed with a 101P3A11 sequence fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of 101P3A11 in prostate cancer tumors, kidney cancer tumors, colon cancer tumors, breast cancer tumors, cancer metastasis pool, bladder cancer pool, and in the normal prostate but not in the other normal tissues.

Northern blot analysis on individual prostate patient tumor specimens is shown in Figure 12A. RNA was extracted from prostate tumors and their normal adjacent tissues (Nat) derived from prostate cancer patients.

Northern blots with 10 pg of total RNA/lane were probed with 101P3A I1 sequence. Results showed expression of 101P3AI 1 in all three patient specimens, and expression is especially upregulated in one of the three prostate tumor tissues.

RNA in situ analysis using anti-sense 101P3AI 1 riboprobe showed significant glandular epithelial and basal cell expression in normal prostate PIN and prostate cancer patients. 101P3Al1 sense riboprobe had little to no staining. The RNA in situ staining in PIN and prostate cancer is shown in Figure 12B and Figure 12C. The staining intensity in the cancer cells was generally higher than that observed in normal glands (Figure 12D and 12E). The RNA in situ results also demonstrate that the expression observed in the prostate tissues is in the glandular epithelia, basal cells, and cancer cells.

00 Endogenous expression of the 101P3All protein is demonstrated in the immunohistochemistry analysis CI of the anti-101P3A 1 (PEPTIDE 1: amino acids 1-14) rabbit polyclonal antibody (Figure 40A-40F). Staining in Sprostate cancer is greater than the staining observed in normal prostate. The staining is localized apically within the luminal epithelia of the normal prostate (Figures 40E and 40F). The staining observed in prostate cancer is 00 also localized apically in low to intermediate grade cancer (Figures 40B and 40C) and throughout all cells of more advanced prostate cancer (Figure 40A). Staining was observed in 19/20 normal prostate patients and in all of the nineteen prostate cancer patients analyzed. The prostate cancer cell line, LNCaP also shows similar staining O (Figures 40D and 40F) in almost all cells.

In addition, the present protocol was used to identify endogenous expression of the 101P3AI 1 protein in 0 prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, and breast cancer.

00 Immunohistochemical analysis was performed with the anti-101P3Al I (PEPTIDE 1: amino acids 1-14) rabbit polyclonal antibody (prostate cancer, Figure 41 A; bladder cancer, Figure 41 B; kidney cancer, Figure 41 C; colon C, cancer, Figure 41D; lung cancer, Figure 41E; and breast cancer, Figure 41F). Specific staining is observed in tumor cells of the six cancers analyzed.

Expression of 101P3AI 1 was also detected in the tumors of two colon cancer patients but not in normal colon tissues (Figure 13), and in five out of six kidney tumors isolated from kidney cancer patients (Figure 14).

The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues of the kidney (isolated from healthy donors) indicates that these tissues are not fully normal and that 101P3A11 is expressed in early stage tumors. In order to assay for androgen regulation of 101P3AI 1 expression, LAPC-9 cells were grown in charcoal-stripped medium and stimulated with the synthetic androgen mibolerone, for either 14 or 24 hours (Figure 15A, Figure 15B, and Figure 15C). Northern blots with 10 pg of total RNA/lane were probed with the 101P3AI I sequences (Figure 15A). A picture of the ethidium-bromide staining of the RNA gel is also presented (Figure 15C). Results showed expression of 101P3A11 is not regulated by androgen. The experimental samples were confirmed bytesting for the expression of the androgen-regulated prostate cancer gene PSA (Figure This experiment showed that, as expected, PSA levels go down in presence of charcoal-stripped serum, and expression is induced at 14 and 24 hours in presence of mibolerone.

Analysis of androgen regulation of 101P3A11 in vivo is shown in Figure 16. Male mice were injected with LAPC-9AD tumor cells. When tumors reached a palpable size, mice were castrated and tumors harvested at different time points following castration. RNA was isolated from the xenograft tissues. Northern blots with pg of total RNA/lane were probed with 101P3A1 lsequences. Size standards in kilobases (kb) are indicated on the side. A picture of the ethidium-bromide staining of the RNA gel is also presented in Figure 16. The results showed that expression of 101P3A I was not affected by androgen deprivation, and therefore, is not androgen regulated.

Example 2: Splice Variants/ Transcript Variants of 101P3A11 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.

87 00 Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding or 3' end) O 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 OO different times. 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 C 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 CN consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length 0 sequences. Each consensus sequence is a potential splice variant for that gene (see, URL Swww.doubletwist.com/products/c 1 agentsOverview.jhtml). Even when a variant is identified that is not a fulllength clone, that portion of the variant is very useful for antigen generation and for further cloning of the fulllength splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs 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 expressed sequence tags, Proc. Natl Acad Sci U S A. 2000 Nov 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see e.g., Proteomic Validation: Brennan, et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug 17;1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR.based Validation: Wellmann S, et 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 beta-defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation: Brigle, et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 101P3AI1 has a particular expression profile related to cancer.

Alternative transcripts and splice variants of 101P3A11 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.

The exon composition of the original transcript, designated as 101P3A1 1 v.1, are: Exon number Start End 1 1 2 91 3136 Using the full-length gene and EST sequences, one transcript variant was identified, designated as 101P3A11 v.2. Compared with 101P3A11 v.1, transcript variant 101P3A11 v.2 has spliced out a fragment from the second exon of variant 1, as shown in Figure 46. All other exons are the same corresponding exons of 101P3AlI v.l. Theoretically, each different combination of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant. Figure 46 shows the schematic alignment of exons of the two transcript variants.

Figure 2 shows nucleotide sequence of the transcript variant (101P3Al Figure 69 shows the alignment of the transcript variant with nucleic acid sequence of 101P3A 11 v.1. Figure 70 lays out amino acid translation of the transcript variant for the identified reading frame orientation. Figure 71 displays alignments of the amino acid sequence encoded by the splice variant with that of 101P3A11 v.l.

Example 3: Single Nucleotide Polymorphisms (SNPs) of 101P3A 1 A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals Nowotny, J. M. Kwon and A. M. Goate, SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct; 11(5):637-641; M.

Pirmohamed and B. K. Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, The use of single nucleotide polymorphisms in the isolation of common disease genes," Pharmacogenomics. 2000 Feb; 1(1):39-47; R. Judson, J. C. Stephens and A.

Windemuth, "The predictive power of haplotypes in clinical response," Pharmacogenomics. 2000 feb; 26).

SNPs are identified by a variety of art-accepted methods Bean, "The promising voyage of SNP target discovery," Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res. 1998 Jul; 8(7):691-697; M. M. She, "Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping teclmologies," 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 00 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, SB. Hines and A. Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol. Diagn. 2000 Dec; 5(4):329-340).

00 Using the methods described above, five SNPs were identified in the original transcript, 101P3A 11 v.1, at positions 441 1430 1532 2774 and 2833 The transcripts or proteins with alternative alleles were designated as variants 101P3A11 v.3, v.4, v.5, v.6 and v.7, respectively. Figure 44 shows Sthe schematic alignment of the SNP variants. Figure 45 shows the schematic alignment of protein variants, Scorresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 Sare not shown in Figure 11. These alleles of the SNPs, though shown separately here, can occur in different OO combinations (haplotypes) and in any one of the transcript variants (such as 101P3AI 1 v.2) that contains the 0sequence context of the SNPs.

Example 4: Production of Recombinant 101P3A1 in Prokaryotic and Yeast Systems To express recombinant 101P3AI 1 in prokaryotic cells, the full or partial length 101P3A1 I 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 101P3A11 are expressed in these constructs, amino acids 1 to 317; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 101P3AI 1, variants, or analogs thereof.

A. In vitro transcription and translation constructs: ECRII To generate 101P3Al1 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad, CA) are generated encoding either all or fragments of the 101P3A1 I cDNA.

The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 101P3A I1 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 101P3A 11 at the RNA level. Transcribed 101P3A1 I RNA representing the cDNA amino acid coding region of the 101P3A1 I gene is used in in vitro translation systems such as the TnT T M Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize 101P3A11 protein.

B. Bacterial Constructs: pGEX Constructs: To generate recombinant 101P3AI proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 101P3A1 I 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 101P3A 11 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxylterminus. The GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission M recognition site in pGEX- 6P-1, can be employed that permits cleavage of the GST tag from 101P3AI I-related protein. The ampicillin resistance gene and pBR322 origin permit selection and maintenance of the pGEX plasmids in E. coli. In one embodiment, amino acids 86-317 are cloned into the pGEX-2T expression vector, the protein is expressed and purified.

00 SpMAL Constructs: To generate, in bacteria, recombinant 101P3AI proteins that are fused to maltose- C, binding protein (MBP), all or parts of the 101P3AI I 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 101 P3A 1I protein sequences with MBP fused at the amino-terminus 00 and a 6X His epitope tag at the carboxyl-terminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and'anti-His antibodies. The 6X His epitope tag is generated by adding 6 histidine codons \0 to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 101P3A1 I. The C pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or pcriplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds. In one 00 embodiment, amino acids 86-310 is cloned into the pMAL-c2X expression vector, the prolcin is expressed and Spurified.

pET Constructs: To express 101P3A1 I in bacterial cells, all or parts of the 101P3A11 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 101P3A1 I protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag 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 the 101P3A 11 protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs: pESC Constructs: To express 101P3A11 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 101P3A I1 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain I of 4 selectable markers, HIS3, TRPI, 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 used to confirm protein-protein interactions of 101P3A11. 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 101P3A1 1 in the yeast species Saccharomyces pombe, all or parts of the 101P3A11 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level expression of a 101P3AI I protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A FlagTM epitope tag allows detection of the recombinant protein with anti- Flag'" antibody.

Example 5: Production of Recombinant 101P3A11 in Higher Eukarvotic Systems A. Mammalian Constructs: To express recombinant 101P3A11 in eukaryotic cells, full or partial length 101P3A11 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 101P3A 11 are expressed in these constructs, amino acids 1 to 318 ofv.l and v.3, amino acids I to 72 ofv.2; or any 5, 6, 7, 8, 9, 10, I1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 101P3A11, variants, or analogs thereof.

00 The constructs can be transfcctcd into any one or a wide variety of mammnlian cells such as 293T cells.

C Transfected 293T cell lysates can be probed with the anti-101 P3A 11 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 101P3A 1 in mammalian cells, the 101P3A I1 ORF was Scloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the 0 cytomegalovirus (CMV) promoter and the SPI6 translational enhancer. The recombinant protein has Xpress

T

and six histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the Sbovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA D stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColEI origin permits selection and maintenance of the plasmid in E. coli.

00 pcDNA3.1/MvcHis Constructs: To express 101P3A 11 in mammalian cells, the 101P3A11 ORF, with a consensus Kozak translation initiation site, was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, C 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 ColE I origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/GFP Construct: To express 101P3A11 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the 101P3A11 ORF, with a consensus Kozak translation initiation site, was cloned into pcDNA3.1/GFP. Protein expression was driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating noninvasive, in vivo detection and cell biology studies. The pcDNA3.1/GFP vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.

The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli. Figure 66 shows expression and detection of 101P3AI.GFP fusion protein. 293T cells were transfected with either pcDNA3.1/101P3A l.GFP recombinant expression vector pcDNA3.1/GFP vector or control pcDNA3.1 vector Cells were harvested 24 hours later and analyzed by microscopy for detection of green fluorescence.

Results show expression of the 101P3A1 I.GFP fusion protein is localized mostly at the cell membrane, whereas expression of the free GFP is throughout the cells. The control vector did not show any fluorescence. We conclude that the 101P3A1 .GFP fusion protein is expressed from the pCDNA3. /101P3A I.GFP construct, and that the fusion protein is localized at the cell membrane.

Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.I/NT-GFP-TOPO spanning the entire length of the 101P3A11 proteins.

Codon optimized 101P3A11: To enhance protein translation of 101P3A 1, the nucleic acid sequence of 101P3A1 I was codon optimized (sll01P3AI The sequence ofcodon optimized sl01P3Al I is listed in Figure 67. The sl01P3A I was cloned into the pcDNA3.1/GFP construct and into the pSRa retroviral vector, to generate the sll01P3Al .GFP fusion protein. The recombinant protein has the Green Fluorescent Protein (GFP) 92 00 0 fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. Figure 68 C1 shows expression and detection of the codon optimized s 101 P3A I.GFP fusion protein. 293T cells were transfected with either pcDNA3.1 vector control (light line), or one of the three different -s pcDNA3.1/sI01P3AI 1.GFP vector clones, 1G2, 2G3, or 3H5 (dark line). Cells were harvested 24 hours later and 00 either analyzed directly for green fluorescence or stained viably using polyclonal anti-101P3Al I antibody and analyzed by flow cytometry. Results show strong expression of the codon optimized s 01P3AI 1.GFP fusion protein at the cell surface of transfected cells.

\O PAPtae: The 101P3A11 ORF, or portions thereof, of 101P3AI1 are cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of the 101P3AI 1 proteins while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated 00 in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of 101P3AI I proteins. The resulting recombinant 101P3AI proteins are optimized for secretion into the media of CN transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the 101P3AI 1 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

ptaO: The 101P3A11 ORF, or portions thereof, of 101P3A1 I are cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generated 101P3AI protein with an amino-terminal IgGK signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 101P3A11 protein was optimized for secretion into the media of transfected mammalian cells, and was used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 101P3A1 I proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: The 101P3AI 1 ORF, or portions thereof, of 101P3A11 are also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G (IgG) Fc (hinge, CH2, CH3 regions) into.

pSecTag2 (Invitrogen, California). This construct generates an IgG I Fc fusion at the carboxyl-terminus of the 101P3A11 proteins, while fusing the IgGK signal sequence to N-terminus. 101P3A 11 fusions utilizing the murine IgGI Fc region are also used. The resulting recombinant 101P3AI 1 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 101P3AI 1 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

The amino acid region 159-202 of the 101P3A11 ORF was cloned into psecFc. The resulting recombinant 101P3Al 1(159-202)-psecFc construct was transfected into 293T and Cos-7 cells, and the expression of recombinant 101P3A1 1(159-202)-psecFc protein assayed by Western blotting (Figure 17). Results show that 101P3A1 l(159-202)-psecFc fusion protein was expressed in the lysates of both 293T and Cos-7 cells. The 101P3A1 l(159-202)-psecFc fusion protein was also secreted and detected in the culture supematants of both cell types.

00 j SRa Constructs: To generate mammalian cell lines that express 101P3A 11, constitutively, the ORF of CN 101P3A1 I was cloned into pSRa constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRa constructs into the 293T-10Al packaging line or co-transfection of pSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus was used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 10P3A 11, 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 IN and ColEI origin permit selection and maintenance of the plasmid in E. coli. Figure 18 shows that 101P3A 11 was 0expressed using the pSRa retroviral vector in the cell line 300.19. The retroviral vectors can thereafter be used Sfor infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-I cells.

00 Additional pSRa constructs are made that fuse an epitope tag such as the FLAG T M tag to the carboxylterminus of 101P3AI 1 sequences to allow detection using anti-Flag antibodies. For example, the FLAG T M sequence 5' gat tac aag gat gac gac gat aag 3' 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 101P3A11 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 101P3A11. High virus titer leading to high level expression of 101P3A11 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The 101P3A1 I coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors.

Alternatively, 101P3All 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 SCaBER, NIH 3T3, 293 or rat-I cells.

Regulated Expression Systems: To control expression of 101P3A11 in mammalian cells, coding sequences of 101P3A11, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 101P3All. These vectors are thereafter used to control expression of 101P3AI1 in various cell lines such as SCaBER, NIH 3T3, 293 or rat-I cells.

B. Baculovirus Expression Systems To generate recombinant 101P3AI 1 proteins in a baculovirus expression system, 101P3A11 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a Histag at the N-terminus. Specifically, pBlueBac-101P3Al I is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodopterafrugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 101P3A I protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 101P3A11 protein can be detected using anti-101P3Al I or anti-His-tag antibody. 101P3A11 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 101P3A11.

00

O

O

C Example 5: Production of Recombinant 101P3A11 in Higher Eukarvotic Systems SA. Mammalian Constructs: STo express recombinant 101P3A1 I in eukaryotic cells, full or partial length 101P3AI 1 cDNA sequences 00 can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 101P3AI I are expressed in these constructs, amino acids 1 to 317 or 318; or any 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 or more contiguous amino acids from S101P3A 11, variants, or analogs thereof.

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

0 Transfected 293T cell lysates can be probed with the anti-101P3A 1 polyclonal serum, described herein.

00 pcDNA4/HlsMax Constructs: To express 101P3A 1 in mammalian cells, the 101P3A 11 ORF was Scloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the C cytomegalovirus (CMV) promoter and the SP 16 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 hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 101P3AI1 in mammalian cells, the 101P3Al 1 ORF, with a consensus Kozak translation initiation site, was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for 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 ColEl origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 101P3AI in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the 101P3A I ORF, with a consensus Kozak translation initiation site, was cloned into pcDNA3. 1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT- GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEI origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of the 101P3AI 1 proteins.

PAPtag: The 101P3A11 ORF, or portions thereof, of 101P3A1 I are cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of the 101P3AI 1 proteins while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated 00 in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of 101P3A11 proteins. The resulting recombinant 101P3AI I proteins are optimized for secretion into the media of Stransfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the 101P3AI proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also 00 contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

DtaS: The 10 IP3AI 1 ORF, or portions thereof, of 101P3A11 are cloned into pTag-5. This vector is Ssimilar to pAPtag but without the alkaline phosphatase fusion. This construct generated 101P3AI 1 protein with San amino-terminal IgGK signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 101P3AI 1 protein was optimized for secretion into Sthe media of transfected mammalian cells, and was used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 101P3A11 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: The 101P3A 11 ORF, or portions thereof, of 101P3Al11 are also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin Gl (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgGI Fc fusion at the carboxyl-terminus of the 101P3A1 1 proteins, while fusing the IgGK signal sequence to N-terminus. 101P3A11 fusions utilizing the murine IgGI Fc region are also used. The resulting recombinant 101P3AI I 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 101P3A11 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

The amino acid region 159-202 of the 101P3A11 ORF was cloned into psecFc. The resulting recombinant 101P3AI l(159-202)-psecFc construct was transfected into 293T and Cos-7 cells, and the expression of recombinant 101P3A1 l(159-202)-psecFc protein assayed by Western blotting (Figure 17). Results show that 101P3A1 1(159-202)-psecFc fusion protein was expressed in the lysates of both 293T and Cos-7 cells. The 101P3A1 l(159-202)-psecFc fusion protein was also secreted and detected in the culture supematants of both cell types.

pSR Constructs: To generate mammalian cell lines that express 101P3A 11, constitutively, the ORF of 101P3AI 1 was cloned into pSRa constructs. Amphotropic and ecotropic retroviruses were generated by transfection ofpSRa constructs into the 293T-10AI packaging line or co-transfection ofpSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus was used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 101P3AI 1, 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 ColEI origin permit selection and maintenance of the plasmid in E. coli. Figure 18 shows that 101P3AI 1 was expressed using the pSRa retroviral vector in the cell line 300.19. 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-I cells.

00 Additional pSRa constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxylterminus of 101P3AI 1 sequences to allow detection using anti-Flag antibodies. For example, the FLAGTM Ssequence 5' gat tac aag gat gac gac gat aag 3' 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 00 proteins of the full-length 101P3Al l proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 101P3A 1. High virus titer leading to high level expression of 101P3A11 is achieved in viral delivery systems \0 such as adenoviral vectors and herpes amplicon vectors. The 101P3A1 I coding sequences or fragments thereof M are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus Spackaging are performed according to the manufacturer's instructions to generate adenoviral vectors.

00 Alternatively, 101P3AI 1 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to Sgenerate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as C SCaBER, NIH 3T3, 293 or rat-I cells.

ReLulated Expression Systems: To control expression of 101P3A 1 in mammalian cells, coding sequences of 101P3A11, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 101P3A 1. These vectors are thereafter used to control expression of 101P3A I in various cell lines such as SCaBER, NIH 3T3, 293 or rat-I cells.

B. Baculovirus Expression Systems To generate recombinant 101P3A11 proteins in a baculovirus expression system, 101P3A11 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a Histag at the N-terminus. Specifically, pBlueBac-101P3Al I is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodopterafrugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supematant and purified by plaque assay.

Recombinant 101P3A 1 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 101P3A11 protein can be detected using anti-101P3Al I or anti-His-tag antibody. 101P3A1 1 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 101P3A1 1.

Example 6 Antieenicity Profiles and Secondary Structure Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9 depict graphically five amino acid profiles of the 101P3A 1 amino acid sequence, each assessment 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 (Deleagc, 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 the 101P3A1 1 protein. Each of the above amino acid profiles of 101P3A I1 were generated using the following ProtScale parameters for analysis: 1) A window size of 9: 2) 00 0 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized S to lie between 0 and 1.

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

\O Average Flexibility (Figure 8) and Beta-turn (Figure 9) profiles determine stretches of amino acids values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in Ssecondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed 00 portions of the protein and thus are accessible to immune recognition, such as by antibodies.

Antigenic sequences of the 101P3A 1 protein indicated, by the profiles set forth in Figure 5, Figure C1 6, Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogcns, either pcptidcs or nucleic acids that encode them, to generate anti-10IP3AI I antibodies. The immunogcn can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 101P3AI 1 protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 317 or 318 .that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 317 or 318 that includes an amino acid position having a value 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 317 or 318 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 317 or 318 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 317 or 318 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 comprise nucleic acids that encode any of the forgoing.

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

The secondary structure of 101P3A11, namely the predicted presence and location ofalpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server http://www.expasy.ch/tools/. The analysis indicates that 101P3A11 is composed 47.95% alpha helix, 21.45% extended strand, and 30.60% random coil (Figure 19A).

Analysis for the potential presence of transmembrane domains in 101P3A11 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server http://www.expasy.ch/tools. The programs predict the presence of 7 transmembrane domains in 101P3A 11, consistent with the structure of a G-protein coupled receptor. Shown graphically in Figure 19A are the results of analysis using the TMpred (Figure 19B) and TMHMM (Figure 19C) prediction programs depicting the location of 00 the 7 transmembrane domains. The results of each program, namely the amino acids encoding the transmembrane 0domains are summarized in Table XXI.

Example 7: Generation of 101P3AI1 Polyclonal Antibodies Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 101P3AI I protein, computer algorithms are employed in design of immunogens that, based on amino acid N sequence analysis are antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles and Secondary Structure"). Such regions would generally be hydrophilic, flexible, in beta-turn conformations, and/or exposed on the surface of the protein (see, Figure 00 Figure 6, Figure 7, Figure 8, or Figure 9 for amino acid profiles that indicate such regions of 101 P3A I).

For example, 101P3A 1 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of the 101P3AI 1 amino acid sequence, such as amino acids 1-23, plus or minus 1-10 amino acids at available termini, and amino acids 159.202, plus or minus 1-10 amino acids at available termini, are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 1-23 of 101P3A 1 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 101P3AI1 protein, analogs or fusion proteins thereof. For example, the 101P3A 11 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.

In one embodiment, a GST-fusion protein encoding amino acids 86-317, plus or minus 1-10 amino acids at available termini, is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 101P3A 11 in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, Brady, W., Urnes, Grosmaire, Damlc, and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used.

These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled "Production of Recombinant 101P3A1 I in Eukaryotic Systems"), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 159-202 is cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified 101P3A11 protein is then used as immunogen.

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

00 0 In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 pg, typically 100-200 0 pg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then g injected subcutaneously every two weeks with up to 200 pg, typically 100-200 pg, of the immunogen in Sincomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization 00 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 TagS 101P3AI 1 encoding amino acids 159-202, the full-length 101P3AI 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled "Production of Recombinant 101P3A 11 in M Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti- 101P3A I1 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific 00 reactivity to denatured 101P3A I protein using the Western blot technique. Immunoprecipitation and flow Scytometric analyses of 293T and other recombinant 101P3A1 1-expressing cells determine recognition of native (c protein by the antiserum. In addition, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 101P3AI 1 are carried out to test specificity.

The anti-serum from the Tag5 101P3A 11 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 peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 8: Generation of 101P3A11 Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 101P3AI 1 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 101P3A11, for example those that would disrupt its interaction with ligands or proteins that mediate or are involved in its biological activity. Therapeutic mAbs also comprise those that specifically bind epitopes of 101P3A1 I exposed on the cell surface and thus are useful in targeting mAb-toxin conjugates. Monoclonal antibodies may also be raised to other antigenic epitopes of 101P3AI 1 including amino acid sequences predicted to be in intracellular regions. These monoclonal antibodies are useful as intrabodies if they disrupt the signaling mechanisms of 101P3A I1, such as the interaction with heterotrimeric G proteins.. Such antibodies are also useful as diagnostic agents for techniques such as immunohistochemistry. Immunogens for generation of such mAbs include those designed to encode or contain the entire 101P3AI 1 protein or regions of the 101P3AI I protein predicted to be exposed to the extracellular environment or hydrophilic cytoplasmic environment, and/antigenic from computer analysis of the amino acid sequence (see, Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, and the Example entitled "Antigenicity Profiles 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 101P3AI 1, such as 293T-101P3A11 or 300.19-101P3AI 1 murine Pre-B cells, are used to immunize mice.

00 0 To generate mAbs to 101P3A11, mice are first immunized intraperitoneally (IP) with, typically, 10-50 pg of protein immunogen or 107 101P3A11-expressing cells mixed in complete Freund's adjuvant. Alternatively, mice are immunized intradermally. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 pg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding 101P3A1 I (sequence is used to immunize mice by direct injection of the plasmid DNA. For example, the predicted first I extracellular loop, amino acids 82-104, or second extracellular loop of 101P3A11, amino acids 159-202, or the Sthird extracellular loop, amino acids 258 275 (in each instance plus or minus 10 amino acids) is cloned into the mammalian secretion vector and the recombinant vector is used as immunogen. In another example the 00 same amino acids are cloned into an Fc-fusion secretion vector in which the 101P3A 11 sequence is fused at the 0amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human IgG Fc region. This recombinant vector is then used as immunogen. Amino acid sequences from intracellular regions may also be used as antigens using similar strategies. These regions include amino acids 50-63, amino acids 121- 146, amino acids 261-275, and amino acids 295-318 (in each instance plus or minus 10 amino acids, except for the C-terminus residue). The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing 101P3A 11.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, Harlow and Lane, 1988).

In one embodiment for generating 101P3A 1 monoclonal antibodies, a Tag5-101P3A I antigen encoding amino acids 159-202 is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 pg of the Tag5-101P3A11 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 TagS antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 101P3A 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 101P3A11 cDNA (see the Example entitled "Production of Recombinant 101P3A 1 in Eukaryotic Systems"). Other recombinant 101P3All-expressing cells or cells endogenously expressing 101P3Al I are also used. Mice showing the strongest reactivity are rested and given a final injection of antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 101P3AI 1 specific antibody-producing clones.

The binding affinity of a 101P3A 11 monoclonal antibody is determined using standard technologies.

Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 101P3AI 1 monoclonal antibodies preferred, for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The 00 g BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and C Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation Sconstants, and affinity constants.

00 Example 9: 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 Sdisclosed protocols PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 S(1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors OO and 1-10 nM '"I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in Ci preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity.

All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and ICso-[HLA], the measured IC5o values are reasonable approximations of the true KD 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

IC

0 s 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 IC$o nM values by dividing the IC 5 s 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 ofpurified MHC.

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

Example 10: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer searches and algorithms for identification of supermotif and/or motif-bearing epitopes The searches performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles" and Tables V-XVIII and XXII TO IL employ the protein sequence data from the gene product of 101P3AI 1 set forth in Figures 2 and 3; the specific peptides used to generate the tables are listed in Table LII.

Computer searches for epitopes bearing HLA Class I or Class 11 supermotifs or motifs are performed as follows. All translated 101P3A I 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 0 produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

SIdentified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the 00 impact of different amino acids at different positions, and arc essentially based on the premise that the overall affinity (or AG) ofpeptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: "AG" a, x a2, x a 3 1 x a, g where aj, is a coefficient which represents the effect of the presence of a given amino acid at a given 0position along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other independent binding of individual side- 00 chains). When residuej occurs at position i in the peptide, it is assumed to contribute a constant amountj to the C free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

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

Biol. 267:1258-126, 1997; (see also Sidney et al, Human Immunol. 45:79-93, 1996; and Southwood et al., J.

Immunol. 160:3363.3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carryingj is calculated relative to the remainder of the group, and used as the estimate of]j. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 supertvpe cross-reactive petides Protein sequences from 10IP3AI 1 are scanned utilizing motif identification software, to identify 9and 1 -mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positivescoring 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 A2-supertype 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 101P3AI 1 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotifprimary 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 A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of 500 nM, often 200 nM, are then tested for binding cross-reactivity to the other common A3supertype 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.

00 0 Selection of HLA-B7 supermotif bearing epitoes The 101P3A1 I protein(s) scanned above is also analyzed for the presence of 9- 10-, or 1 1-mer g peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA- S B*0702, the molecule encoded by the most common B7-supertype allele the prototype B7 supertype allele).

00 Peptides binding B*0702 with ICs 5 of 500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules B*3501, B*5101, B*5301, and B*5401).

Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.

IO

Selection ofAl and A24 motif-bearing epitopes 0 To further increase population coverage, HLA-Al and -A24 epitopes can also be incorporated into 00 vaccine compositions. An analysis of the 101P3A11 protein can also be performed to identify HLA-A1- and SA24-motif-containing sequences.

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

Example 11: Confirmation of Immunogenicity Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology: Target Cell Lines for Cellular Screening: The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.I restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% heat inactivated FCS. Cells that express an antigen of interest, or transfcctants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures: Generation ofDendritic Cells PBMCs are thawed in RPMI with 30 pg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytcs are purified by plating 10 x 106 PBMC/well in a 6-well plate. After 2 hours at 37"C, the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFc 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 2 0 0.250x106 PBMC are processed to obtain 24x10' CD8' T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30pg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/l% AB serum at a concentration of 20xlO0cells/nl. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (1401l beads/20xl06 cells) and incubated for 1 hour at 4"C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at 100xl06 104 0 cells/ml (based on the original cell number) in PBS/AB serum containing 100 l/ml detacha-bead® reagent and pg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are Swashed 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 00 cell concentration of 1-2x10 6 /ml in the presence of 3pg/ml 2- microglobulin for 4 hours at 20*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 lxl0 cells/ml) are co-cultured with S0.25ml of CD8+ T-cells (at 2x10 6 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7.

SRecombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.

CK Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days 00 after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5x10 6 cells/ml and irradiated at -4200 rads. The PBMCs are plated at 2x10 6 in 0.5 ml complete medium per well and incubated for 2 hours at 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 presence of 3 lg/ml B 2 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37*C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998).

Seven days later, the cultures are assayed for CTL activity in a "Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNy ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL lytic activity by S"Cr release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) "Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 1 Og/ml peptide overnight at 37*C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200jCi of SICr 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 non-specific lysis). Target cells (100 pl) and effectors (100pl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37'C. At that time, 100 pl of supernatant are collected from each well and percent lysis is determined according to the formula: [(cpm of the test sample- cpm of the spontaneous "Cr release sample)/(cpm of the maximal "Cr release sample- cpm of the spontaneous "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 (samplebackground) 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.

0 In situ Measurement of Human IFNY Production as an Indicator of Peptide-specific and Endogenous Recognition El Immulon 2 plates are coated with mouse anti-human IFN monoclonal antibody (4 pg/ml 0.1M NaHCO 3 pH 8 overight at 4 0 C. The plates are washed with Ca 2 Mgz-free PBS/0.05% Tween 20 and blocked with 00 PBS/10% FCS for two hours, after which the CTLs (100 pl/well) and targets (100 pl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptidepulsed or endogenous targets, are used at a concentration of lx106 cells/ml. The plates are incubated for 48 hours Sat 37 0 C with 5% CO 2 SRecombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200pg/100 microliter/well and the plate incubated for two hours at 37 0 C. The plates are washed and 100 pl of biotinylated 00 mouse anti-human IFN.gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are Sadded and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6x with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The'reaction is stopped with 50 microliter/well IM H 3

PO

4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5x10 4 CD8+ cells are added to a T25 flask containing the following: Ix10 6 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x10' irradiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (anti-CD3) at 30ng per ml in RPMI-1640 containing human AB serum, non-essential amino acids, sodium pyruvate, 25pM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 2001U/ml and every three days thereafter with fresh media at 50IU/ml. The cells are split if the cell concentration exceeds lxl06/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10,3 and 1:1 in the "Cr release assay or at 1xl10/ml in the in situ IFNy assay using the same targets as before the expansion.

Cultures are expanded in the absence ofanti-CD3' as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5x10 4 CD8' cells are added to a T25 flask containing the following: Ixl06 autologous PBMC per ml which have been peptide-pulsed with 10 pg/mi peptide for two hours at 37C and irradiated (4,200 rad); 2xl0' 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, Lglutamine and gentamicin.

Immunogenicity of A2 supermotif-bearing peptides A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific 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 101P3Al 1. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes 106 00 and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously K expressing the antigen.

c Evaluation ofA*03/A 1 immunogenicity 00 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 supermotifpeptides.

Evaluation of B7 immunogenicitv DO Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation ofA2-and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, HLA-A 1, HLA-A24 etc. are also confirmed using similar 00 methodology CK Example 12: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs .HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in Ihe identification and preparation of highly cross-reactive native pcptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

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.

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, ifA*0201 binding capacity is maintained, for A2supertype cross-reactivity.

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

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, bind at an ICs 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-reactivity by T cells specific for the parent epitope (see, Parkhurst et al., J. nmunol. 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 to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

Analogine of HLA-A3 and B7-supermotif-bearing peptides 00 O Analogs ofHLA-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- Ssupertype molecules are engineered at primary anchor residues to possess a preferred residue S, M, or A) at C position 2.

00 The analog peptides are then tested for the ability to bind A*03 and A*l I (prototype A3 supertypc alleles). Thosc peptides that demonstrate 5 500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

SSimilarly to the A2- and A3- motif bearing peptides, peptides binding 3 or more B7-supertype alleles can Sbe improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue I, L, or (71 F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. Immunol. 157:3480-3490, 00 01996).

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

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay.

Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

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

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 101P3AI -expressing tumors.

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

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

Example 13: Identification and confirmation of 101P3All-derived sequences with HLA-DR binding motifs 00 O Peptide epitopes bearing an HLA class II supermotifor motif are identified and confirmed as outlined Nc- below using methodology similar to that described for HLA Class I peptidcs.

Selection of HLA-DR-supermotif-bearing epitopes.

To identify 101P3Al l-derived, HLA class II HTL epitopes, a 101P3A I antigen is analyzed for the 00 presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions 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 0ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR- OO 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 C'K 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 DRI, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 101P3Al l-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: DRI, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 11, DR2w2 p2, DR6wl9, 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 DR4wl5, DRSwl 1, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 101P3AI -derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 motif peotides Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 101P3A1 I 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 l1pM or better, less than I pM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

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

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

Example 14: Immunogenicitv of 101P3A11-derived HTL epitopes 109 00 This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate 00 transgenic mouse models. Immunogenicity is determined by screening for: in vitro primary induction using normal PBMC or recall responses from patients who have 101P3A1 I-expressing tumors.

Example 15: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic M backErounds to determine breadth of population coverage SThis example illustrates the assessment of the breadth of population coverage of a vaccine composition 00 comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

SIn 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=l-(SQRT(1-af)) (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=l -(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*(l-A)). Confirmed members of the A3-like supertype are A3, Al 1, 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, B*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, A is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when Al and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is An analogous approach can be used to estimate population coverage achieved with combinations of class II motifbearing epitopes.

Immunogenicity studies in humans Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., 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 With a sufficient number of epitopes (as disclosed herein and from the art), an average population CN coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see Osbome, 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 00 comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is

O

Example 16: CTL Recognition Of Endogenously Processed Antigens After Priming This example confirms that CTL induced by native or analoged peptide epitopes identified and selected 00 as described herein recognize endogenously synthesized, native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA- C< A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on S'Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on "Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 101P3A1 I expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 101P3AI 1 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/K transgenic mice, several other transgenic mouse models including mice with human Al 1, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others transgenic mice for HLA-Al and A24) are being developed. HLA-DRI and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 17: Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 101P3A1 1derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 101P3AI -expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bcaring epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTUHTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes 111 00 0 obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

SCell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA- A2.1/Kbchimeric gene Vitiello et al., J. Exp. Med. 173:1007, 1991) 00 Ivitr CTL activation: One week after priming, spleen cells (30x 106 cells/flask) are co-cultured at 37C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10xl06 cells/flask) in 10 ml of culture flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

0 Assayfor cytotoxic activity: Target cells (1.0 to 1.5x10') are incubated at 37 0 C in the presence of 200 pl of sCr. After 60 minutes, cells are washed three times and resuspended in RIO medium. Peptide is added where Srequired at a concentration of 1 pg/ml. For the assay, 10 4 sCr-labeled target cells are added to different 00 concentrations of effector cells (final volume of 200 pl) in U-bottom 96-well plates. After a six hour incubation period at 37 0 C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Csl Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release 100 x (experimental release spontaneous release)/(maximum release spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, "Cr release data is expressed as lytic units/10' cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour "Cr release assay. To obtain specific lytic units/l 06, the lytic units/I 0 obtained in the absence of peptide is subtracted from the lytic units/I 06 obtained in the presence of peptide. For example, if 30% "Cr release is obtained at the effector target ratio of 50:1 5x10' effector cells for 10,000 targets) in the absence of peptide and 5:1 5x10 4 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)-(1/500,000)] x I0 6 18 LU.

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

Example 18: Selection of CTL and HTL Epitopes for Inclusion in a 101P3All-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 101P3A1 I clearance. The number of epitopes used depends on observations of patients who spontaneously clear 101P3A11. For example, if it has been observed that patients who spontaneously clear 101P3AI -expressing cells generate an immune response to at least three epitopes from 101P3A I antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class I epitopes.

00 g Epitopes are often selected that have a binding affinity of an ICo 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 pepides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.

SIn order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif 00 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 SMonte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, \of population coverage.

O When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to 0generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are 00 similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained CN 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 mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-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 101P3Al 1, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatbst number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 101P3A 1.

Example 19: 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-A I and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 101P3A i1, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 101P3A I1 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing 113 O epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected SCTL 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 Ii protein may be fused to one or more HTL epitopes as described in the art, wherein 00 the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

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

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with 00 principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonuclcotides that can, for example, average about 70 nuclcotides in length with nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide cpitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using die following conditions: for 15 sec, annealing temperature (50 below the lowest calculated Tm of each primer pair) for 30 sec, and 72*C for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 pg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 pl reactions containing Pfu polymerase buffer (lx= 10 mM KCL, 10 mM (NH4) 2 S0 4 20.mM Tris-chloride, pH 8.75,2 mM MgSO 4 0.1% Triton X-100, 100 ig/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gelpurified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 20: 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 ofpeptide-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 00 Sconcentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, Kageyama el al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro Sassessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, 00 as detailed in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity ofa DNA minigene construct containing at least one HLA-A2 supermotifpeptide to induce CTLs in vivo, HLA-A2.

1 /Kb transgenic mice, for example, are immunized 0 intramuscularly with 100 pg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA g immunization, a control group of animals is also immunized with an actual peptide composition that comprises 0multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

00 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 S activity in a 51 Cr release assay. The results indicate the magnitude of the CTL response directed against the A2restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

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

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized intramuscularly with 100 pg ofplasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene).

The HTL response is measured using a 'H-thymidine incorporation proliferation assay, (see, Alexander et al.

Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein Bamett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, Hanke et Vaccine 16:439.445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson 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 peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10' 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 00 without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for pcptidc-spccific activity in an alpha, beta and/or gamma IFN ELISA.

00 It is round that the minigcnc utilized in a primc-boost protocol clicits greater immune responses toward the HLA-A2 supermotifpeptides than with DNA alone. Such an analysis can also be performed using HLA-Al I 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 C CTL Responses Using a Prime Boost Protocol." 0 00 Example 21; Peptlde Compositions for Prophylactic Uses Vaccine compositions of the present invention can be used to prevent 101P3A11 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 101P3A11-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 pg, generally 100-5,000 pg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of 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 101P3A 1-associated disease.

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

Example 22: Polyepitopic Vaccine Compositions Derived from Native 101P3A11 Sequences A native 101P3AI polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify "relatively short" regions of the polyprotein that comprise multiple epitopes. The "relatively short" regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, "nested" epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, it has a high concentration of epitopes. As noted herein, cpitope motifs may be nested or overlapping frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

00 O The vaccine composition will include, for example, multiple CTL epitopes from 101P3AI 1 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 Cll one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties 00 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 Stherapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment Sprovides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown.

Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple 00 peptide sequences that are actually present in native 101P3A 1I, thus avoiding the need to evaluate any junctional Sepitopes. 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 23: Polvepitopic Vaccine Compositions From Multiple Antigens The 101P3A1 1 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 101P3AI 1 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 101P3A11 as well as tumor-associated antigens that are often expressed with a target cancer associated with 101P3AI 1 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 24: Use of Peptides to Evaluate an Immune Response Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 101P3Al 1. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetrameric complexes ("tetramers") are used for a cross-sectional analysis of, for example, 101P3A11 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 101P3A 11 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, 32-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is 117 00 added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is 0 referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for minutes and resuspended in 50 pl of cold phosphate-buffered saline. Tri-color analysis is performed with the 00 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 SA*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by Sflow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted SCTLs, thereby readily indicating the extent of immune response to the 101P3AI I epitope, and thus the status of S exposure to 101P3AI1, or exposure to a vaccine that elicits a protective or therapeutic response.

0O Example 25: 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 101P3A1 1-associated disease or who have been vaccinated with a 101P3A 1 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed.

The vaccine may be any 101P3A 1 vaccine. PBMC are collected from vaccinated individuals and HLA typed.

Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 pg/ml), and Hepes (10mM) containing 10% 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 ofT cell help during the first week of stimulation.

In the microculture format, 4 x 10' PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 pl/well of complete RPMI. On days 3 and 10, 100 pi of complete RPMI and 20 U/ml final concentration ofrIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flatbottom plate and restimulated with peptide, rIL-2 and 10' irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific "Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et Nature Med. 2:1104,1108, 1996; Rehermann et al., J.

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

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

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

00 Cytolytic activity is determined in a standard 4-h, split well 5 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target ratios of 20-50:1 on day S14. Percent cytotoxicity is determined from the formula: 100 x [(experimental release-spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent S(2% Triton X-100; Sigma Chemical Co., St. Louis, MO). Spontaneous release is <25% of maximum release for Ci all experiments.

00 The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been 0stimulated by previous exposure to 101P3A1 I or a 101P3Al 1 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96well flat bottom plate at a density of 1.5x 10 cells/well and are stimulated with 10 pg/ml synthetic peptide of the invention, whole 101P3A 1 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 IL-2. Two days later, 1 pCi '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 'H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3 H-thymidine incorporation in the presence of antigen divided by the 3 H-thymidine incorporation in the absence of antigen.

Example 26: Induction of Specific CTL Response in Humans A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows: A total of about 27 individuals are enrolled and divided into 3 groups: Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 pg of peptide composition; Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 pg peptide composition; Group 11I: 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 rcversibility.

00 Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after CN injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

00 The vaccine is found to be both safe and efficacious.

Example 27: Phase II Trials In Patients Expressing 101P3A11 O Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 101P3Al 1. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 101P3A 11, to establish the safety of inducing 00 a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, by the reduction and/or shrinking of lesions. Such a study is C' 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. Drugassociated adverse effects (severity and reversibility) are recorded.

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

All of them have a tumor that expresses 101P3Al 1.

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 101P3A 1-associated disease.

Example 28: 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 ofa DNA vaccine in transgenic mice, such as described above in the Example entitled "The Plasmid Construct and the Degree to Which It Induces Immunogenicity," can also be used for the administration of the vaccine to humans.

Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled "Construction of"Minigene" Multi-Epitope DNA Plasmids" in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 pg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5xl0 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 00 0 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 Sprotective immunity against 101P3AI 1 is generated.

00 Example 29: Administration of Vaccine Compositions Using Dendritic Cells (DC) Vaccines comprising peptide epitopes of the invention can be administered using APCs, or S"professional" APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a M 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 00 to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, Srespectively, of the target cells that bear the 101P3A11 protein from which the epitopes in the vaccine are derived.

C-i For example, a cocktail of epitope-comprising peptides is administered ex vive to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Monsanto, St.

Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, Nature Med. 4:328, 1998; Nature Med 2:52, 1996 and Prostate 32:272, 1997). Although 2-50 x 106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC.

For example, PBMC generated after treatment with an agent such as Progenipoietin T are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 100.

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 ProgenipoietinT mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 106 DC, then the patient will be injected with a total of 2.5 x 10' peptide-loaded PBMC. The percent DC mobilized by an agent such as ProgenipoietinT is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex vive activation of CTL/HTL responses Alternatively, ex vivo CTL or HTL responses to 101P3A1 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 30: An Alternative Method of Identifying and Confirminr Motif-Bearing Peptides Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single 00 Stype of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g.

101P3A11. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence 00 determined, by mass spectral analysis Kubo et al., J. Immunol. 152:3913, 1994). Because the majority ofpeptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

SAlternatively, cell lines that do not express endogenous HLA molecules can be transfected with an f 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 101P3A1 I to isolate peptides corresponding to 101P3AI that have 1 been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to 0 binding to the single HLA allele that is expressed in the cell.

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

Example 31: Complementary Polynucleotides Sequences complementary to the 101P3AI l-encoding sequences, or any parts thereof, arc used to detect, decrease, or inhibit expression of naturally occurring 101P3A 11. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, OLIGO 4.06 software (National Biosciences) and the coding sequence of 101P3A11. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 101P3A 1encoding transcript.

Example 32: Purification of Naturally-occurring or Recombinant 101P3A11 Using 101P3A11- Specific Antibodies Naturally occurring or recombinant 101P3A1 I is substantially purified by immunoaffinity chromatography using antibodies specific for 101P3A11. An immunoaffinity column is constructed by covalently coupling anti-101P3Al I 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 101P3A 11 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 101P3AI 1 high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/101P3AI 1 binding a buffer ofpH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 33; Identification of Molecules Which Interact with 101P3A11 00 101P3AI 1, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, C, Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multiwell plate are incubated with the labeled 101P3A 1, washed, and any wells with labeled 10IP3A11 complex are assayed. Data obtained using different concentrations of 101P3A 11 are used to calculate values for the number, 00 affinity, and association of 101P3A I with the candidate molecules.

Example 34: In Viva Assay for 101P3AI1 Tumor Growth Promotion The effect of the 101P3Al 1 protein on tumor cell growth can be confirmed in vive by gene overexpression in a variety of cancer cells, including prostate, kidney, colon and bladder. For example, SCID Smice can be injected subcutaneously on one flank with I x 106 prostate, kidney, colon or bladder cancer cells 00 (such as PC3, LNCaP, SCaBER, UM-UC-3, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839 cells) containing tkNeo empty vector or 101P3A 1.

C At least two strategies can be used: Constitutive 101P3A 1I expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems.

Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and is followed over time to validate that 101P3Al 1-expressing cells grow at a faster rate and that tumors produced by 101P3A1 -expressing cells demonstrate characteristics of altered aggressiveness enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Figure 21 compares subcutaneous growth of control 3T3-neo and 3T3-101P3A1 1 cells. One million cells stably expressing neo or 101P3A11 were injected subcutaneously in SCID mice along with matrigel. Tumor volume was evaluated by caliper measurements. This experiment demonstrates that expression of 101P3A11 in NIH 3T3 cells is induces tumor formation in 6/6 mice. In an experiment comparing the effect of a strong oncogene such as Ras to that of 101P3A11, we showed that 101P3AI 1 induced tumor growth of 3T3 cells in a more rapid and aggressive manner that "V-Ras (Figure 55). The results indicated that expression of 101P3AI I is sufficient to induce tumor formation in vivo. Figure 42 shows demonstrates that 101P3A1 I induces orthotopic growth of tumors. Additionally, SCID mice were implanted with the same 3T3-101P3A I1 cells orthotopically in the prostate to determine if 101P3Al I has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs or lymph nodes. This experiment (figure 57) shows that while control 3T3-neo cells fail to induce tumor formation in the prostate of SCID mice, significant tumor growth was seen in cells expressing 101P3A11. In an analogous manner, cells can be implanted orthotopically in the bladder, colon or kidney. (Saffran, et al., PNAS 10:1073-1078; Fu, et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., etal., Anticancer Res., 1997. 17: p. 3239-3242; Peralta, E. etal., J. Urol., 1999. 162: p. 1806-1811). The tumor enhancing effect of 101P3A 11 was also observed when 101P3AI 1 is expressed in prostate cancer cells such as PC3 and introduced into the prostate of SCID mice (figure 58). A 2.5 fold increase in tumor weight is observed in tumors expressing 101P3AI relative to control cells.

00 O Expression od 101P3A11 also enhances tumor growth and progression in the tibia os SCID mice.

Clinical studies have repeatedly shown that prostate cancer may become metastatic to the bone. In order to investigate the contribution of 101P3AI 1 to bone tropism and tumor growth in the bone, control and 101P3A11expressing cells were compared for their ability to induce tumor growth in the tibia of SCID mice. Experiments 00 in figures 61 and 62 show that injection of 101P3Al I expressing 3T3 or PC3 cells into the bone ofSCID mice results in increase tumor growth and tumor formation relative to control cells.

Furthermore, these assays is useful to confirm the anti-101P3A1 I inhibitory effects of candidate therapeutic compositions, such as for example, 101P3AI 1 antibodies or intrabodies, and 101P3A11 antisense S molecules or ribozymes, or 101P3AI directed small molecules. In Figure 22, we depict the effect of a small Smolecule, pertussis toxin (PTX) on tumor formation by 3T3-101P3A I cells. In this experiment, SCID mice were i injected with 3T3-101P3A11 alone or in conjunction with PTX. Each mouse was given 5 doses of PTX at 3-4 00 0 days interval. Tumor volume was evaluated by caliper measurements. Figure 22 shows that PTX inhibits tumor C growth in a dose dependent manner. Delivery of PTX at shorter intervals, such as 5 time per week, resulted in a larger rate of inhibition of tumor growth, with 70% inhibition of tumor growth observed after 25 days (Figure 56). Similarly, treatment with the G-protein inhibitor suramin inhibits the growth of 3T3-101P3A11 tumors (Figure 60). In addition to demonstrating that 101A3PI I plays an important role in tumor growth, Figures 21 22 and M9 identify a signaling pathway associated with 101P3A11 and indicate that 101P3AI 1 produced its effect on tumor growth by activating an adenylate cyclase dependent pathway.

Example 35: 101P3A11 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo The significant expression of 101P3AI 1 in cancer tissues, together with its restricted expression in normal tissues, makes 101P3A11 an excellent target for antibody therapy. In cases where the monoclonal antibody target is a cell surface protein, as is 101P3Al 1, antibodies have been shown to be efficacious at inhibiting tumor growth (See, Saffian, et al., PNAS 10:1073-1078 or on the www at pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target is not on the cell surface, such as PSA and PAP in prostate cancer, antibodies have also been shown to recognize and inhibit growth of cells expressing those proteins (Saffran, et al., Cancer and Metastasis Reviews, 1999. 18: 437-449). As with any cellular protein with a restricted expression profile, 101P3A11 is a target for T cell-based immunotherapy.

Accordingly, the therapeutic efficacy ofanti-101P3Al 1 mAbs in human colon, kidney, bladder and prostate cancer mouse models is modeled in 101P3AI 1-expressing kidney, colon, bladder or prostate cancer xenografts or cancer cell lines, such as those described in the Example entitled "In Vivo Assay for 101P3A 11 Tumor Growth Promotion", that have been engineered to express 101P3A11.

Antibody efficacy on tumor growth and metastasis formation is confirmed, in a mouse orthotopic prostate, colon, bladder or kidney cancer xenograft model. The antibodies can be unconjugated, or can be conjugated to a therapeutic modality, as appreciated in the art. It is confirmed that anti-101P3Al 1 mAbs inhibit formation of 101P3A11-expressing kidney, colon, bladder and prostate tumors. Anti-101P3Al 1 mAbs also retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice. These results indicate the utility of anti-101P3Al 1 mAbs in the treatment of local and advanced stages of cancer. (See, Saffran, D., et al., PNAS 10:1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698) 124 00 Administration of anti-101P3A I mAbs retard established orthotopic tumor growth and inhibit CK metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 101P3Al I is an attractive target for immunotherapy and demonstrate the therapeutic potential Sof anti-101P3Al 1 mAbs for the treatment of local and metastatic kidney, colon, bladder and prostate cancer.

00 0_ Similar studies manifest that 101P3A11 is safe and effective when used in combination with other therapeutic modalities such as surgery, radiation therapy, hormone therapy or chemotherapy.

SThis example demonstrates that unconjugated 101P3A11 monoclonal antibodies effectively to inhibit the IN growth of human bladder tumors grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

00 Tumor inhibition using multiple unconjugated 101P3A11 mAbs Materials and Methods 101P3A11 Monoclonal Antibodies: Monoclonal antibodies are raised against 101P3A 11 as described in the Example entitled "Generation of 101P3A11 Monoclonal Antibodies (mAbs)." The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 101P3A11. Epitope mapping data for the anti-101P3A 11 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 101P3A11 protein.

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

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein- G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20 0 C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of UM-UC3, J82, CaKil, 769P, CaOvl or PA tumor xenografts.

Cell Lines The bladder, kidney and ovary carcinoma cell lines, UM-UC3, J82, CaKil, 769P, CaOvl and PAl as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in DMEM supplemented with L-glutamine and 10% FBS.

A UM-UC3-101P3A11, J82-101P3A 11, CaKil-101P3AI 1, 769P-101P3AI 1, CaOvl-101P3Al 1, PAl- 101P3A11 and 3T3-101P3Al 1 cell populations are generated by retroviral gene transfer as described in Hubert, et al., Proc Natl Acad Sci U S A, 1999. 96(25): 14523.

Xenograft Mouse Models.

Subcutaneous tumors are generated by injection of I x 10 6 cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For bladder orthotopic studies, an incision is made through the abdomen to expose the bladder, and tumor cells (5 x 10') mixed with Matrigel are injected into the bladder wall in a 10-pl volume. To monitor tumor growth, mice are palpated and 125 00 S blood is collected on a weekly basis to measure BTA levels. For kidney and ovary orthopotic models, an incision is made through the abdominal muscles to expose the kidney or the ovary. Tumor cells mixed with Matrigel are S injected under the kidney capsule or into the ovary in a 10-ul volume (Yoshida Y et al, Anticancer Res. 1998, S18:327; Ahn et al, Tumour Biol. 2001, 22:146). To monitor tumor growth, blood is collected on a weekly basis

OO

0 measuring G250 and SM047 levels. The mice are segregated into groups for the appropriate treatments, with anti- 101P3A1 I or control mAbs being injected i.p.

Anti-101P3Al 1 mAbs Inhibit Growth of 10P3A 11-Exopessin Xenoeraft-Cancer Tumors MD The effect of anti-101P3Al 1 mAbs on tumor formation is tested on the growth and progression of bladder, kidney and ovarian cancer xenografts using UC3-10IP3A11, J82-101P3A 1, CaKil-101P3Al 1, 769P- C 101P3A 1, CaOvl-101P3A1l and PAl-101P3All orthotopic models. As compared with the s.c. tumor model, 00 the orthotopic model, which requires injection of tumor cells directly in the mouse bladder, kidney and ovary, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse S health, and subsequent death (Saffran, et al., PNAS supra; Fu, et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, J Cell Biochem, 1994. 56(1): p. The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder, kidney or ovary, and 2 days later, the mice are segregated into two groups and treated with either: a) 2 00-500gg, ofanti-101P3Al I Ab, or b) PBS three times per week for two to five weeks.

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

Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-CK20 for bladder cancer, anti-G250 for kidney cancer and SM047 antibody for ovarian cancer models (Lin S et al, Cancer Detect Prev. 2001;25:202; McCluggage W et al, Histopathol 2001, 38:542).

Mice bearing established orthotopic tumors are administered 1000g injections of either anti-101P3Al 1 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-101P3Al 1 antibodies on initiation and progression of prostate and kidney cancer in xenograft mouse models. Anti-101P3Al 1 antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-101P3All mAbs demonstrate a dramatic inhibitory effect on the spread of local bladder, kidney and ovarian tumor to distal sites, even in the presence of a large tumor burden. Thus, anti- 101P3Al 1 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 36: Therapeutic and Diagnostic use of Anti-101P3Al1 Antibodies in Humans.

Anti-101P3Al 1 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-101P3All mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 101P3AI 1 in carcinoma and in 126 00 Smetastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti- C 101P3AI 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-101P3Al I mAb specifically binds to carcinoma cells. Thus, anti- S 101P3AI 1 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 Sdetection of localized and metastatic cancers that exhibit expression of 101P3AI 1. Shedding or release of an ND extracellular domain of 101P3A 1 into the extracellular milieu, such as that seen for alkaline phosphodiesterase (Meerson, N. Hepatology 27:563-568 (1998)), allows diagnostic detection of 101P3A 1 by anti- S101P3A11 antibodies in serum and/or urine samples from suspect patients.

00 Anti-101P3Al I antibodies that specifically bind 101P3AI 1 are used in therapeutic applications for the treatment of cancers that express 101P3A I1. Anti-101P3Al I antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-101P3A 11 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, kidney cancer models AGS-K3 and AGS-K6, (see, the Example entitled "I01P3AI 1 Monoclonal Antibody-mediated Inhibition of Bladder, Kidney and Ovarian Tumors In Vivo"). Conjugated and unconjugated anti-101P3Al 1 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

Example 37: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-101P3A11 Antibodies In vivo Antibodies are used in accordance with the present invention which recognize an epitope on 101P3A11, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 101P3A 11 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-101 P3Al I antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-101P3All 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- 101P3Al I antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

II.) Monotherapy: In connection with the use of the anti-101 P3A 11 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.

00 0 III.) Imaging Agent: Through binding a radionuclide iodine or yttrium (I131, Y) to anti- 101P3AI 1 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing S101P3AI 1. In connection with the use of the anti-101P3Al I antibodies as imaging agents, the antibodies are 0 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 In)-101P3Al 1 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 101P3AI 1 IN (by analogy see, Divgi et al. J. Nail. 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 00 Dose and Route of Administration As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-101P3A I1 antibodies can be administered with doses in the range of 5 to 400 mg/m 2 with the lower doses used, in connection with safety studies. The affinity of anti-101P3Al 1 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti.

101P3Al I antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-101P3Al 1 antibodies can be lower, perhaps in the range of 50 to 300 mg/m 2 and still remain efficacious. Dosing in mg/m 2 as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-101 P3Al 1 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP) Overview: The CDP follows and develops treatments of anti-101P3Al I 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-101P3Al l antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 101P3A 1 expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 101P3A11, Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-101P3Al 1 antibodies are found to be safe upon human administration.

00

O

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SExample 38: Human Clinical Trial Adiunctive Therapy with Human Anti-101P3All Antibody C and Chemotherapeutic Alent(s) A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti- 101P3AI 1 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 ofanti-101P3Al I antibodies when utilized as an adjunctive therapy to an Santineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, N is assessed. The trial design includes delivery ofsix single doses of an anti-101P3AI antibody with dosage of antibody escalating from approximately about 25 mg/m 2 to about 275 mg/m over the course of the treatment in accordance with the following schedule: 00 0 DayO Day 7 Day 14 Day21 Day28 mAb Dose 25 75 125 175 225 275 mg/m 2 mg/m mg/m 2 mg/m 2 mg/m 2 mg/m 2 Chemotherapy (standard dose) Patients are closely followed for one-week following each administration of antibody and chemotherapy.

In particular, patients are assessed for the safety concerns mentioned above: cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 101P3Al 1. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-101P3A 1 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 39: Human Clinical Trial: Monotherapy with Human Anti-101P3All Antibody Anti-101P3Al 1 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase 11 human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-101P3A 11 antibodies.

Example 40: Human Clinical Trial: Diagnostic Imagine with Anti-101P3All 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 ofanti-101P3AI I antibodies as a diagnostic imaging agent.

The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J.

Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

00 O Example 41: Identification of 101P3A11 sequences involved in ligand binding.

O As shown in Figure 4, the transmembrane regions of 101P3A I and mouse olfactory receptor S (ORS25)predicted using the TMHMM algorithm are highlighted in gray. The amino acids of ORS25 predicted by SFloriano, et al. to be involved in binding of the ligand hexanol and/or involved in the formation of the ligand 00 binding pocket are italicized and bolded in Figure 4, and are listed below. (Floriano, et al, 2000, Proc. Natl.

Acad. Sci., USA, 97:10712-10716) Leu 131 Ala 230 SVal 134 lie 231 Val 135 Gly 234 00 Gly 138 Thr 284 Thrl39 Phe 287 C Ser 193 Gin 300 Ser 197, Lys 302 Phe 225 Sequences of 101P3A 11 involved in ligand binding are identified based on homology to mouse olfactory receptor S25. Shown is the amino acid alignment of 101P3A11 with mouse olfactory receptor S25 depicting the predicted transmembrane domains of each GPCR. The amino acids of S25 involved in the recognition and binding of its ligand hexanol or that lie in the proximity of the binding pocket (Floriano, et al, 2000, Proc.

Natl. Acad. Sci., USA, 97:10712-10716), are also shown. These amino acids lie close to or within the transmembrane domains ofORS25. Accordingly, the structurally homologous regions of 101P3A1 I are involved in the binding of its cognate ligand. These regions encode the amino acids of the first extracellular loop and of the amino terminal end of transmembrane domain 3 (amino acids 82-112), the amino acids at the carboxyl terminal end of transmembrane domain 4 and into the second extracellular loop (amino acids 160-185), the amino acids at the end of the second extracellular loop and into transmembrane domain 5 (amino acids 186-212), and the amino acids at the carboxyl terminal end of transmembrane domain 6, the third extracellular loop, and the amino terminal end of transmembrane domain 7 (amino acids 250-280). Thus, ligands of 101P3AI 1 are identified that interact with at least 3 of the following regions of 101P3A1 1: amino acids 82-112, amino acids 160-185, amino acids.186-212, and, amino acids 250-280.

Example 42: Homology Comparison of 101P3A11 to Known Sequences The 101P3AI 1 protein of Figure 3 has 318 amino acids with calculated molecular weight of 35.2 kDa, and pi of 8.7. 101P3A11 is predicted to be a cell surface protein. Cellular localization was demonstrated by FACS analysis and immunofluorescence in cells engineered to express 101P3A11, as shown in figure 64 (panel Immunofluorescence staining ofpermeabilized cells revealed that 101P3A1 I becomes internalized and localizes then to the cytosol (Figure 64).

101P3A11 shows best homology to rat olfactory receptor RAlc (gi 3420759, http://www.ncbi.nlm.nih.gov) sharing 59% identity and 76% homology with that protein. 101P3A1 1 also shows homology to human prostate specific GPCR (gi 13540539) and human olfactory receptor 51112 (gi 14423836), 00 sharing 59% identities/ 77% homology, and 53% identities/ 69% homology with each, respectively (Figures 23- C 25). More recent studies have identified a mouse homolog of 101P3A11, namely MOR 18-1, (gi 18479284, Figure 65). MORI8-1 is a mouse olfactory receptor that shares 93% identity and 96% homology with the S101P3A11 protein.

00 In addition to 101P3AI 1 variant 1 used predominantly in the studies listed below, 101P3AI I has 2 additional variants. Variant 3 is a SNP of variant I and exhibits a mutation at position 104, with an exchange of isoleusine to methionine at that position. Variant 3 is expected to be localized to the cell surface, in a manner similar to variant 1, and exhibits the same motifs and transmembrane domains as variant 1 (table XXI). Variant 2 of 101P3A11 is 72 amino acids long, contains 2 transmembranes and localizes predominantly at the cell surface S with some cytoplasmic localization.

00 Sequence and motif analysis indicate that 101P3A1 I belongs to the family of olfactory receptors.

Bioinformatic analysis revealed 101P3AI1 to be a 7 transmembrane protein, with strong domain and structural C"1 homology to G-protein coupled receptors (GPCRs) (see Table XXI, TM Pred, Sosui, Pfam, Blocks, Print).

Proteins that are members of the G-protein coupled receptor family exhibit an extracellular amino-terminus, three extracellular loops, three intracellular loops and an intracellular carboxyl terminus. G-protein coupled receptors are sevcn-transmembrane receptors that are stimulated by polypeptide hormones, neurotransmitters, chemokines and phospholipids (Civelli O et al, Trends Neurosci. 2001, 24:230; Vrecl M et al Mol Endocrinol. 1998, 12:1818). Ligand binding traditionally occurs between the first and second extracellular loops of the GPCR.

Upon ligand binding GPCRs transduce signals across the cell surface membrane by associating with trimeric G proteins. Their signals are transmitted via trimeric guanine-nucleotide binding proteins (G proteins) to effector enzymes or ion channels (Simon et al., 1991, Science 252: 802). Signal transduction and biological output mediated by GPCR can be modulated through various mechanisms including peptide mimics, small molecule inhibitors and GPCR kinases or GRK (Pitcher JA et al, J Biol Chem. 1999, 3;274:34531; Fawzi AB, et al. 2001, Mol. Pharmacol., 59:30).

Recently, GPCRs have also been shown to link to mitogenic signaling pathways of tyrosine kinases (Luttrell et al., 1999, Science 283: 655; Luttrell et al., 1999 Curr Opin Cell Biol 11: 177). GPCRs are regulated by phosphorylation mediated by GPCR kinases (GRKs), which themselves are indirectly activated by the GPCRs (Pitcher et al., 1998, Ann. Rev. Biochem. 67: 653). Olfactory GPCRs transmit their signals by activating the cAMP pathway via adenylate cyclase resulting in downstream signaling to protein kinase A, and by activating the phospholipase C pathway by generating inositol 1,4,5-trisphosphate (IP3) and diacyl-glycerol (DAG) (Breer, 1993, Ciba Found Symp 179: 97; Bruch, 1996, Comp Biochem Physiol B Biochem Mol Biol 113:451). IP3 results in an increase in intracellular calcium, while DAG activates protein kinase C.

Recent studies have associated GPCRs with cellular transformation. In particular, KSHV G proteincoupled receptor was found to transform NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV- GPCR-transgenic mice (Schwarz M, Murphy PM. J Immunol 2001, 167:505). KSHV-GPCR was capable of producing its effect on endothelial cells and fibroblasts by activating defined signaling pathways, including the AKT survival pathway (Montaner S et al, Cancer Res 2001, 61:2641). In addition, KSHV-GPCR induced the activation of mitogenic pathways such as AP-1 and NFkB, resulting in the expression of pro-inflammatory genes (Schwarz M, Murphy PM. J Immunol 2001, 167:505). Other GPCR associated with tumor formation include G2A, and the PAR-1, which has been found to induce transformation of NIH 3T3 cells (Whitehead I et al, Oncogene 2001, 20:1547).

00 SThis information indicates that 101P3AI 1 plays a role in the transformation of mammalian cells, induces C'I mitogenic responses including activation of various signaling pathways, and regulate gene transcription by transmitting cell surface signals to the nucleus, see also, the Example entitled, "In Vivo Assay for 101P3A1 I 's Tumor Growth Promotion".

00 Accordingly, when 101P3AI 1 functions as a regulator of cell transformation, tumor formation, or as a modulator of transcription involved in activating genes associated with inflammation, tumorigenesis or proliferation, 101P3A 11 is used for therapeutic, diagnostic, prognostic and/or preventative purposes, in manners sO analogous to or that track other GPCRs as discussed herein and in the art..

00 Example 43: Identification and Confirmation of Potential Signal Transduction Pathways Many mammalian proteins have been reported to interact with signaling molecules and to participate in C1 regulating signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, GPCRs have been reported to activate MAK cascades as well as G proteins, and been associated with the EGFR pathway in epithelial cells (Naor, et al, Trends Endocrinol Metab. 2000, 11:91; Vacca F et al, Cancer Res. 2000, 60:5310; Della Rocca GJ., et al, J Biol Chem. 1999, 274:13978). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 101P3A 11 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 101P3AI1, including phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999,274:801; Oncogene. 2000, 19:3003; J. Cell Biol. 1997, 138:913).

Using Western blotting and other techniques, the ability of 101P3A11 to regulate these pathways is confirmed. Cells expressing or lacking 101P3A11 are either left untreated or stimulated with cytokines, androgen and anti-integrin antibodies. Cell lysates were analyzed using anti-phospho-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, P3K, PLC and other signaling molecules. Using such techniques, we showed that 101P3A I1 alters the tyrosine phosphorylation pattern of NIH 3T3 cells (Figure 26) indicating that 101P3A11 is regulating protein kinases and phosphatases. In the experiment, data shown in Figure 26, control 3T3-neo and 3T3-101P3A11 cells were either treated with 0.5 or FBS and whole cell lysates were analyzed by anti-phosphotyrosine Western blotting. Expression of 101P3AI 1 resulted in reduced phosphorylation of several proteins in NIH-3T3 cells, while inducing the phosphorylation of proteins at 79-81 and 28-32 kDa.

Using anti-Phospho-ERK antibodies, we demonstrated that expression of 101P3A11 induced ERK phosphorylation in the prostate cancer cell line PC3 (Figures 27A and Figure 27B), and that ERK phosphorylation in 101P3A1 I expressing cells was regulated by GPCR ligands. In this experiment, control PC3-neo cells and PC3-101P3A 1 cells were left untreated FBS) or were stimulated with 10% FBS, lipophosphatidic acid (LPA), gastrin releasing peptide (GRP), leukotriene (LKB4) or platelet activating factor (PAF). The cells were lysed and analyzed by Western blotting using anti-Phospho-ERK (Figure 27A) or anti-ERK (Figure 27B) mAb. The results showed that expression of 101P3A1 I mediated significant ERK phosphorylation by FBS, LPA, GRP and PAF, while LKB4 resulted in a more modest level of ERK phosphorylation in PC3-101P3AI I cells. In contrast, none of the GPCR ligands induced significant ERK phosphorylation in PC3-Neo cells, demonstrating the specificity of GPCR ligands-mediated responses in 101P3A11 expressing cells. The ERK overlay demonstrated 132 00 0 equal loading, supporting the specificity of this data. In order to delineate the signaling pathway by which C 101P3A 1I mediates ERK phosphorylation in cancer cells, it was confirmed which of the two pathway inhibitors: MEK inhibitor PD98059 or the p38 inhibitor SB203580 regulate 101P3A I mediated ERK phosphorylation S(Figure 28). To obtain this data, PC3-neo and PC3-101 P3A I cells were treated with media alone or in the 00 presence of PD98059, SB203580, or genistein were stimulated with FBS or GRP. Cells were lysed and analyzed by Western blotting using anti-Phospho-ERK or anti-ERK mAb. Treatment with 10% FBS or with GRP induced the phosphorylation of ERK in PC3-101P3Al I but not in control PC3-neo cells. 101P3AI 1-mediated ERK \phosphorylation was inhibited by the MEK-I inhibitor PD98059 but not the p38 inhibitor SB203580 or genistein.

SThe ERK overlay demonstrated equal loading, supporting the specificity of the results. These results were 0 confirmed by those obtained in two additional sets of experiments. The inhibition of 101P3AI 1-mediated ERK OO phosphorylation by PD98059 demonstrates that 101P3A 11 activated the classical MEK-ERK cascade, a pathway O associated with mitogenesis, proliferation and tumorigenesis.

CN' Results in Figures 26-28 indicate that 101P3A I1 regulates the activity ofkinases, including ERK, and phosphatases. In order to confirm the association of 101P3A I with phosphatase activity, the effect of the protein phosphatase inhibitor sodium orthovanadate on 101P3A I1 mediated ERK phosphorylation was determined (Figure 29). PC3-neo and PC3-101P3A 1 cells were grown in media alone or in the presence of sodium orthovanadate (Na3VO4), and were stimulated with 0.1% or 10% FBS. Cells were lysed and analyzed by Western blotting using anti-Phospho-ERK or anti-ERK mAb. Treatment with Na3VO4 resulted in a increase in ERK phosphorylation in PC3-101P3A11 cells, compared to a two-fold increase in PC3-neo cells.

Results in Figure 29 confirm the contribution of protein phosphatases to 101P3A 11 mediated signaling.

Several GPCRs have been shown to transactivate receptor tyrosine kinases associated with the cell membrane, such as the EGF receptor (EGFR) (Pierce et al, J Biol Chem. 2001, 276:23155; Nath, et al, J Cell Sci. 2001, 114:1213). In order to determine whether 101P3A1 I signaling results in the activation of EGFR, we compared the effect of the EGFR inhibitor, AG1517, on EGFR- and 101P3AI 1-mediated ERK phosphorylation (Figure 30). In Figure 30, PC3-neo and PC3-101P3A 11 cells were grown in media alone (0.1% FBS) or in the presence of AG1517. The cells were stimulated with 0.1% or 10% FBS, GRP or EGF, lysed and analyzed by Western blotting using anti-Phospho-ERK or anti-ERK mAb. Treatment with 10% FBS, GRP and EGF induced ERK phosphorylation in PC3-101P3A11 cells. ERK phosphorylation by EGF was completely inhibited by AG1517. O11P3A1 mediated ERK phosphorylation in cells treated with 10% FBS was partially inhibited by AG1517. Data in Figure 30 indicate that some cross talk occurred between 101P3A1 I and EGFR signaling pathways.

In addition to activating the ERK cascade, 101P3All activated a parallel MAK pathway, namely p38. In Figure 31A and Figure 31B, PC3-neo and PC3-101P3A1 I cells were grown in 1% or 10% FBS. Cells were lysed and analyzed by Western blotting using anti-Phospho-p38 (Figure 31A) or anti-p38 (Figure 31B) monoclonal antibody (mAb). Our results demonstrate that expression of 101P3AI 1 mediated p38 phosphorylation in cells treated with 10% FBS. Equal loading was demonstrated in the p38 overlay.

Results shown in Figures 26-30 and Figure 31A-31B confirm that 101P3AI I activates several signaling pathways in cancer cells, including the ERK and p38 cascades. In addition to MAPK, 101P3A11 signaling was associated with protein phosphatase activity and EGFR transactivation. These signaling pathways have been associated with cell growth, survival and transcriptional activation, all of which play an important role in tumor initiation and progression. When 101P3Al 1 plays a role in the regulation of signaling pathways, whether 133 00 individually or communally, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

To confirm that 101P3A 1I directly or indirectly activates known signal transduction pathways in cells, S luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These S 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.

S1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation C 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress 00 4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis p53-luc, p53; SAPK; growth/differentiation/apoptosis 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.

Signaling pathways activated by 101P3A1 I are mapped and used for the identification and validation of therapeutic targets. When 101P3AI 1 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 44: 101P3A11 Functions as a GPCR Sequence and homology analysis of 101P3A 1 indicated that 101P3AI is a member of the olfactory receptor family of GPCR. Olfactory receptors are known to regulate biological responses by activating adenylate cyclase. In order to confirm that 101P3AlI functions as a GPCR and mediates the activation of adenylate cyclase, cAMP accumulation in PC3 and PC3-101P3A 11 cells were compared (Figure 32). Control PC3 and PC3- 101P3A 11 cells were grown in a low concentration of fetal bovine serum (FBS) for 14 hrs in the presence or absence of pertussis toxin (PTX). The cells were stimulated with 0.1% or 10% FBS, washed in PBS and lysed using a lysis buffer provided by Amersham Pharmacia. Intracellular concentration of cAMP was measured using a commercially available enzyme immunoassay (EIA) according to the manufacturer's recommendations (Amersham Pharmacia).

Each assay was performed in duplicate. Calculations of cAMP concentrations were based on OD450 of the standard curve. Expression of 101P3All induced a four-fold increase in cAMP accumulation in the absence of stimulation. Treatment with 10% FBS further enhanced cAMP accumulation in PC3-101P3All cells to nearly seven-fold over control PC3-neo cells. 101P3AI1 mediated cAMP accumulation was inhibited by PTX. These results were confirmed by two separate sets of experiments. Results shown in Figure 32 demonstrate that 101P3Al functions as a GPCR in prostate cancer cells and exhibits classical GPCR characteristics, such as cAMP accumulation that is inhibited by PTX.

Since adenylate cyclase activity modulates intracellular levels of cAMP and induce downstream signaling events such as activation of protein kinase A, calcium and ERK MAPK signaling (Pierce et al, 00 O Oncogene; 2001, 20:1532), we determined that PTX, an inhibitor of adenylate cyclase signaling, prevents 0 101P3A 11-mediated ERK phosphorylation along with inhibiting cAMP accumulation (Figure 33 and Figure 34).

PC3-neo and PC3-101P3AI 1 cells were grown overnight in 0.1% FBS in media alone or in the presence of pertussis toxin (PTX). Cells were stimulated with 0.1% or 10% FBS (Figure 33) or 10% FBS, EGF or GRP 00 (Figure 34). Cells were lysed and analyzed by Western blotting using anti-Phospho-ERK mAb. Expression of 101P3A 1 mediated ERK phosphorylation by 10% FBS in PC3 cells, which was inhibited by PTX (Figure 33 and Figure 34). In contrast, GRP and EGF.mediated ERK phosphorylation was relatively unaffected by PTX (Figure M\ 34), demonstrating the specificity of 101P3A11 mediated responses. These results were replicated in additional -experiments. In addition to inhibiting ERK phosphorylation and 101P3A 1-mediated signaling, PTX had a 0 marked effect on the proliferation ofPC3-101P3Al I but not control PC3-neo cells (figure 53). Figure 53 shows 00 that PTX inhibited the proliferation f 101P3AI I expressing cells in a dose dependent manner, and confirms that the GPCR function of 101P3AI is important for tumor growth.

C, GPCR transmit their signal by activating trimeric G proteins. Once GPCRs are activated, the associated Ga subunit binds GTP, dissociates from the receptor and participates in downstream signaling events (Schild, D., and Restrepo, D. Physiol Rev. 1998, 78:429-66). In order to determine that inhibition of Ga subunits has an effect on 101P3AI 1 mediated cell growth, the effect of two Ga inhibitors on the proliferation of3T3-101P3AI 1 cells was investigated. Control 3T3 and 3T3-101P3A 1 cells were grown in the presence or absence ofsuramin or its derivative NF 449 (Sigma). Cells were analyzed for proliferation 72 hours later (Figure 35). The experiment was performed in triplicate. The data showed that suramin and NF449 inhibited the proliferation of 3T3-101P3A11 cells by 60% and 80%, respectively. This response was 101P3Ai I specific as suramin and NF449 had no effect on the proliferation of control 3T3 cells. Similarly, inhibition of G protein activation by suramin and NF449 in PC3 cells inhibits the proliferation ofPC3-101P3AI 1 cells grown in 5% FBS (Figure 52).

In parallel with the inhibitory effect of suramin and NF449 on PC3-101P3AI 1 proliferation, we demonstrate their inhibitory effect on 101P3A11-mediated signaling in prostate cancer cells (Figure 51). As shown in figures 27 and 28, treatment with FBS iduces ERK phosphorylation in 101P3AI 1-expressing cells. This ERK phosohorylation and activation was inhibited by suramin and NF449 in PC3-101P3AI 1 and 3T3-101P3A 11 cells.

ERK phosphorylation was inhibited in a dose dependent manner in PC3-101P3A 11 cells treated with suramin.

Thus, as 101P3A 11 is involved in GPCR activity, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

GPCRs can be activated by a variety of ligands, including hormones, neuropeptides, chemokines, odorants and phospholipids. In the case of olfactory receptors, individual olfactory receptors may recognize multiple odorants, and can are activated by a diverse array of molecules. These ligands and molecules recognized by a receptor (as described above) are small molecules as described herein.

In order to identify 101P3AI 1 (small molecule) ligand(s), the possibility that epithelial cells may be secreting 101P3A11 activators was investigated (Figure 36A and Figure 36B). Prostate cancer epithelial cells, (PC3, PC3-101P3A 11, LAPC4 2 hT), normal prostate cells (PrEC), fibroblasts (3T3, 3T3-101P3A 11), and human kidney epithelial cells (293T) were grown in the presence or absence of FBS. Cell supernatants were collected and used to stimulate PC3 and PC3-101P3AI 1 cells. Cell lysates from resting and supernatant treated PC3 and PC3-101P3A11 cells were lysed and analyzed by Western blotting with anti-Phospho-ERK (Figure 36A) and anti- ERK (Figure 36B) mAb. As shown in Figure 36A and Figure 36B, supernatants form normal prostate cells, PrEC, and prostate cancer cells, PC3, PC3-101P3AI I and LAPC42hT, induced the phosphorylation of ERK in 135 00 S PC3-101P3A 1I but not control PC3 cells. In contrast, no specific ERK phosphorylation was observed using C'i supematants from 3T3 or 293T cells. Our results show that prostate cells, grown in the absence of serum, produce one or more factors that contribute to the activation of 101P3A 11 mediated signaling events. Thus, as 101P3A I1 S responds to stimuli and functions in signaling and GPCR activity, it is used as target for diagnostic, prognostic, 00 preventative and/or therapeutic purposes.

Example 45: Inhibitors of 101P3A11 GPCR Function \As mentioned in the Example entitled "Homology Comparison of 101P3A 1 to Known Sequences," GPCRs are activated by ligand binding to the extracellular loops, resulting in the activation of trimeric G proteins 0 and the initiation of several signaling cascades. Using this information, several therapeutic and small molecule 00 strategies are utilized to inhibit GPCR activation or downstream signaling events.

One strategy inhibits receptor and ligand binding. Recent studies using several types of GPCRs, have C- demonstrated the effectiveness of this strategy (Fawzi AB, et al. 2001, Mol. Pharmacol., 59:30). Using a compound named SCH-202676, they inhibited agonist and antagonist binding to GPCRs by allosterically hindering ligand-GPCR interaction. Using this and even more specific allosteric (small molecule) inhibitors, signal transduction through 101P3A11 is inhibited, thereby providing therapeutic, prognostic, diagnostic and/or prophylactic benefit.

A second approach is to inhibit G alpha subunit activation. Activation of GPCRs results in the exchange of GTP for GDP on the G alpha subunit of the trimeric G protein. Inhibition of Ga activation prevents the activation of downstream signaling cascades and therefore biological effects of GPCR. One molecule used to inhibit GDP exchange on Ga subunits is Suranim (Freissmuth M et al, 1996, Mol. Pharmacol, 49:602). Since suranim functions as a universal Ga inhibitor, it prevents the activation of most Ga subunits. Using techniques described, for example and without limitation, in the present Examples entitled "In Vivo Assay for 101P3AI I Tumor Growth Promotion; "Identification and Confirmation of Potential Signal Transduction Pathways," "101P3A11 Functions as a GPCR," and "Regulation of Transcription", small molecules are identified that selectively inhibit the Ga subunit that associates with 101P3A11, thereby providing therapeutic, prognostic, diagnostic and/or prophylactic benefit.

A third approach is to inhibit Ga subunit association with GPCR. In order for trimeric G proteins to be activated following GPCR/ligand interaction, it is necessary for them to associate with their corresponding GPCR.

Mutational analysis has mapped the interaction of Ga to the first and third intracellular loops of GPCRs (Heller R at al. 1996, Biochem. Biophys. Res. Commun). Several studies have used synthetic (small molecule) peptides corresponding to the intracellular sequence of loops 1 and 3 as inhibitors (Mukherjee, et al. 1999, J. Biol.

Chem.). Using such short peptides that serve as receptor mimics, they are used to compete for binding of Ga subunits to 101P3AI 1 and thereby provide therapeutic, prognostic, diagnostic and/or prophylactic benefit.

Thus, compounds and small molecules designed to inhibit 101P3A1 I function and downstream signaling events are used for therapeutic diagnostic, prognostic and/or preventative purposes.

Example 46: Involvement in Tumor Progression The 101P3A11 gene can contribute to the growth of cancer cells. The role of 101P3A 1 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, colon, bladder and kidney cell 00 0 lines, as well as NIH 3T3 cells engineered to stably express 101P3A 11. Parental cells lacking 101P3Al I and cells expressing 101P3A 11 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). Using such a technique, we demonstrated (see Figure 37) that 101P3AI 1 imparts a growth advantage on NIH 3T3 cells. 3T3- 00 neo and 3T3-101P3A1 I cells were grown in 0.5% or 10% FBS and analyzed 48 hours later. The assay was performed in triplicate. Expression of 101P3A11 resulted in 6-fold increase in proliferation relative to control 3T3 cells grown in 0.5% FBS. In addition, 101P3AI 1 imparts a growth advantage to PC3 cells as shown in \figures 54 and 63. PC3 cells grown in 0.5% and 10% FBS were compared to PC3-101P3A11. Figure 54 shows that expression of 101P3AI 1 enhances the proliferation of PC3 cells under both conditions. The effect of S101P3Al I was also observed on cell cycle progression. Control and 101P3AI -expressing cells were grown in OO low serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells were analyzed for BrdU and propidium Siodide incorporation by FACS analysis. Figure 63 shows that expression of 101P3AI 1 enhances cell cycle entry C" in both 3T3 and PC3 cells.

To confirm the role of 101P3A11 in the transformation process, its effect in colony forming assays was investigated. Parental NIH-3T3 cells lacking 101P3A I1 were compared to NIH-3T3 cells expressing 101P3A11, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res.

2000;60:6730). The results are shown in Figure 43, where 101P3A11 induces colony formation of over 100 fold increase relative to neo resistant controls. We previously showed that expression of 101P3A1 I in NIH 3T3 cells induces the growth of these cells in soft agar (129-24usul), indicating that 101P3A1I participates in the process of transformation.

To confirm the role of 101P3Al 1 in invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive a Transwell Insert System assay (Becton Dickinson) (Cancer Res.

1999; 59:6010)). Control cells, including prostate, colon, bladder and kidney cell lines lacking 101P3Al I are compared to cells expressing 101P3A11. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane analog the 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.

101P3A1 I can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 101P3A1 I are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the GI, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 101P3A11, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 101P3AI I can play a critical role in regulating tumor progression and tumor load.

When 101P3A11 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

00 0Example 47: Involvement in Angioeenesis Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan Folkman, Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as (lie tissue culture assays endothelial cell tube 00 formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 101P3AI 1 in angiogenesis, enhancement or inhibition, is confirmed For example, endothelial cells engineered to express 101P3A11 are evaluated using tube formation and proliferation assays. The effect of 101P3AI I is also confirmed in animal models in vivo. For example, cells M either expressing or lacking 101P3A1 I are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques.

00 101P3AI 1 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes Example 48: Regulation of Transcription The cell surface localization of 101P3A11 and its similarity to GPCRs indicate that 101P3A 11 is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, by studying gene expression in cells expressing or lacking 101P3A 1. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 101P3AI I-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000.

83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELKl-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.

Thus, 101P3A11 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 49: Involvement in Cell Adhesion Cell adhesion plays a critical role in tissue colonization and metastasis. 101P3A 1 can participate in cellular organization, and as a consequence cell adhesion and motility. To confirm that 101P3A11 regulates cell adhesion, control cells lacking 101P3AI I are compared to cells expressing 101P3A 1, using techniques previously described (see, Haier 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 101P3A 11 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 138 00 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 101P3AI 1 is involved in these processes. Thus, it serves as a diagnostic, prognostic, preventative and/or therapeutic modality.

00 Example 50: Protein-Protein Association Several GPCRs have been shown to interact with other proteins, thereby regulating signal transduction, gene transcription, transformation and cell adhesion (Sexton PM et al, Cell Signal. 2001, 13:73; Turner CE, J S Cell Sci. 2000, 23:4139). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 101P3A11. Immunoprecipitates from cells expressing 10 IP3AI 1 and cells lacking 101P3AI I are compared for specific protein-protein associations.

00 Studies are performed to confirm the extent of association of 101P3AI I with effector molecules, such as receptors, adaptor proteins and paxillin, kinases, phsophates and Ga proteins. Studies comparing 101P3A1 I positive and 101P3A1 I negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 101P3A1 I-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 101P3A 1, 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 101P3A 1.

Thus it is found that 101P3AI I associates with proteins and small molecules. Accordingly, 101P3Al land these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 51: Biological effect of Anti-101P3A11 Antibodies.

The generation of anti-101P3A11 polyclonal Ab (pAb) using an amino-terminal peptide encoding amino acids 1-14 (MVDPNGNESSATYF; SEQ ID NO: as antigen was reported in our Priority Application. The effect of this antibody on 101P3A1 I mediated ERK phosphorylation (Figure 38) and cAMP accumulation (Figure 39) was determined. 293T cells were transfected with control or 101P3A1 I cDNA. Cells were allowed to rest overnight, and treated with anti-101P3Al 1 or control Ab in the presence of 0.5% or 10% FBS. Cells were lysed and analyzed by Western blotting with anti-Phospho-ERK and anti-ERK mAb. Figure 38 shows that expression of 101P3AI 1 induces ERK phosphorylation in cells treated with 0.5 or 10% FBS. Anti-101P3Al 1 pAb reduced the phosphorylation of ERK in 293T-101P3A11 cells treated with 0.5% FBS. The ERK overlay demonstrated equal loading, supporting the specificity of this data.

In order to confirm that anti-101P3Al 1 pAb has a detectable effect on cAMP accumulation, PC3 and PC3-101P3All cells were grown in 0.1% FBS and treated with anti-101P3Al 1 pAb. Cells were analyzed for cAMP content as described in Figure 32. Expression of 101P3AI I induced a 5-fold increase in cAMP accumulation in PC3 cells, which was partially inhibited by PTX. Treatment ofPC3-101P3Al I cells with anti- 101P3A 1 pAb resulted in a 4-fold increase in cAMP accumulation in PC3-101P3A11 but not control PC3 cells.

139 00 Results shown in Figure 38 and Figure 39 indicate that anti-101P3Al I pAb produces a measurable biological i effect in cells expressing 101P3AI 1. Accordingly, 101P3A1 I is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

OO

,0 Example 52: 101P3A11 Promoters A eukaryotic cells promoter is a short DNA sequence located in the 5' region of a gene. It provides binding sites for RNA polymerase and associated transcriptional cofactors, which upon assembly promotes O transcription of the gene. In humans, most genes are transcribed by RNA polymerase II. The promoter DNA sequence normally contains binding motifs for RNA polymerases and its associated cofactors and activators including a TATA-box, cap signal, CCAAT-box and the GC-box. A eukaryotic cell enhancer is DNA sequence 00 where transcriptional factors and their associated coactivators or suppressors bind and interact with promoterbound RNA polymerase to regulate the expression of the gene located next to the promoter. While a promoter(s) CN, locates close to the transcription starting site(s) of a gene (usually 25-30 base pairs), enhancers can be found up to 50,000 base pairs in either direction to the transcription starting site(s) of the gene. There are many different gene regulatory proteins, namely transcription factors and their associated coactivators and cosuppressors that bind to specific enhancer sequences and regulate gene expression. These proteins, upon interaction with specific DNA regulatory sequences and with each other, allow each gene to be regulated up or down in different tissues and cell types. Chapter 9. "Control of gene expression" in Molecular Biology of the Cell. 3 r d ed. Ed. by Alberts et al., (New York and London) Garland Publishing, 1994).

Tissue specific gene expression is associated with the presence of specific combinations of transcription factors and their associated coactivators and suppressors, the presence of specific binding sites present in the DNA regulatory region of the gene for these factors, and the activation or inactivation of signaling pathways that modulate their relative activity. For example, prostate specific expression of prostate specific antigen, (PSA, or human kallikrein is dependent on the presence of androgen receptor binding elements in defined 5' upstream enhancer and promoter sequences of the gene and intact androgen receptor signaling pathway (Pang S, et al., Cancer Res 1997 Feb 1; 57(3):495-9). It is also dependent on the presence of other cis-acting DNA regulatory elements in the promoter region (Zhang J, et al., Nucleic Acids Res. 1997, Aug. 1; 25(15):3143-50.) and on the expression of other transcription factors, such as the prostate specific ets-like transcription factor (Oettgen P, et al., J Biol Chem. 2000, Jan. 14; 275(2):1216-25).

With the accumulation of data and knowledge on human gene expression, promoters and enhancers are identified using different algorithms and computer programs (Chapter 8 "Gene Prediction" in Bioinformatics Sequence and Genome Analysis, ed. by David W. Mount. Cold Spring Harbor Laboratory Press, 2001).

Accordingly, we identified (Table LV) promoters in a 5.04 kB 5' upstream genomic region of the 101P3A11 coding sequence using Neural Network Promoter Prediction computer program (http://www.fruitfly.org/seqtools/nnppAbst.html; Reese, M.G. and Eeckman, F.H. (1995) Novel Neural Network Algorithms for Improved Eukaryotic Promoter Site Recognition. Accepted talk for The Seventh International Genome Sequencing and Analysis Conference, Hyatt Regency, Hilton Head Island, South Carolina, September 16-20, 1995), indicated by the underlined sequences in Table LIV. Using a program called SIGNAL SCAN (http://bimas.dcrt.nih.gov/molbio/signal/; Prestridge, D.S. (1991) SIGNAL SCAN: A computer program that scans DNA sequences for eukaryotic transcriptional elements. CABIOS 7, 203-206.), which searches a comprehensive database of regulatory element binding sites, we found numerous transcriptional binding sites for 140 00 various known transcription factors in the 5.04 kB sequence 5' to the 101P3A1 I gene, suggesting the presence of C specific enhancer regions that may mediate tissue specific 101P3AI 1 transcription. Such transcription factors include, but are not limited to, NFAT, NF-1, NF-E, CP2, API, AP-2, Spl, OCT-I, OCT-2, NFKB, CREB, CTF, TFIIA, TFIIB, TFIID, Pit-I, C/EBP, SRF, and various steroid receptors, such as glucocorticoid receptor (GR) and 00 androgen receptors (AR) (Mitchell P J and Tijan R (1989) Science 245: 371). Comparison of the 5 kB upstream sequence of the 101P3A1I gene to the 5 kB upstream sequence of the PSA gene, 5 homologous regions were found that are important for prostate cell specific expression. Table LVI shows the alignment of the these Ssequences and also indicates predicted transcription factor binding sites common to both sequences identified CO using SIGNAL SCAN.

O

Experimentally, one defines the regions in the 5' genomic upstream regions of the 101P3A11 gene using various methods well known in the art, such as deletion and mutation analysis of the putative regulatory regions 00 fused to a transcriptional reporter gene such as luciferase or chloramphenicol acetyl-transferase. These transcriptional reporter vectors are then introduced into cell lines, tissues, or transgenic animals to analyze the tissue and cell type specificity of transcription and expression of the reporter gene. To identify transcription factors and proteins that interact with specific 101P3A11 transcriptional regulatory sequences, one employs one or more of various techniques known in the art such as DNAse footprinting, gel mobility shift assays, and DNA/protein affinity chromatography. Various techniques concerning use of promoters are set forth, U.S.

Patent 5,919,652 which concerns embodiments of nucleic acid compositions that comprise prostate specific antigen (PSA) promoter alone or in combination with a cytomegalovirus (CMV) promoter and related uses.; and, U.S. Patent 6,110,702 which concerns PSA positive regulatory sequence (PSAR) and related uses.

Once regulatory sequences are identified that mediate 101P3A 11 tissue-specific expression, these sequences are employed in various gene therapeutic strategies for cancer, such as driving tissue-specific expression of a toxic gene or a cell suicide gene. Such cell suicide strategies are currently employed using the PSA-promoter enhancer using the thymidine kinase/ganciclovir system (Suzuki S, Tadakuma T, Asano T, Hayakawa M. Cancer Res. 2001 Feb 15;61(4):1276-90). Unlike PSA, which is an androgen regulated gene, 101P3A11 does not exhibit androgen regulated expression. Thus, identification and use of regulatory sequences of the 101P3AI I gene that mediate, prostate-specific, but androgen insensitive gene expression, is useful for the treatment of both early stage androgen sensitive and late stage androgen insensitive or hormone refractory prostate cancer.

Example 53: Generation of PHOR-1 monoclonal antibodies The use of agents to identify the presence of PHOR-1 in biopsy specimens or to neutralize the effect of PHOR-1 has a beneficial effect in diagnosis, prognoosis, prophylaxis and/or therapy. One particularly useful class of anti PHOR-I agents is antibodies, in particular monoclonal antibodies (mAbs) to PHOR-I. Anti PHOR-1 Abs, such as mAbs, are generated that react with the epitopes of the PHOR-I protein such that they either indicate it's presence, disrupt or modulate it's biological function (for example those that would disrupt the interaction with ligands or proteins that mediate or are involved in it's biological activity) or are able to carry a toxin to the cell which is expressing PHOR-1.

The term anti PHOR-I antibody as used herein is to be understood to cover antibodies to any epitope of the PHOR-1 gene product. Diagnostic mAbs, e.g. those used for imaging or immunocytochemistry, comprise those that specifically bind epitopes of PHOR-I protein and thus demonstrate its presence. Therapeutic mAbs 00 S include thbse that are useful for diagnosis but also comprise those that specifically bind epitopes of PHOR-I C"I exposed on the cell surface and thus are useful to modulate growth and survival of cells expressing PHOR-1 by disrupting the function of a cell expressing PHOR-1 and/or disrupting the interaction of cells expressing PHOR-I S and the ligand for PHOR-1.

00 Preferred antibodies which form one aspect of the invention include but are not limited to antibodies named X18(1)4, X18(1)10, X18(1)23, X18(4)7 or prefixed by the number X20 and X47 and derivatives thereof, the production of which is described herein. Hybridomas,respectively, that produce these antibodies were sO deposited with the ATCC on 14 May 2002.

Pathological conditions which are characterized by the presence of PHOR-1 expression include, but are not restricted to, neoplasms of tissues such as. those listed in Table I. One aspect of the invention provides a 00 method of detecting the presence of PHOR-I. A further aspect of the invention provides a method of treatment of conditions characterized by the presence of PHOR-1, comprising administering an effective amount of an anti r PHOR-1 antibody. The administration ofanti-PHOR-1 antibody is particularly advantageous in the treatment of conditions characterized by the presence of PHOR-1.

The antibodies against PHOR-1 for use according to the invention can be from any species, and can belong to any immunoglobulin class. Thus, for example, the anti PHOR-1 antibody for use according to the invention can be an immunoglobulin G, Immunoglobulin M or immunoglobulin A, Immunoglobulin E,.

The anti PHOR- antibody can be from an animal, for example mammaliam or avian origin, and can be for example ofmurine, rat or human origin. The antibody can be a whole immunoglobulin, or a fragment thereof, for example a fragment derived by proteolytic cleavage of a whole antibody, such as F(ab') 2 Fab' or Fab fragments or fragments obtained by recombinant DNA techniques, for example Fv fragments.

Particularly useful antibodies for use according to the invention include humanized or fully human anti PHOR-I antibodies and fragments thereof. These antibodies are produced by any suitable procedure including, but not restricted to, mammalian cell and bacterial cell fermentation systems.

The anti PHOR-1 mAbs are prepared by immunological techniques employing PHOR-1 antigens. Thus, for example, any suitable host can be injected (immunized) with a suitable reagent which makes PHOR-1 available as an immunogen. Immune cells from the host, for example splenocytes or lymphocytes, are recovered from the immunized host and immortalized, using for example the method of Kohler et al, Eur. J. Immunol 6, 511 (1976), or their immunoglobulin genes can be isolated and transferred to an appropriate DNA vector for expression in an appropriate cell type. The resulting cells, generated by either technique, will be selected to obtain a single genetic line producing a single unique type of antibody more commonly known as a monoclonal antibody. Antibody fragments can be produced using techniques such as enzymatic digestion of whole antibodies e.g. with pepsin (Parham, J. Immunol 131:2895 (1983)) or papain (Lamoyi and Nisonoff, J. Immunol Meth.

56:235 (1983)), or by recombinant DNA techniques.

Suitable hosts for the production of Mab's to PHOR-1 include mice, rats, hamsters and rabbits. For example, mice are immunized with a number of different reagents which make PHOR-1 available as a source of antigenic material (immunogen). The route and timing if the immunizations will depend on the source and/or embodiment of the immunogen. Sources of immunogen for PHOR-1 include, but are not restricted to, peptide, protein, fusion protein, DNA, RNA, cells or cell membranes. These can be used separately as immunogens or in combination to produce a specific immune reaction to PHOR-1. The use and application of these various immunogens is described fully in the accompanying examples.

142 00 C, EXAMPLE 54: Generation of antibodies to PHOR 1 using peptide encoding the first 23 N' terminal amino acids of PHOR-1 as the immunoeen.

SIn one embodiment eptides encoding the first 23 amino acids of PHOR-I 00 (MVDPNGNESSATYFILIGLPGLE) (SEQ ID: were generated. These were, synthesized by SigmaGenosys using their custom peptide services. The peptide was synthesized with the addition of a Serine-Glycine-Serine- Glycine-Cysteine (SGSGC) C-terminal linker sequence and then coupled to KLH through the C-terminal cysteine S residue. In this orientation the N-terminal PHOR-I sequence remains free for antigenic recognition.

Balb/c mice were immunized intraperitoneally with 10pg of peptide every 2 weeks over a 4 week period. The initial immunization was given i.p. in Complete Freunds Adjuvant (CFA) and the subsequent two 00 immunizations were given i.p. in Incomplete Freunds Adjuvant (IFA).

To determine the specificity of the response following immunization, mice were bled 10 days after the rC final immunization. Reactivity was determined by Enzyme Linked Immunosorbent Assay (ELISA) using non- KLH conjugated (free) peptide as a target. All five mice had very high titers to the antigen.

Two mice with the highest titers were given a final boost of 10 pg peptide in PBS and sacrificed for fusion 3 days later. Spleen cells from the immunized mice were fused with mouse Sp2/0 myeloma cells using PEG. 1500 according to standard protocols (Kohler et al, Eur. J. Immunol 6: 511 (1976)). Fused cells were plated in 10 96 well microtiter plates and hybridomas were selected using HAT media supplement. Supematants from fusion wells were screened 10-17 days later by ELISA against PHOR-1 peptide. Twenty-one positive hybridomas were identified; these hybridomas are set forth in Table L Table L Clone number O.D. Clone number O.D.

X20(5)1 0.157 X20(5)ll 0.172 X20(5)2 0.511 X20(5)12 0.159 X20(5)3 0.310 X20(5)13 0.244 X20(5)4 0.735 X20(5)14 1.204 X20(5)5 0.160 X20(5)15 0.245 X20(5)6 0.322 X20(5)16 0.220 X20(5)7 0.179 X20(5)17 0.225 X20(5)8 0.173 X20(5)18 0.186 X20(5)9 0.170 X20(5)19 0.176 X20(5)10 1.171 X20(5)20 0.224 X20(5)21 0.502 EXAMPLE 55: Generation mAbs to PHOR 1 Using DNA Immunization.

Therapeutic mAbs to PHOR-1 comprise those that react with PHOR-1 epitopes that disrupt or modulate the biological function of PHOR-1, for example those that disrupt its interaction with ligands or proteins that mediate or are involved in its biological activity. Structural modeling and experimental binding data of the murine olfactory receptor S25 indicates that amino acid residues at the junction of extracellular loop I and transmembrane domain 3, the region of extracellular loop 2 between transmcmbrane domains 4 and 5, and the 143 00 S region of extracellular loop 3 between transmembrane domains 6 and 7 are involved in the binding of the ligand C1 hexanol (Floriano, et al, 2000, Proc. Natl. Acad. Sci., USA, 97:10712-10716).

SThus, in one embodiment, a vector was constructed that encodes the amino acids of extracellular loop 2 S (159-202) between transmembrane domains 4 and 5 of PHOR-1 fused at the C-terminus to the human 00 immunoglobulin GI (IgG) Fc (hinge, CH2, CH3 regions). This construct was used in a DNA based immunization strategy.

Five Balb/c mice were immunized intra-dermally (ID) at the base of their tail. Three immunizations S were given to each mouse of 100g of DNA in PBS over a two week period. To increase the immune response, each mouse was given an i.p. boost of 2 pg of PHOR-I-Fc protein in tissue culture media 10 days after the final DNA immunization. Bleeds were collected 10 days after the final immunization and reactivity in the sera to the 00 middle loop of PHOR-I was tested by ELISA using PHOR-1-Fc fusion protein as a target (test In parallel the sera were also tested on an unrelated human Fc fusion protein (test Specific reactivity to the PHOR-I portion C of the fusion protein was indicated.

All mice were sacrificed and fusions and hybridoma selection was carried out as described in Example 54. Hybridoma supematants were screened 10-17 days later by ELISA using PHORI-Fc protein as target.

PHOR-1-Fc positives were subsequently cross screened on irrelevant Fc proteins to identify PHORI specific clones. A total of 16 positives specific for PHORI-Fc but not reactive to other Fc fusion proteins were identified, these hybridomas are set forth in Table LI.

Table LI Clone number O.D. Clone number O.D.

XI(l)l 0.557 XI(l)lI 0.672 Xl(l)2 0.511 Xl(1)12 1.209 Xl(1)3 0.610 Xl(l)13 0.244 XI(1)4 0.735 Xla(2)1 1.109 0.860 Xla(2)2 .654 Xl(l)6 0.322 Xla(2)3 0.220 XI(1)7 0.779 XI(1)8 0.473 Xl(1)9 0.770 0.541 Example 56: Generation of Monoclonal Antibodies specific to Amino Acids 86-310 of PHOR-1 A fusion protein was constructed that encodes amino acids 86-310 of PHOR-1 fused at the amino terminus to glutathione-S-transferase (GST). This fusion protein, GST-PHOR-1, encompasses sequences that proceed transmembrane domain 3 through transmembrane domain 7 so that all of the extracellular loops of PHOR-I are represented and the only extra-cellular domain that is not represented is the N' terminal. The fusion protein was purified from induced bacteria using standard glutathione affinity chromatography and used to immunize five mice folldwing the protocol of Example 54. The PHOR-1 specific titer of the sera was determined 00 0 cN 00 O O following the fourth immunization (bleed 2) using a fusion protein composed of amino acids 86-310 of PHOR-1 fused to maltose binding protein (PHOR I-MBP), see Table LlI below and Figure 49. The sera from each mouse specifically recognized PHOR-I protein in 293T cells transfected with an epitope tagged PHOR-1 cDNA as assessed by Western analysis (Figure 48). When screened on 293T-PHORI cells compared to 293T-neo cells by FACS several of the sera were positive indicating generation of antibodies specific to cell associated PHORI, see Figure Table LII Mouse Titer,Bleed 2 1 lxlO 2 lxl0 6 3 2x 4 lxl0 5x10 Two mice with high titers were given a final boost of MBP-PHOR-I fusion protein in PBS and sacrificed for fusion 3 days later. Fusion and hybridoma growth selection was carried out as in Example 54. Hybridomas were screened by ELISA against GST-PHORI and cross-screened against MBP-PHOR-1 to identify PHOR-1 sequence reactive clones. 48 hybridomas were identified that exhibited specific reactivity to MBP-PHOR-1.

These hybridomas are set forth in Table LIII.

Table LIII Number Clone number O.D.

1 X18(1)1 0.425 2 X18(1)2 0.445 3 X18(1)3 0.573 4 X18(1)4 0.228 X18(1)5 0.218 6 X18(1)6 0.333 7 X18(1)7 1.459 8 X18(1)8 0.260 9 X18(1)9 0.253 X18(1)10 0.282 I11 *X18(1)11 0.362 12 X 18(1)12 0.343 13 X18(1)13 0.261 14 x 18(1)14 0.773 X18(0)15 0.631 16 X18(1)16 1.427 17 X18(1)17 0.372 18 X18(1)18 0.657 19 X18(1)19 0.677 X 18(1)20 0.338 21 X 18(1)21 0.398 22 X 18(1)22 0.232 23 X18(1)23 0.560 24 X 18(1)24 0.554 X 18(1)25 0.442 26 X1 8(4)1 0.848 27 X18(4)2 0.420 28 X18(4)3 0.230 29 X18(4)4 0.333 X18(4)5 0.389 31 X18(4)6 0.264 32 X1 8(4)7 0.358 33 X 18(4)8 0.669 34 X18(4)9 0.429 X18(4)10 0.253 36 X 18(4)11 0.277 37 X18(4)12 0.536 38 X18(4)13 0.662 39 X18(4)14 0.344 X18(4)15 0.256 41 X 18(4)16 0.212 42 X18(4)17 0.304 43 X18(4)18 0.531 44 X 18(4)19 0 .286 X18(4)20 0.472 46 X 18(4)21 0.770 47 X18(4)22 0.877 48 X 18(4)23 0.450 00 0 Four hybridomas reactive to MBP-PHOR-1 exhibited strong specific reactivity to PHOR-1 protein C- expressed in cells. This was demonstrated by Western analysis of 293T cells transfected with the epitope tagged PHOR-1 cDNA (Figure 47). The positive clones are indicated in bold, namely 18(1)4; 18(1)10; 18(1)23; and, 18(4)7. Hybridomas expressing respectively, 18(1)4; 18(1)10; 18(1)23; and, 18(4)7 were deposited with the 00 ATCC on 14 May 2002.

Example 57: Activation of 101P3All.

NO It is possible to measure the constitutive and ligand-mediated activation of 101P3A 11 using the cAMP accumulation assay mentioned in example 44 above or by measuring the binding of the GTP analog, namely [35S]GTPyS binding is generically applicable to all GPCRs; and occurs proximal to the membrane 00 surface, where the GPCR is located. Preferably, a GPCR:Fusion-Protein is utilized for these assays. The assay utilizes the ability of G protein-coupled receptors to stimulate [35S] GTPyS binding to membranes expressing the relevant receptors. Therefore, the assay may be used to directly screen compounds and antibodies for their effect on the activation of 101P3AI 1.

A scintillation proximity assay can be utilized to monitor the binding of[ 35S] GTP7S to membranes expressing 101P3AI l-Gs-Fusion Protein (expressed in 293 or 3T3 cells). In brief, membrane proteins are incubated with [35 S]GTPyS and GDP for 60 minutes. The assay plates are-counted in a scintillation counter.

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 (WWW.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

00

TABLE

Normal Tissues: 00 Prostate Ovary (by RT-PC 1: Tissues that Express 1OIP3AII When Malig nant Ronly) Rectum Prostate Colon C1 Kidney 00 Breast Uterus Cervix Stomach nt Tissues: Metastases Pool TABLE 11: Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Lcu leucine; S Ser serine Y Tyr tyrosine C Cys cysteine W Tip tryptophan P Pro proline H His histidine Gin glutamine R Arg arginine I le isoleucine Met methionine Thr threonine, NAsn asparagine Lys lysine Val valine Ala alanine DAsp aspartic acid Glu glutamiic acid G Gly glycine 00 TABLE III: Amino Acid Substitution Matrix Adapted from the GCG Software 9.0 BLOSUM62 amnino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See URL 00 www.ikp.unibc.chimnual/blosum62.html) A C DE F G H- I K L M N P Q R S T V W Y.

4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C 6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F 6 -2-4 -2-4 -3 0-2-2 2 0 -2 3 -2 -3 G C18 -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 1 1 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1I L -2 -2 0 -1 -1 -1 1 -1 -1 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 Q -1-1 -3-3 -2 R 4 1 -2 -3 -2 S 0 -2 -2 T 4 -3 -1 V 11 2 W 7 Y TABLE IV HLA Class I/lI MotifslSupermotlfs TABLE IV lILA Class I Supermotifs/Motifs SUPERMOTIFS -POSITION POSITION

POSITION

2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primry Anchor) Al TIL YMS A2 LIVMA T2

IVMATL

A3 VSMATLI A24 YFWIVLMT

FYWLM

B7 P

VILFMWYA

B27 RHK FYL WMI YA ED

_F"WYLIMVA

B58 ATS _________FWYLJVMA B62 QLIVMP ________FWYMIVLA Al TSM Al DEAS Y A2.1 LMYQ1A T

VL!MAT

A3 LMVISATFCGD All VTMLISAGNCDF

YH

A24 YFWM

FLIW

A*3101 MVTALIS A*3301 MVALFIST A*6801 AVTMSLI

RIC

B30702 P

LMFWYAIV

B3*350l P _________LMFWYIVA B51 P ________LIVFWYAM B*5301 P

IMFWVYALV

B3*5401 P ________ATIVLMFWY Bolded residues are preferred, italicized residues arc 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 lILA Class II Supermotif 1 6 9 W, F, L A, V,1, L,P, C, S,T A, V, 1, L, C, S,T,M, Y 2008200363 18 Jan 2008 TABLE IV HLA Class 11 Motifs MOTIFS P anchor 1 2 3 4 5 P anchor 6 7 8 9 DR4 preferred FMYL)'VW M T I VSTCPA4L!M MH MIH deleterious W R WDE DRI preferred MFLIVVWf PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MffLJVWY M W A IVrMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 10 anchor 1 2 3 1 anchor 4 5 1P anchor 6 motif a LIVMFY D preferred motif b Lrv'MFAY DNQEST KRH preferred DR MFL!VWY VMSTACPL! Superniotif Italicized residues indicate less preferred or "tolerated" residues 2008200363 18 Jan 2008 TABLE IV liLA Class I Supermotifs POSITION: 4 5 6 7 8 C-termninus

SUPER-

MgTIFS I Anchor TIL VMS 1V Anchor 1P Anchor

FWY

10 Ancho A3 preferred P j rh YFW YEW YEW P 1V Anchor VSMATLJ (415) RK deleterious DE DE P (515) A24 1P Anchor P Anchor YF9WIVLMT FlY WLM 137 preferred FWY 10* Anchor FWY FWY l 0 Anchor LIVM P VILFMWYA deleterious DE DE G QN DE (415) (415) B27 P Anchor l 0 Anchor pj{K FYLWMIVA B410 Anchor 11 Anchor ED FWYLIMVA B58 1P Anchor 10 Anchor ATS FWYLIVMA B62 1P Ancho PO Anchor QL!VMP FWYMIVLA Italicized residues indicate less preferred or "tolerated" residues 2008200363 18 Jan 2008 TABLE IV lILA Class I Motifs POSITION: 1 2 3 4 5 6 7 8 9 C-terminus or C-terminus Al preferred GFY Il*Anchor DEA YFW P DEQN YFW l 0 Anchor 9-mer W STM y deleterious DE RHKLIVMP A G A Al preferred GRHK ASTCLIVM l 0 Anchor GSTC ASTC LIVM DE l 0 Anchor 9-mer DEA4S y deleterious A RMKDEPY DE PQN RHK PG GP

FW

Al preferred YFW l 0 Anchor DEAQN A YFWQN PASTC GDE P l 0 Ancbor 1 0-mer STM y deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A Al preferred YEW STCLIVM l 0 Anchor A YFW PG G YEW I'Anchor 1O-mer DEA4S

Y

deleterious RJFiK RHXDEPY P G PRHK, QN

FW

A2. I preferred YEW I *Anchor YFW STC YEW A P 1 0 Anchor 9-mer LMJVQAT VLIMA T deleterious DEP DERKH RKH DERKH Italicized residues indicate less preferred or "tolerated" residues 2008200363 18 Jan 2008 TABLE tV HOLA Class I Motifs, continued POSITION: 1 2 3 4 S 6 7 8 9 C-Terminus A2.1 preferred AYFW l 0 Anchor LVEM G G FYWL I *Anchor 1O-mer LM!VQA vim VLIMA T

T

deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK M 0 achor YEW PRHKYFW A YFW P l 0 Ancbor LIMSA KYRHiFA

TFCGD

deleterious DEP DE All preferred A I*Anchor YFW YFW A YEW YFW P l 0 Anchor VTLMIS KR YH

AGNCDF

deleterious DEP A G A24 preferred YFWRHK I 0 Anchor STC YFW YFW I Anchor 9-mer YFWAI FLIW deleterious DEG DE G QWP DERH G AQN

K

A24 preferred l 0 Anchor P YFWP p I *Anchor lO-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 preferred RHK l 0 Anchor YEW P YEW YEW AP l 0 Anchor MVTAL!S RK deleterious DEP DE ADE DE DE DE A3301 preferred l 0 Anchor YEW AYFW l 0 Anchor MVALF1 RK

ST

deleterious GP DE Italicized residues indicate less preferred or "tolerated" residues 2008200363 18 Jan 2008 TABLE IV lILA Class I Motifs, continued POSITION 1 2 3 4 5 6 7 8 2 C-Terminus A6801 preferred YFWSTC l 0 Anchor YFWLTV YEW P l 0 Anchor AVTMSLI M

RIC

deleterious GP DEG RHK A B0702 preferred RIHKFW I*Anchor RHX REIK RHK RHK PA l 0 Anchor y P

LMFWYA[V

deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV l 0 Anchor FWY FWY l 0 Anchor MP

LNMFW'Y!V

A

deleterious AGP G G 1 preferred LIVMFW I *Anchor FWY STC FWY G FWY I *Anchor Y P LIVFWYA4M deleterious AGPDER DE G DEQN GDE

I{KSTC

B5301 preferred LIMFW l 0 Anchor FWY STC FWY LIVMFWY EW Il*Anchor Y p

IMFWYAL

deleterious AGPQN G RHiKQN DE B5401 preferred FWY I *Anchor FWYL LIVM ALWV FWYAP 1 0 Anchor P IVM

ATIVLMF

WI,

deleterious GPQNDE GDES RHKDE DE QNDGE DE

TC

Italicized residues indicate less preferred or "tolerated" residues. The information in this Table is specific for 9-mers unless otherwise specified.

able V-1OlP3A11-V1-A1-9mers [StartjSubsequencel Score je 211T] GLDSLLISF J125.00]IE 1 [2 NVD)PNGNES ]5.00o 3 22 EA 213 DSLLISFSY ]1(3.7501 5 42 VLGNLTIIY 1 2.5011 6 26 HVCAVFIFY J250 7 1 __2IMES soA12.S1 58eI LHEPMYIFL 12.250J 9 NESSATYF 220 LSMVHRFSK I j] 11 STVLLAMAF 1.2501 12 191~[ CDIRVVI1. 000[ 13 F1 59 MAPLPVFIK 11.0001 14 LISTSSMPK 1 .000j 15 13 RSNILSHSY 0.516 79 SSMPK4LAI 0.7 17 71 GIDILISTS 110.5001L 18 135IHATVITmPR~~so] 19 l'i LLAAMAFDRY 1osol 20 117~I VLLAMAFDR Ios] 21 (51NLTIIYIVR 1051 22 [REAA1I 0 O 23 181 YCLHQDVMK 0.40011L 24 138_ VJ LPT 110.4001[ 25 [184 H1 DV0.375] 26 139J LThPRVTKI 1.5] 27 6 1NGNESSATY j0.2501 28 53 RTEHSLHEP 1(T~j 29 IFIGLSMVHR ]Lo.200 30~ f 7 SLHEPMYIF 3 I23 CVSHVCAVF 110.200 1 32 146 1KIGVAAVVR 10.20011 I126 YVAICHPLR 10201______ 2 57 ]ALMAPLPVF 020I 35 249 JjAVFIFYVPF 10. 200-[ 36 288J VLNPIVYGV I0.200f 37 16 ~APLPVFIKQi0151 38 1241LPVILANIY 10. 12 39 I96 QFDACLLQI i 0. 1251 40 [121 ATYFILIGL 10251 41 272 SPLPVILAN 42 284 43PLP iT~ 18 LMAPLPVFI~o 0 44 Table 1O1P3A11-V1-A1-9mers IStartJ Subsequence Scr ~e.ID Num] .9 ESSATYFIL 0IO.075]j4 NSTTIQFDA]1005] 48 IijfISFSYLLIL 11~~1 49 lF 51_HSL-HEP-MYI 10. 0751 291FSYLLILKT 0.075hI 71_ DSPLPVILA 0.075 52 78 TSSMPKM~LA 110.07511 14 13 PVFIKQLPF 57.001 ___YIFLCML.SG 11.00 I46 ][LTIIYIVRT 11 05 60 3 j182 3(CLHQDVMKL 0.05[ 61 3 f rT~[FIKQLPC 0: i 62 3 289 3[ YGVK 1 ~j 63 269_ RRDSPLPVI LO 64__ ___LISFSYLLII] T] 65 1EAQAKAFGTT I.iTo]L 66 3 [TT[VAICHPLRHJ 61 1 67 1 66][LCMLSGIDI 1 68 [4[MLAIFWFNS I 69 ___DACLLQIFA 1000~ 7 155 1[ EHSLHEPMY 110.050 1[ 71 ___ILANIYLLV I: ool 72 1~~1GTCVSHVCA I0. 050 1 73 1211LGLEQF 7.4 FTT1LLQIFAIHS F136 ATVLTLPRVJ 10. 050 76 I283 j[LLVPPVLNP 0o.osl 050 77 15 [AALMAPLPV o0.05011 78 FIFYVPFIG o1.osoll 26T1 SMVH'RFSKR I1~sj 80 I99 ACLLQIFAI 11.5 81 11 SATYFILIG 050 82 NVVYGLIVI 10.o050lf_ I_ LCSLIA-Iv 10n05011 83 I~h MLSGIDILI 11.80J VT h AVLGNLTI 0.050I 8 IISAIGLDSJ 01.050 87 20 IIVIISAIGL 10.050 ii 88 28 SFSYLLILK 1.s) 89 1I~IIKLCDIRV11.001 9 'Table V-11P3AJ1-V1-A1-9mers [StartjlSubseqaence[ Score JSeq._ID Num 1 AIGLDSLLI 1Ij s] 91 F27 VCAVFIFYV Ios][ 92 ___TTIQFDACL] 0. 050J[ 93 __TKEIRQRIL] 0.045i 95 CSLYLIAVL 11 9 MPK Irj 31 97 F~TIESTVLLAMA] F 98 24 [VSHVCAVFIJ 92 STTiQFDAC 11.051 100 ITable V:1OI1A1-V2-A1-9mer I rjSubsequence SreSeq ID. Numl 1 I1 VLASGVTLR 0.1001 101 I11 SSWPISICW 0.0751 102 ISIWFLLC .0751 H10 SICWLLrz0.05:10 28 LLCSTQLSM 000 105 ___LASGVTLRC 000 106 ___RCPSSWPIS 020 107 1771AVLASGVTL I 108 3 YLIAVLASG 110,0201 109 11 jj CPSS.2 110 [1 1I SLYLIAVLA 0O.02 Ill E16 CPSSWPISI 10.0131 112 1 27 _FL-LCSTQLS J0. 010 113 51 IVLSGVT 10.010] 114 VTlLIAVLASGVT0.0101 115 14 11LRCPSSWPI .0 116 __21 PISICWFLL H0.005[117 1 V 19 ISCWF0 .005 118] 0.003F119 _I [2 11LYLIAVLAS JijjjJ 120 12 VTLRCPSSW Ii~j1 121 SGVTIJRCPS 1003 122 ASGVTLRCP 0.002 13 17 11PSSWPISIC 10.002 14 I ICWFLLCST 0.005 29 1LCSTQLSI4E12 IF 2 51 CWFILLCSTQ 10.o0011 127 2671 WFLLSTQL 10.ooNJ 128 13 0.000_ 129 Iable V: 1O1P3A11-V3-A1-9mers I StatSbseuene Sore Seg ID Nuil Ii~QFDCLLQ 012 130 F37EIDACLLQMFA 0F. 0501[ 131 141ALLQMFAI 0.05013 _jLQMAIH~ 1341 L~IFDACLLQMFI 1-05I36Z I 1_MFAHSLSG '0.0031 137]3 V 1 LQMFAIHSL0.02 j- FTable VI; 1O1P3A11-V1-A1-10mers [Start jSubsequence I. Num FTI7LM.APLPVFI1Isoo 4 1 7 rIMVDPNGNESSII1 o 140T- 1~9I[SMPKLAF 13.0001 141 El-fcII92vv 2.5001 142 1 VTFIAVIGNLIIY I3~p~j 143 [F 23 LEEAQFWLAFI.5 14 4 ____TKIRQRILR 1220 145 [3 [RTEHSLHEPM 1220 146 r[587ILHEPMYIFLC 2250 1.47 ~IVLNPIVYGVK 200 149 I IGLSMVHRFSK 1.00 1 74 ILISTSSMPKl 10001 12 22IGLEEAQFWLAL 1 153 11 J !ESTVLLAMAFI.70 154 24 jVHVAFIF 0.5 155s ?V285V FIII.2 156 1 391 11 ACDDIRVNVV 10.500O~ 157 117 1 LAAHRY] iS58 21 1 GD LIS!FJ 10.-5001 %FDR 160 171 R IIISTSSJ 16 3 Wh1 LAFPLCSLYL .0 163 ET GNEISSATYFI 11045 L 164 ITTl TVIJTLPRVTK .400 165 56 1 HSLHEPMYIF 11030 166 139 1 LTLPRVTKIG 0O.250] 16 F 0.00 169 24J[LVPVLNPIV 10 170 2 48 j[CAVFXFYVPFII0.200H 173__ able VI: 102P3A11-V1-A1-10mers rrabe VI 1013A11V1-A-lomrs Table VI: 1O1PA11-V1-Al-lmers Start Subsequence Score Seg ID. Num 719 IGLPGLEEAQF 0.200 174 26 iLSMVMRFSKR I0.150 I[75 162 IKLP 0.125 177 44 GNUTIIR0.2 7 771 STSPKML 0 .1TJ25E 179 245 IISHVCAVFIFY 0.1251 180 1 [LP'LP~~o2121 LDSLLISFSY 0.12 181 1 182 257 ][FIGLSMVHRF1 0. 1001 183 232 ][TREAQAKAFGII.9 184 9 ][ESSATYFILIIIF 185 SSATYFILIG 186i~,.

69 LSGIDILIST 0.075 1~j8I 271DSLPIFUT0.075 L188_ 78 TSSMPKY.UAI 0.075 189 272 SPLPVILANI 0.050 90 216 [LISFSYLLIL 0.05 19 273 ][PLPVILNIYIIO 192EI 224 ILKTVLGLTRJI0.050 193 1L26 YVAICHPLRHIO.050 194 106 H AIHSLSGMESJI.5 195 1T99 IIVVYGLIVIIS 10.050 196 269 RPSPLPVIL 0. 05 0 197 94[TIQFDACLLQ~j.5 198 2S [EAQFWLAFPL]I. 50 199 9 STTIQFDACL 200oso 215 1[LLISFSYLL1I0.05020o1 39 LALN 110.0501 204 205 V~IISAIGLDS] 0.050 205 159 jfMAPLPVFIKQ 10.050 206 68 IMLSGIDILIS ]O.050~ 207 36 [SLYLIAVLGN 10.050 20 100 ICLLQIFAIHS 10.050 209 SLHEPI4YIFIII0.050 1 651FC4SGD1000 21 203 ILIVIISAIGL 0.050 212 276 VILANIYLLV 0.05 261 JSMVHRFSKRR 0,0501 1 208 [SAIGLDSLLI 0.050 215~ ILSHSYCIHQ 0.0501 217 290 INPIVYGVKTK 0.-0501 218 S~~trtSubsequenceIScore ISeq ID. NUm 196] RVNVVYGLIV o ioo 1 1 SATYFILIOL 0.05 [98] DACLLQIFAI0.5 22 I222 LLILKTVLG3L1005 23 181 JYCLHQDVMKL 0.050 Jl2241 229 WL50LiQAK 0.05 F103 JIQIFAIHSLSG 200 227 209 AIGLDSLLIS 0.5 228 35 CSLYLIAVL. 0.030 229 110ILSGMEFWSTV 0I.025 230 2 j[GTCVSHVCA 232 [172 FCRSNILSIHSY 0.025 23 270 JRDSPLPVIJA 0.02-51 3 6 jfGNESATF 0.025 ~~lcrvGLTREA1 251 236 S 13I ATVLTLPRVT 0. 0251 238 rable VI:101P3A11-V2-A1-10mers Str]subsequence Score lSeq ID. Newl 19 ]jSSWPISICWF 0.150 239 71 1o 240 SCFC 231 F28 QLCTLSM 10.0-501 -242 1 2 ISLYLIAVLASH0.0501 243 8 ]VLA.VTLRC 10. 05 0 1__4 16 RPSPISI 0..050 245 1 jCSLYLIAVLA 0.030 24 6 R IAVLASGVTL H 0.0201 247 10 jASGVTLRCPS 0.015H 248 21]WPISICWFLL 0.013 249 [T]GVTLRCPSSW 010 250 12411 SICWFLLCSTE 2510Z~Z 4 YLIAVLASGV HO.o1011 15ILRCPSSWPIS 01.010 5 hi LAVI.~qsVr 0.1 254 f18 JjPPwI-T. SICW 11 22 PIXWLC0.0051 256 1 HJSGVTLRCPSS 0.005 J 257 [17 1'ICMPSS -sc 0.003 258 (~TDLCSQLSM 11.00 259 14J~TLRCPSS WPI pjf 260 fable VI:101P3A11-V2-AI-lmers ~Starts-ubse uence Fco eqe ID. NewJ R 25IICWFLLCSTQ 261 9 .0 11 262 13 LYIVAG1001 263 [~~fWFLCTQL 10.001r 264 ICWFLLCSTQL 0JI 071 265 WPISICWFL 000 266j1 13 11 VTLRCPSSWPI 0.0 267l j Iable VI:1O1PA11-V3-Al-lmers IStartjsubse uencel Ecoeq I. um I2 [QFDACLLQMFJ 0. 1 268 4 DACLLQKFAI 0.0501 269 6 [LLQMFAIHS 0.050 270 1..LJQMF11AIHSLS9JG 021 271 7j LLQMFAIHSL 010 273 ___IQFDACLLQMj 2.0 7 4~ 1 3 JFDACLLQMFA 10. 003 L_275 Ia I[IQMFAIHSLS 0.0 276 110 jMFAIHSLSGI4 10. 001 277 trable VII: 1O1P3A11-V1-A2-9mers iStartjfSubaeuc ScreSe ID INumj TTifSLLISFS-YL 825.977H 278 28 LPVG 2198 29 F 18 I ~CDDIRV J243.432] 28 0 I~T WLAFPLCS 22.0141 281 247 ]I VCAFIFYV Q215.1921 28 2 S22 11 G.LEEAQFWLj 178.8151 283 ___777 ILANIYLLV 1Lk7 7. 3 58 J 284 221 TTIV- 14 9071[ 285 215 ]jLLISFSYLL] 13.0L12861 1 15 11 FILJIGLPGIJ1I114.952Zi87-- LMAPLPVFI ]7050 L2e88 FjNIYLLVPPV 2890387] -89 [109 SLSGMESTV j~6.5 290 [8 JjKMLAIFWFNl 1~5.2] 291 CLHQDVMKL 11 49.134 ]1 292f 61AQFWLAFPL]~ 4 6.480 293 ___LILKTVLGL 42.94[ 294 [276 I VIAIYLL 4244[ 295 1_38_1 YLIAVLGNL 1129.3821 29 6 171 CMLSGIDIL H126.3771 9 ITable VII:1O1P3A11-V1-A2-9mers [startjlSubsequence[ Score Seq. ID Num' F119 J1 LAMAFDrRYV] 25.398 1 298 F202EGLIVIISAI ][399J 299 [30771 RLFHVATHA F[18.382][ 300 [81MLSGIDILI ]17.736[ 301 [~6lAIFWFNSTT ][14.407] 302 [oIYIVRTEHSL ]13 .1ST! 303 [F-175 NIL SHSYCL ]o.6 304 [T~IQLPFCRSNI 0.433][ 305 flFIPMYIFLCML ]943)[ 306 10!LQIFAIHSL ]8.6J[ 307 25 VPFIGLSM4V IF 6.56 12 iMAFDRYVAI ii5.605 309~ i IMLAIFWFNS 1 1 19 VVYGLIVII 3~ 4.811 161 PLPVFIKQL7] 4.08j 3-12 305 IL HAT 13.659 313 21 jLISFSYLLI ]E3.58 314 156 11 AALMAPLPV 113.574 1 284 LVPPVLNPI 356 316 99 ACLLQIFAI]331] 317 17L1 AVLGNLTII 315] 318 204 JjIVIISAIGL 11 3.178 1_ 319 94 TIQFDACLL 2.37F 320 FPLCSLYLI 266 321 ___ATVLTLPRV 222 322 IIATYFILIGL 218 323 ___SGMEST VLL 215 324 1~~I LTLPRVTKI 209 325 [~iIYPFGLM 21 0 326 34 RILRLFHVA 196 327 ILCSLYLIAVJ 1.775 328 LIAVLGNLTH 1.742 329 7T1911 FSYLLILKT 330 44 GNLTIIYIV 111.584 ]~331 [-h1 AIADRYVA II1.4711_ 332 ILGNLTI IYI ]1.465 ]1 333 209 AIG D 1.35 334 2 1 I YGLIVI ISA 127 33.5 282 1LVPIJ .6 336 93 TTIQFDACL 117IL NESSATYFI7 1..11 338 242 0TCVSHVCAV 104 339 1987 NVVYGLIVI 081 340 217 ISFSYLLIILLA3 341 191 1ACDDIRVNV]f j 342 7 00 00 00 Table VII:1O1P3A11-V1-A2-9mers Start Subse ence Score Seq. ID Num 105 If FAIHSLSGM 0.730 343 244 VSHVCAVFI 0.637 344 79 SSMPKMLA 0.580 345 117 VLLAMAFDR 0.544 346 112 G4ESTVLLA 0.

5 28 347 273 PLPVILANI 0.528 348 CSLYLIAVL 0.4 87 349 101 LLQIFAIHS 0.481 350 SGIDILIST 0.459 351 143 RVTKIGVAA 0.435 352 151 AVVRGAALM 0.435 353 66 LCMLSGIDI 0.428 f 354 251 FIFYVPFIG [0.415 355 46 LTIIYIVRT 0.405 356 63 YIFLCMLSG 0.401 357 113 MESTVLLAM 0.378 358 169 LPFCRSNIL 0.360 359 LAIFWFNST 0.334 360 227. TVLGLTREA 0.322 361 154 RGAALMAPL 0.321 362 197 VNVVYGLIV 0.316 363 285 VPPVLNPIV 0.316 364 128 AICHPLRHA 0.314 365 150 AAVVRGAA_ 0.297 366 208 SAIGLDSLL 0.297 367 77 STSSMPKML 0.297 368 57 SLHEPKYIF 0.288 369 207 ISAIGLDSL 0.267 370 157 ALMAPLPVF 0.260 371 IAVLGNLTI 0.246 372 36 SYLIAVLG 0.238 373 18 IGLPGLEA 0.230 374 275 PVILANIYL 0.226 375 187 VMKLACDDI 0.220 376 100 CLLQIFAIH 0.215 II able VII:101P3A11-V2-A2-9mers Start Subsequence Score Seq ID. um WPISICWFL 26.460 378 4 11 LIAVLASGV 16.258 379 1 SLYLIAVLA 15.898 380 28 LLCSTQLSM 8.446 381 6 AVLASGVTL 6.916 382 24 ICWFLLCST 1.579 383 27 FLLCSTQLS 1.268 384 able V1I:101P3A11-V2-A2-9mers Start Subseuence Score I[Seq ID. Num 3j YLIAVLASG ]10.7881[ 385 21 PISICWFLL 0.637 386 26 WFLLCSTQL 0.252 387 8 LASGVTLRC 388 22 ISICWFLLC _f 389 23 SICWFLLCS 0.090 390 16 IfCPSSWPISI 0.068 391 7 VLASGVTLR] 0.058 392 14 LRCPSSWPI 0.018 393 5 IAVLASGVT 0.009 394 11 GVTLRCPSS 0.007 395 [2 VTLRCPSSW 390.007 I 13-1 TLRCPSSWP 390.006 18 1 SSWPISICW 0.004 398 177 PSSWPISIC 390.0011 10[ 1 SGVTLRCPS F50.000 I 400 29 LCSTQLSME IfT01 401 1 15 RCPSSWPIS FT00l 402 121 LYLIAVLAS 1 J00[ 403 Fs wpsISICwF 1[TiI 404 1 ASGVTLRCP 01-0 I1 405 25 CWFLLCSTQ O.L2.OOQf 406 able VII: 11P3A11-V3-A2-9mers Start Subsequence Score Se ID.Num 7 If LQMFAIHSL I31.3341 407 411 ACLLQMFAI Ir 33 408 LLQMFAIHS 0.481 409 5 CLLQMFAIH 015 410 8 1LS 0.19911 411 3 1 DACLLQMFA 110.02811 412 1 QFDACLLQM 110.003 413 2 IIFDACLLMF If .ooi1f 414 9 MFAIHSLSG H. -000 415 able VIII:1O1P3A11-V1-A2-lomers Start ISubsequencel Scr seg. ID Num I..57 1( SLHEPMYIFL 11722.58311 416 118 If LLAMAFDRYV 494.237 417 214 SLLISFSYLL 300.355 418 4 IfVLGNLTIIYI 224.357 419 157 IfALMAPLPVFI212.307 420 251 FIFVPFIGL 194.9871 421 276 VILNIYL L O.2311 422 222 LLILKTVLGL][83.527 I TableVIII:102P3A11-V1-A2-10mer5 Start ISubsequence TIFrlI n 7 101 LLQIFAIHSL 1 83.527 424 140 TLPRVTKIGV 69.552 425 254 YVPFIGLSMVjf64.3881[ 426 IQFDACLLQIJ[62.741[ 427 246 HVCAVFIFYV]57.690 428 63 YIFLCMLSGII56.155 429 6 I FLCMLSGIDII47.9911 430 249 AVFIFYVPFI 42.72711 431 283 LLVPPVLNPI][40.792 432 138 VLTLPRVTKI 40.792)[ 433 38 YLIAVNLT 34.279 434 f3i KAFGTCVSHV] 28.772 F 435 67 CMLSIDILII27.879 436 135 AQAKAFGTC126.797 E 37 F215 JLLISFSYLLII26.6041 43 1 83 J[ KMLAIFWFNS 26.114 439 F-847J MLAIFWFNST 24.070 440 22 J[GLEEAQFWLA 18.5761 441 NLTIIYIVRTI17.14o 442 219 FSYLLILKTV 15.371]_ 443 304 RILRLFHVAT 14.407 444 143 RVTKIGVAAV]13.9971 445 167 KLPFCRSNI 13.69e 446 182 CLHQDVMKLA .11.4261 447 120 AMAFDRYVAI 11.302 448 LAFPLCSLYL10.2641 449 109 SLSGMESTVL 8.7591 450 168 QLPFCRSNIL 8.759 451 228 VLGLTREAQA] 8.446 452 302 RQRILRIFHV 7.149 53 190 LACDDIRVNV 6.733 454 206 IISAIGLDSL 5.628 455 181 YCLHQDV?4KL1I 5.459 456 86 IAIFWFNSTTI 5.308 .457 243 CVSHVCAVFI 5.021 4S8 203 LIVIISAIGL 4.993 459 230 GLTREAQAf(A 4.968 460 L292 IVYGVKTKEII4.966 461 216 LISFSYLLILI4.709 462 160 APLPVFIKQL 4.510 463 284 LVPPVLNPIV 4.242 464 280 NIYLLVPPVL 3.854 465 26 AQFWLAFPLC 3.541 466 33 PLCSLYLIAVI 3.519 467 299 KEIRQRILRLIL 3.344 468 ill able VIII:101P3A11-V1-A2-10mers StartjSubsequence Score Seq. ID Num 75 LISTSSMPKM 469 201 YGLIVIISAI 2.666 470 196 RVNVVYGLIV 2.495 471 39 LIAVLGNLTI 2.439 472 241 GTCVSHVCAV 2.222 473 12E]LILKTVLGLT 1.927 474 121 MAFDRYVAIC 1.678 475 21 PGLEEAQFWL 1.485 476 8 NESSATYFIL 1.482 477 17 LIGLPGLEEA 1.309 478 274 LPVILANIYL 1.304 479 221 IYLLILKTVLG 1.268 480 [TT PVLNPIVYGV 1.139 481 111 SGMESTVLLA 1.132 482 92 STTIQFDACL 1.127 483 100 CLLQIFAIHS 1.048 484 279 ANIYLLVPPV 1.044 485 128AICHPLRHATI 1.025 486 [T5 GAALMAPLPV 0.966 487 I0 FNSTTIQFDA 0.865 488 198 NVVYGLIVII 0.861 489

I

3 J REAQAKAFGT 0.840 490 129 ICHPLRHATV 0.772 491 43 LGNLTIIYIV 0.728 492 191 ACDDIRVNVV 0.702 j 493 VT SATYFILIGL[ 0.682 1 494 85 LAIFWFNSTT 0.669 495 188 MKLACDDIRV 0.608 496 282 YLLVPPVLNP 0.583 497 272 SPLPVILANI 0.580 9 36[SLYLIAVLGN 0.548 499 112 GMESTVLLAM 0.528 500 149 0VAAVVRGAAL F[0.504 501 163 IPVFIKQLPF][ 0.448 502 117 .11 VLLAMAFDRY][ 0.436 503 151 AVVRGAALMA 0.435 504 66 LCMLSGIDIL 0.405 505 213 DSLLISFSYL 0.404 506 113 MESTVLLAMA 0.378 507 211 GLDSLLISFS 0.377 508 1 ][MMVDPNGNES 0.375 509 29 WLAFPLCSLY 0.343 510 32 1 FPLCSLYLIAII 0.339 It 511 60 iEPMYIFLCMILj[ 0.338 512 93 TDACLLS 0.297 513 ~able VIII :lOlP3All-V1A210mrs Start IISubeg-ue-n-eI score SeI~ D Num1 [SMPKI4LAIF1,:]~ 0.2961 jZ I RLFHVATHAS i Table VIII: 1O1P3A11-V2-A2 -10mers Start Subsequencel score IISe ID Num] 4 YLAVLSGV31993911 516 28 FLLCSTQLSM 84551 517 3 8 VLASGVTLRC if 8.446 I S 1 21 1WPISICWFLL 6.325 II 519 34 TLRCPSSWPI 5. 95 52 24 43 SIWLCTj 520 2 7LYLIjAvLAs~l0.5 8[ 5E 22 [6 ][IAVLASGVLf 0.504 523 IfSWPISICWFL1I0.1-221 524 ]IICSLYLILAII 0.120 ]LIAVLASGV~rI[ 0.093 526 1 6 I RCPSSWPISI 0.068] 527 29 LLCSTQLSME [.0.058 If 528 19 IfSSWPISI CWF 0 .051] 529 1 17 ]CPSSWPISICII 0.031]f 530 22 JPISICW.FLLC1 0.029 11 531 7 jAVLASGVTLR If0.011 532 26 ]CWFLLCSTQLIL0.01 533] 23 ]ISICWFLLCSII007 Ls34 I 13 IfV'LRCPSSWPj 53 1[ IcWFLLCSTQj IF0.0011 3 27 WFLLCSTQL~sfl 0.001 _f 538 11 1 SGVTLRCPSSj( 0. 000 Ifj 539] 1 ASGVTLRCPSII.0] 540 9 ILASGVTLRCPi 0.000 If 541 3 ILYLIAVLASG 1 0.000 If 542 ILRCPSSWPISI O.ooo] 5 4 3 18 0 PSSWPISICW 11~ 0]00 5 able VIII :1OlP3A11-V3-A2-l0mers Start Sus eque;-c~Scor eq ID. Num 7 1 LLQMFAI HSL 18 3 -7 545] L1 11 IQ)FDACLLQM 29.8677I[ 546 CLLQFAIHSI[] ij 547 3 IfFDACLLQMFA[10. 175I 549 4 ][DACLLQMFA1 0. 145 550 a[LQMFAIHSLS:] 0.048 551 I MFI!SLSGM110.0n13-41[ 552 ACLLQMFAIH~r 001& 7 1IQFDACLLQT~MF fo001 5 ,4 1 fablc IX:.1O1P3Ai1-V1-A3-9n1CrG E230 11 GLAQAK]j 0 j 555 -1 13 i VTLPRVTK]30.000j 556 211 ifGLDSLLISF 118.000j 558 261 ifSMVHRFSKR 118.o]559 117 ifVLLAM4AFDR 13.o00 56 45 I YIVR12.000J 561] 11 LMFDRY H200 562 ___LLISFSYLL 18101 53 2 F GLEEAQFWL j 8.100_1 .564 j[VLGNLTIIY l~oj 6 157 1ALMAPLPVF 1670 566 202 1LIVI ISAI if6.075 567 288 VLNPIVYG;V 14.05011 568 __75 LISTSSMPK 114.000 I 6 GMESTVLLA ~(3.00,1f 570 1 1 iT VCAVFIF 13. 600 II 5711 182 ]182CLHQDVMKL 572_ 249 ]1AVFIFYVPF 1300i 573 o [SKPKI4LAIF 113. 000 II 574 MLSGIDILI 12.70011 575- ___SLLISFSYL 12.700]I 576 fMPLPVFIK 12.70011 577 307_ RLFHVATHA if1.500 57 29 I1WLAFPLCSL_[I 51 ,79 Q 16111 PMYIFLCML 1 1.3501 580 I~ f CLQIAI 11.350I 581 15 CMISGIDIL 11.5 I 582 [TfKMLAIFWFN]IL~i 583 146_ KiGVAAvvR 1 1.200 11_ 584 165][ FIKQLPFCR 1.2001 585 [T~TJLACDDRV_11.20011 58 221__ YLILTVLM Jro.90goo 587 F5Isaj 7LM'APLPVFI If0.900 sea8 I -9T- VVYGLIVII f 0.757 589J 3=2 ATYFILIGL _9 3 ][YLIAVLGNL 11.0 f 591 0.600 -I 9 257 JrFIGLSMVHR ]0.600i 593 27-7:] ILANIYLLV 1[0.60011 594 168] QLPFCRSNI JI060[ 18 ~VKLACDDI 1O60I 596_ 00 00 00 Table MI OMPA11-V1-A3-9mers [Table IX:101P3A1l-V1-A3-9mers Start~jSubeqneJ SoejSeq. ID Mum 22 MVHRFS KRR 10.600]i 59 22 (LILKTVLGL 110.5-401I 598 21 PIVYG3VKTK 1.411 599 LSMVHRFSK 10411 600 283 J[LLVPPV P 10405 1 601 276 J[VILANIYLL- 104051 602 126_ YVAICHPLR 603 84 MLAIFWFNS 'F0.36011 604 115 1[STVLLAI4AF 1f0.3001 6 0 5L; 305 ]1 ILRLHAT 030 606 280 r1 NIYLLVPPV ].0lIL 607 109_ SLSGMESTV I .0J 60 18 ~YCLHQDVMK ]030] 0 243 IfCVSHVCAVF I[0.3ooJ[ 610 IfLAFPLCSLY ]03J[ 611 :26 IfAQFWLAFPL 10.270 ]1612 19 8 I NVVYGLIVI 110.2701( 613 175 11NIL-SHSYCL_[o.270[ C614. 1 101 IfLLQIFAIHS 615 A139 IfLTLPRVTKI 1 Ti] 616 297 IfKTKEIRQRI 1(.03 5 17 41 If AVLGNLTII11023i 1 -284 J] LVPPVLNPI 1[0.203] F 619 _176 1f ILSHSYCLHI 620 1631 Pf VFIKQLPF][20] 621 1I IVIISAIGL 10o.180] 622 6I SLYLIAVLG 10.iso--][ 623 86 AIFWFNSTT 624 48 If IIYIVRTE-Hl ][.15011 625 _93__1 TTIQFDACL7 [0.135] 626 2737 PLPVILANI 1(0.13SJ 627 217 I SFSYLLIL 10.135[ 628 161 IfPLPVFIKQL ]0.13] 629 FILIGLPGL ]0 15] 630- 209 IfAIGLDSLLI 0.120 11 631 1 216 IfLISFSYLLI 0.12011 632 299 IfKEIRQR ILR 10.1081[ 633 If YIVRTESL 1I~ogo 634 304 IfRILRLFHVA _l.90[ 635 19 [f GLPGLEEAQ 10.090][ 636 135-11 RATVLTLPR:1o] el 637 J 224 I1 ILKTVLGLT 1I0.068[ 638 218 SFSYLLILK l0.060] 639 94 TIQFDACLL 0.0601( 640 2 74 14 LPVILANIY If006 0]1 641.

Start iSubsequence IF oe- 1241 YPILN So e q. I) N uw] 254 YPILM10.060 If 642 I15 2 VVRGAALMA 111 0 060IF 643 30 fEIRQR ILRL 110.054 J[ 644 F-1 J0 SSATYFILI I1Ti[ 645 1 251 FIFYVPFIG j045( 646 1282 IfYLLVPPVLN If0.0451( 647 238 ][KAFGTCVSHI 0.045] 648 32_ FPLCSLYLI 649 99 ACLLQIFAI IL0. 041] 650 12 LQIFAIHSL 10.041] 651 J I 13] DSLLISFSY 11[0 043] 652 33_1 PLCSLYLIA 653 -4-71LTIIVRUT 0R.0341 654 [rable IX:101P3A11-V2-A3-9mers [t]SubsequenceSor Seq ID. Num] [7jVLASGV'rLR 112.000 655 F-T] SLYLIAVLA 11[1._5007 656 [28 IfLLCSTQLSM__][-0400 1 657_ 6 AVLASGVTL ]joooI 658 13 IYLIAVLASG 659 J r2 1 FLLCSTQLS H 0.066 660 ___WPISICWFL 10011 6 r23 ](SICWFLLCS 662 .06] 663 ___TLRCPSSWP 11.00] 664 22_ ISICWFLLC J[.o7] 665 18_ SSWPISICW H 0 022]1 666 ___LIAVLASGV]1.21 667 _3 ___PISICWFLL 0. 0181 668 ___VTLRCPSSW ](OTiT1] 669 ___GVTLRCPSS ][TTI 670 ___LASGVTLRC _____09_1671 ___ICWFLLCST 1000 672 31 19_ SWPISICWF 1(0.0031 673 3 L 4I LRCPSSWP~I 1(0.031 674__ 2 fWFLLCSTQL 1001 675 J F- ALSV].0 676 J 1 fRCPSSWPIS i ool 677 J 2 fLYLIAVLAS 678 11LCSTQLS4E 110o.o0o00 679 77 Iswii PSW Ifoooj 680D 1251 CWFLJCSTQ 0f.000jL 681 1 fSGVTLRCPS If 0.000 [f 682 19JASGVTLRCP 110.0001F 683 rable IX:1O1P3All-V3-A3-9mers [Start ISubsequencel FScoreI Seq ID.Num] CLLQMFAIH 0.9001 64 8 IfIH IT 0. 0 685 a I QMFAIHSLSF7 8 4 IfALLQMFAIS[p.40iI6aa6 2 H1 CLLQMFI110.0031 6898_ IFDACLLQM 0.0031 691 3 f FALSGF 0.000 692 Fable X:101P3A11-V1-A3-lomers Start I[Subsecrence Score Seq. ID Numi 158 I[MAiLPVFIK 405.000 693~ 259 IGLSMVHRFSK18.0016941 7 ILI STS SMPK I60. 0 0 01 695 17VLLAMA.FDRYJJ18.000 694 288 VNIYV 13501 697 19 If LPGLEEAQFI 9. 000 J[ 698 21SMVHRFSKR~j9.00 699 21 SLLISFSYLL]8.0 700 224 GLEEAQFWLA][610] 701 24 ILKTVLGLTR 8.000 70 222 IfLLILKTVLGLI 5.400 703J 137 ITVLTLPRVTKJ[ 4.0 704 187 JI VMLC DDIRJ 4.000 Jr 705 29 WLAFPLCSLYJ[4.000 I[ 706 283 ILPVNIi3081_ 707 249 ]AVFIFYVPFI[ 2.700 L[ 708 112 ]IGMESTVLLAM[ 2.700 1[ ___09 251 jFIFYVPFIGLJ 2.700 710 67 J cMSGIDILII F2.700 71 116 ]ITVLLAAFDR J[1800712 42 VLGNLTIIYIJ L1.800 If_ 733 57 ]ISLHEPMYIFLJL1.800 j~714 18VLTLPRVTKI][80j 715 411AVLGNLTIIYJ1B~ 716 215 JLLISFSYLLI]r 1.600 717 83 j K4LAIFWFNSJi 1.620 718 217 ISFSYLLILK150j 719 FcLID111 1.200 720____ 101 LLQIFAIHSLI 0.900 721 157 MAPLPVFIII0.9001 722 109]JSLSG?4ESTVL 110.900[ 723 184 Rf ILAIFWFNSTI[ 0.900 IL 724 Frable X:101P3A11-V1-A3-lomers Star Subsequence I Score IfSeq. ID u 36]~ SLYLIAVLONJl 0.900 If 725j 305 ][IRLH 1Tj 0.6001 726 168 ][QLPFCRSNILI[ 0.600 j 727 230 Jr LTREAQAKAJI 0.600 728 J 120 (IAMAFDRYVAI] 0.600 729 j r ][37 FGLMVR 0.600 Jr 730 216 1 LISFSYLLIL 0. 540 731 63 11 YIPLCMLSGI[ 0.K450 J[ 732 L2A0NIYLLVPPVLJ 0.5 -[733 S290 Jj1'PIVYGVKTKJI 0.450 7 IfNLTIIYIVRTJ 0.450 735 rT~]TLPRVTKIGV[ 3 IIPLPVILAIY J[040~ 737 1 i7ISMPK4LAIFw][0.0 738 1 100ICLL IFAXHS 11 0. 360 If 739 r 132 J PLRAVT[ 0.360 If 740 8 fAIFWFNSTTI 11O.3001[ 74 1 1246 IfHVCAVFIFYVJ[0.70I 742 1 282 IQFDACLLQIJ 07204[ 3 ___YLLVPPVLNP[ 0.270 ]f 744 [199 IVVYGLIVIIS[ 0774J 1[194 [DIRVNVVYGL[023 746 1292 I VYGVKYTKEI][ 0.225 J 747 J 1182 IfCLHQDVMKLAI i0.225 J[ 748 228 jvLGLrEQ 0.0[ 74 681MLSGIDILIS FO 0180 752 J 277 HILANXYLLVP[ f 012j 753 1 1 33 IPLCSLYLIAVI 07504L~ 211 JrGLDSLLISFS 755oI~s 295 JrGVKTKEIRQRf 0.180 If 756 38 JrYLIVGLI 0.1501- 757 19 rNVVYGLIVII If0.135 L 758 238 IfKAFGTCVSHVII~3I 761 167 IfKQLPFCRSNI 0.2 f 762 196 IRVNVVYG;LIVII0.120I 763 I176I[ILSHSYCLH F. 2 764 39~ LIAVLG4LNTII 0.120 Jj 765 144 IGNLTIIYIVR 0.108 1f 766 56 HSLHEPMYIF 10.101 II 767 92 ISTTIQFDACL 0090 7 68 244IVHVC F0.090 76971 Table X:lO1P3A11-V1-A3-10mers [Srubsequence Score- Seq. ID umJ [121 jMAFDRYVAICj009 770 206 ]IISAIGLDSL ]0.090 771 [81 ][PKrILAIFWF ii0.090] 772 J 276 ifVILANIYLLV 0.090 773~ [41 GTCVSHVCAvII- 0.090 774 ILAFPLCSLYL 0.9 775o 260 LSMVHRFSKR- 0.090 776] 248 I1AVFIF~rVPFl 0.090 L.777] 26 IIAQFWLAFPLC i 0.090 77?8] 1156][AALMAPLPVF 0.068 781 1 ~]AVVRGAA.LMA 0.060[ 782 243 783 162 ]LPVFIYQLPF[ 0.060 J[ 784 [iiRV'rKIGVAAVf 0.060] 785 [1IMMVPNGNES if0.060 786 J 245[SHVCAVFIFYi 0.054 788 81 YCLHQDVIKLj 0.054 789 93 TIQFDCLI] .045 I 9 04RILRLFHAT 004 791 3 47j TIIYIVRTEH 0.04 792 [able X:1O1P3A11-V2-A3-lomers 1 [Startj Subsequence Score Seq 17. um] 14 TF RCPSSWPI 1.800 793] 7 AVLASGVTiR 1.800 79 2 ISLYLIAV~iLA 1.2001 795 18 1FLLCSTQLSM-[, 0600 1 796 181 VLASGVTLRC 0. 60 0[797 3 4 ZI IAVLASGV 0.300 ~798 I19 ~fSSWPISICWF 79091 112 IGVTLRCPSSW 80006 22 jfPISICWFLLCf~.3 80p1 16~ RCPSSWPISIII.3 802 29 LLCSTQLSME 0.31 803 [24 SICWFLLCST].s 8057 [6 ifIAVLASGVL 0.009 806 17 1CPSSWPISIC 0.005[ 807 [26 ][CWFLLCSTQL]0.003 808 ]LIAVLASGVT 10.003 80 2 SWISICWF 10.003 810 L123 11IILc 0.003 a 811 I[able lOlP3AI1-V2-k3-lomers [Start, Subsequence Score Seq ID. NumJ [13 ]VTLRCPSSWP0.2 81 1] ISYIAL 0.002~ 813] [25 jjICWFLLCSTQ 0.001 81 18 IPSSWPISICW 0.00 815 [10~ ASGVTLRCPS 0.000o~ 816 3 LLALS .o 817 LRCPSSWp I7S 0. 000 820 [::j[LASGVTLRCP_00 0Io76-1 2 [rable X: 1O1P3A11-V3 A3-lomers [trtubsequence Score Seq ID. Numi 7 LIF__SLIMO90 822 1CLQ IS0.3601 823 IhlQMFAIHSLSG I0.2001I 824 J IQFDACLLQM ~I1I 825 F CI L iF 09 1 826 12I1FDACLLQNF 0.0031 828 Ia ILQMFAIHSLS 0.00311 829 VIFDACLLQ=MFA 0.000= 830 jf MFAIHSLSGM 0 000~jj 831-- Lrable XI: O1P3A11-V1-A3 -9mers j~tatI1~juence ScorelSeq. ID Num] 20 GLTREAQAK 1.2001 832 75fLISTSSMPK 0.800 833 15 fMAPLPVFIK Jj. 600 834 E126 J[YVAICHPIJR J 835 218_ SFSYLLILK 10.400 836 ___VLTLPRVTK 0.400 837 117 ZIFDR 0.3 838 J YCLHDV__ 0.300 839 11 KIGVAAVVR 0.40 840___ ___FIKQLPFCR 1.2401 84 262 ifMVHRFSKRR 0.200 842 45 [NLTIIYIVR .16~0 843 3 20 LSMVHRFSK 0.120l 8 4 4 3 KERRILR 0.120 846 135 AVTP .8 4 2574 [16 RVNVVYGLI 0.0601 849 Table XI: 101P3A11-V1-A9-g~r- StartJ Subsequence Score Seq. ID Num 143 RVTKIGVAA 0.060 850 198 NVVYGLIVI 0.060 851 204 IVIISAIL 0.060 852 289 LNPIVYGVK 0.040 853 254 YVPFIGLSM 0.040 854 12 ATYFILIL 0.040 855 199 IVYGLIVII 0.040 856 152 VVRGAALMA 0.040 857 249 AVFIFYVPF 0.040 BS8 246 HVCAVFIFY 0.040 859 22 GLEEAQFWL 0.036 860 26 AQFWLAFPL 0.036 861 302 RQRILRLFH 0.036 862 291 PIVYGVKTK 0.030 863 297 KTKEIRQRI 0.030 864 115 ]TVLLAMAF 0.030 865 151 AVVRGAAM 0.030 866 41 AVLGNLTI 0.030 867 241 GTCVSHVCA 0.030 868 112 GMESTVLLA 0.024 869 189 KLACDDIRV 0.024 870 211 GLDSLLISF 0.02 871 307 RLFHATHA 0.024 872 243 CVSVCAVF 0.020 873 51 IVRTEHSL 0.020 874 284 LVPPVLPI 0.020 874 202 GLIVIISAI 0.018 876 125 RYVAICHPL 0.018 877 304 RILRLFHVA 0. 018 __8:7h [.36 ATVLTLPRV 0.015 879 139 LTLPRVTKI 0.015 880 L93 TTIOFDACL 0.015 881 276 VILANIYLL 0.012 882 223 LILTVLGL 0.012 883 215 LLISFSYLL 0.012 884 175 NILSHSYCL 0.012 885 295 GVKTKEIRQ 0.012 886 238 KAFGTCVSH 0.012 887 231 LTREAQAKA 0.010 888 144 VTKIVAAV 0.010 889 99 ACLLOIFAI 0.00 890 102 LQIFAIHSL 0.009 891 163 PVFIKQLPF 0.008 892 252 IFYVPFIGL 0.008 893 _I2 CLHDVMKL o.oo0j 894 able XI:lO1P3A11-V1-A3-9mers Start Subse e Seq. ID Num 216 LISFSYLLI 0.008 895 42 VLGNLTIIY 0.008 896 288 VLNPIVYGV 0.008 897 66 LCMLSGIDI 0.008 898 225 LKTVLGLTR 0.008 899 68 MLSGIDILI 0.008 900 277 ILANIYLLV 0.008 901 209 AIGLDSLLI 0.008 902 120 AMAFDRYVA 0.008 903 280 NIYLLVPPV 0.008 904 57 SLHEPMYIF 0.008 905 48 IIYIVRTEH 0.008 906 157 ALMAPLPVF .008 907 188 MKLACDDIR 0.006 908 294 YGVKTKEIR 0.006 909 27[ PVILANIYL 0.006 910 67 CMLSGIDIL 0.006 911 148 jVAAVVRGA 0.006 912 IYLLVPPVL 0.006 913 YLLILKTVL 0.006 914 15 FILIGLPG L 0.006 915 214 SLLISFSYL 0.006 916 100 CLL][FAH 0.006 917 127 VAICHPLRH 0.006 918 32 fPLCSLYLI 0.006 919 247 VCAVFIFYV 0.006 920 F4_0 IAVLGNLTI 0.006 921 1561 AALMAPLPV 0.0061-- 50 YIVRTEHSL 0.006 923 38 YLIAVLGNL 0.006 924 STSSMPKML 0.005 925 226 KTVLGLTRE 10.00 926 l SMPKMAIF10.004L-27 29 WLAFPLCSL 0.004 928 109 SLSGMESTV 0.004 929 13011 LAFPLC 1 04 930 187 VMKLACDDI 0.004 931 able XI:1O1P3A1-V2-A11-9mers Start Subsequence Score Seq ID. Num 7 VLASGVTLR 0.080 932 6 1AVLASVTL 0.030 933 12 VTLRCPSSW 10.015l 934 28 LLCSTQLSM 0.0083 935 I-ThIM 0008 936 fable XI:101P3A1-V2-Ai.1-9mers [Start ISubsequence] Score ISeq ID. _NumJ 11 GVTLRCPSS 10.006! 93 WPISICWFL 1.01 938 16 [CPsswPISI 0 01 939 4 LIAVLASGV ]I0.0041 940 26 WFLLCS TQL ]k0.0031 941 1 21 PISICWFLL ]10.01i][ 942 2 IfLYLIAVLAS 0I.0011l 943 23 ICF LC oKOj 944 18 SSPSIW 0spsc olooi] 945 27 _j FLLCSTQLS 0.001 i[ 946 ]1 RCPSSWPIS Io Fil 947 I F_3 If _YLIAVLASG. 1001 4 1 J 14 L PSSWPI H0.0001 950 8 11 LASGVTLRC 0.0001 951 24 I0F LCS 17R 5 IAVLASGVT 953 29 IfICSTQLSME 000 954 19 IfSWPIrSICWF 0FO. 000 955 22 IIFLLC 10.001 956 I WLCT .01 957 1 SGVTLRCPS 000 -OI_958 9 VLRC 0.000 9 1 ][PSSWPisISIC 0.00 960 ableXI:11P3A11-V3-A11-9mers IstartiSubsequence SoeSeq ID._ N um] 7 LQMFAIHSL F012 961 4 ][LLPAI 0.009 962] CIiIL MFIZL2963 1I] QFDACLLQM 0.0041 964 3I ACMFA 0.001j 965 8 ]M 9 HSLII 0.0011 966 1 6 LLQMFAIHS 0.0011 96 9 SLSG]F0.000 968 2 DCLM 0.00 969 [rable XII :1O1PA11-VI-A11-lomers Us Start IlSubsequenceliscorerSeq. ID NumjI Table XII:101P311-Vl-All-l0mers I~S~tartSubsequence -Score Seq. ID Num 288 ]VwPIVYGVK ]0.400 975 1 L180 SYCLHQDVMK 0.400 976 15RYVAI CHPLR 0.360 977 164 iVFIKQLPFCR 0.180 978 224 I ILKTVLGLTR 0.160 97 290 1NPIVYGVKTKD0 150 980 295 fGVKTKEIRQR 0.120 981 217 ISFSYL 0.8 8 [293 VMKACIR 0.080 984Zi [44fGNLTIIYIVRI0.072 986 FFY 0. 0601 987____ 148 IfVAAVVRGAA .060 891 [143 IfRVTKIGV1tb7 990 26 RMSR .6 9 41 RAVLGNLTIIY 0.060 992 302 IfRQRILRLFHVI [05 h hI99 12 IYVAICHPLRI0. 0 94F 249 [AVFIFYVPFI ]J.0401 995 229 LGLTREAQAK 10031 996~~: 241 11 GT~gr170 3 9 53 IRTEHSLHEPMI0.3 998 198 IfNVVYGLIVIII 0 IT 99 .167 If KQLPFCRSNI F.0271 1000 134 IRHATVLTLPRI 1001 11 fGMESTVLLMj~ 95 IIQFDACLLIIIO.024 1003 22 -GLEEAQFWLA 1.041 004- l 77 STSSMPKMLA 100 1005 :254 YVPFIGLSMV 0. 1006 2 92 ]C 0 21 1007 284 INP~v~7~tZ100 243 CVSHVCAVFI 0O.020 1009 251 if FI FYVPFIGL 10.-0 16 1 100- 93_ TTIQFDACLL 0.015 101 186 ]DVMKLACDDI 110.012 1012 222 jfLLILKTVLGfl 1 1013 2 3 0 I GLTREAQAKA ]10 .0121 1014 1 155 fGAALM4APLPV 0.012E_1015 214 JSLLISFSYLL 0.0121 1016 253 IfFYVPFIGLSM 0.012 1017 238 IfKAFGTCVSHV] [Pi12 i 1-01 276 HfVILANIYLLV 0]L.0121. 1019 29JGLSMVHRFSK 3.600 97 1377 IVTPTK 13. 0001 971 1.16 1f TVLLAMAFDRI. 1.80 158 LMAPLPVFIK I1.200 973 74 Ifl ILISTSSMPK m_ 97 trable XII: 1O1P3A11-V1-A11-lomersraeXI1P31--1-lms rable XII-10MA11-VI-Ali-10mers I usqec score Seq. ID 1NumJ 0.0121 1020 19]GLPGLEEAQF 0.012 1021 L2 LVI SAI GLJ2] 122J 92 ISTTIQFDACL 0.010 1024 144 IVTKIGVAAVV 0O.010l 1025 [LAFPLCSLYL 0..008 1026F- ]SMPKIILAIFW 0.0081 02 12803[NIYLLVPPVL 0.008 1028 r63 YIFLCNI.SGIl 1029r~~~- 57 SLHEPMYIFL 0.006 1030 119 ILAMAFDRV 0.00 8 13 1 199 JVVYGLIVIIS 10.0 08 1.032 -216- LIFYLLI L0.081 03 42 VLGNLTIIYI 11.08 03 255]ILIVGLTIII.008 1038 140 TLLPVI 10. 008 1039 298 JjTKEIRQRILR 10.00 1040 FLCMLSGIDI 10.008 1041 256 I~GLSVH 0.006j 1044 1 145 TKIGVAAVVR 10. 0061 1045]1 so IYIVRTEHSLH 0C 162 LPVFIKQLPF 0.006 1047 47 TIIYIVRTEHII.6060 14 265 RFSKRRDSP 1.06F 0.

81 [MPKMLAIFWF 10.00611 1~050 37 [LYLiAvLGNLIIo-.00-6 I1 lZ] 220 [SYLLILK'rvLJI0.006 1052 25]PVILANIYLL110. 0061 [105 175S] NILSHSYCLHII0.006 1054] 181 rYCLHQDVMKLII0.006 10i5II 283 ILLVPPVLNPII 10061 1056 32 jFPLCSLYLIA 10061 1057 2T PVLNPIVYGV 1.0.06i 058I 235SJ AQAKAFGTCV 11.006 1059 27 JLPVILANIYLII0. 006 1060 117 J VILAMAFDRY0.0 1062i~ 208 1 SAIGLDSLLI 10. 0061 1063 1KI4LAI FWFNS 0L. 0 05 1064 Start Sus neSoe- T r 299 31KEIRQRILRL 0.0 1065 2317D LTREAQAKAF 0.005 106 6 226 IKTVLLTREA IEOSF 1067 35 ILRLFHATH 1 -041 1068 rable XII :1O1PA11-V2-AII-lomers Start jSubsequence Score Seq ID .NumJ 7 IAVLASGVTLRHO0.60011 .1 0 12 IGVTLRCPSS$ O06t 1071 16 IfLPSWIS .02 172 J 28 IFLLTQLSM 2.1 1073 j21 WPISICWFLLI0.009 1074 14 TLRCPSSWPIIO08 1075 4 YLALAG 0O.0061 1076 6 If AVLA 0.003 1077] 2 11 SLYL 10.021 107 13 [VTLRCPSSWP 000 1079 1[VLASGVTLRC 0.01 108 1 26 ICWFLLCSTLI 0.00 182 1I SSWPISICWF OF._0011 08 0.b 001[1084 __IR LIAVI ASG' 085_I 24 IfSICWFLLCSTI 0.000- 29 IILLCSTQLSMEI.01 107 !gHIW 1088 1 CS[YIAU0 D0 089 F2771 IWFLLCSTQLS 0. 0 00 1090 7 ICPSWISLC0.000 1093 180000 109477 11ISGVTLRCPSS 10.0 1095 15ILRCPSSWPIS 10. 0001 10.96 10IfASGVTLRCPS 000 1097 9 II LASGVTLRCP 10. 0001 1098 [Table XII: 1O1P311-V3-A11-l0meraS7 IstartIlSubsequencej Score ISeg ID. Numi 1IIQFDACL.LQM 0.024 I 10.99] 1 QMFAIHSL 0.0041 1100) 2IQFDACLLQMF 000 1102 10 IfMFAIHSLSGM 0.00 1103 00 00 00 Fable XII:101pA11-V3-Al1-lomer Start [Subsequence ScreSeq ID. Num rn DAQMFAI0N0021 1104= rn AIHS LSG .0 10 rn CLLQMFAIHS 0.01 1106 Frn7 LOMFAIHSLS01 1107 if 3 FDACLLQMFA 10. 0001 1108 [Table XIII:1O1P3A11-Vl-A24-9mer-S 1-- ____~luseuec Score Seq. ID Numj F125 1[ RYVAICHPL__8O.O01109 E I J[ YLLvPPVL J420.0001F11101 23 VYGVKTKEI I1 I500 f ii 1 AFPLCSLYL 1130.00011[ 1112 1 180_ SYCLHQDVM 1[5ooI 1113 52__7 IFYVPFIGL ][24.000J[ 1114 8 9j WFNSTTIQFJ]~~] 1115 s SYLLILKTV ][oso[ 1116 154 RGAALMAPL ]9.0 r 1117 [62 MYIFLCMLS [9.000 1118 [253 FYVPFIGLS 1119 L38_1 YLIAVLGnLJ 8.400 12 12501 VFIFYVPFI 750 12 6 IFLCMLSGI 7.500 112 2 1 491 IYIVRTEHS 11230 37 LYLIAVLGN 7.500 112 1221 GLEEAQFWL 7.200 1 1125 1 2141 SLLISFSYL 17.200] 1126 11 1SGMESTVLL 7. 1127 V 11SAIGLDSLL 11.20) 1128 N 3S 11CSLYLIAVL 1 7.200 Jr 1129 122111 YLLILKTVL 117.200 1130 120011 VGLIVIIS_1200J 1131 150 AAVVRGAAL 116.000 1132 1671 CMLSGIDIL N6.000 1133 1f13111 HPLRHATVL j_.00J 1134 1151 FILIGLPGL 1 .00J 1135 11751 NILSHSYCL 116.000 1136 1 931 TTIQFDACL] 6.0 1137 -71T LLISFSYLL 116,0 1138 102 1LQIFAIHSL 600 1139 7 6.000 ]1 1140 94_J_ TIQFDACLL [60 1141 276r VILANIYLL 600 1142 I VIISAIGL r 6.000 '_14 223 11LILKTVLGL I 600 1144 Table XlII:1O1P3A11-V1-A24-9mers ~SatISubsequence SoejSeq. ID Num] 12~I ATYFILIOL]~560I 1145S 7 IFWF'NSTTI f5.000 j 1146 3 9jjQFDACLLQI 1 5.000 11 1147 3 [TI[LPFCRSNIL 4.800 1.148 J 26 AQFWLAFPL 4.800 J1 1149 182 11 115007 3I 11911RVNVVYGLI J 4.200 r7 1151 1271KTKEIRQRI 11 4.032 11 1152 ESSATYFIL Jr 4. 000 1153 1 4 4.000 1154 FSKRRDSPL J400Jr 1155 _PCS 4.000] 1156,# 11 rLSGMESTVL 114.005~i7 27 ISFSYLLIL Ir400 r 15 f207 J[ISAIGLDSL ~[4.000 1 1159 1 _SPKI 'F4.00011 1160 STVLLAMAF J[360 1161 284iJ LVPPVLNP 3.024 1162 ___GNESSATYF Jr3 3.000 1163 (17kAIZAPLPVF 300 1164 80__ SMPKNLAIF Jr3.000 11i6-6 CVSHVCAVYF r280 L1167 ___SLHEPMYIF 2.40 168~ 211_ GLDSLLISF J1 2.400 [1169 I 20 rGLIVIISAI] Jr210111170 1 ___AVFIFYVPF Jr2.0001 1Z 71 f2 JrLPGLEEAQF] 2.0007 1172i [19[LTLPRVrKI 1. 9 80M13 1791 SSMPKMLAI Jr1.600 1 174 IT] AVLGNLTII J i jo 1i75 ][FPLCSLYLIJ if1.500_I 1 316 6 1LCMLSGIDI71 .0 1177 ___ACLLQIFAj r11.0 1178 11IAVLGNLTI 1 r00J 11791 ___NVVYGLIVI' 1.500 1180 ___HSLHEPMYI 1.50 1181.

QLPFCRSNI 50 0 Jj 1182 [..(NLIY 1.440 1183___ T7iL 1.400 1185 601EPMYIFLCM 1.260 1186 I10 SjS!RATY II 1.200 1187 12 AF1VA .200 1 18 199 VVYGLIVII 1.20 118.9 able XIII:1O1PA11-V1-A24-9mers StartJISubsequence]~ Score jjSeq. ID NumJ 270 ]jRDSPLPVIL ]91i. 152jj 1190 254 IIYVPFIO3LSM]i4 J1. 05-0- 1191 216 JJLISFSYLLI JJ1.000 Jf 1192 244 JjVSHVCAVFI ]i.ooo[ 1193 1871 VMKLACDDI 1~.000J 1194 209 JjAIGLDSLLI 1~.000j 1195 195 IRVNVVYGL 0 .840] 1196 164 J[VFIKQLPFC 0.750 Er 1197 j [151 AVVRGAAJM jj0.750 1198] 73 DILISTSSM jj0.750 J[ 1199 105 IfFAIHSLSGM J 0.750 j[ 1200 158 IfLMEPMYIFL Jf0.720 1201 -13J TYFILIGLP Jr 0.600 jj 1202 28 TKEIRORIL 1 0.600 1 1203 PVILANIYL 1r 0.600 1204 161. IfPLPVFIKQL 1 0.6001 1205 27 QFWLAFPLC ft0.600- 1206 76 1][STSSPKJ 0.550 I 1207 83 KM!2LAIFWFN 0.50-4- 1208 able XIII: 1O1PA11-V2-A24-9mers StartJ[Subaeque nce score Se[(ID. Num 26 WFLLCSTQL 30.000 [1209 WPISICWFL 18.40011210 2 J[ I A (7.5001 1211 6 JrAVLASGVTL (6.0001 19 JrSWPISICWF 300 123 16 1(SPII 1. 000 Il 1214 28 LLCSTQLS.M 6~."oj 1215 21 PI[ WFLL 0.4 0 1216 is RPSWIS 0 -0 1217 SGVTLRCPS 18 0 1 1218 22 ISICWFLLC Jro _.18oJ0 1219- 27 JrFLLCSTQLS Pi] 1220 ssws SSPSIW[.T16 J 221 12~ VTLRCPSSW 0f. is[ 312 23 1 JJ LYLIAVLA ]j0.140] 1225 4 ]JLIAVLASGVT1I0.1201[ 1226 14 LRCPSSWPI 1I0.120] 1227 24 ICWFLSTJ010( 28 23J SICWFLLCS 0.100 1 11 QVTLRCPSS 0.0D13 3 YLIAVLASG] 0.0211 1231 Table XIII: 101P3A11-V2-A24-9mers IStartlSubsequencej Score [Seg ID. Num] 25 J[CWFLLCSTQ JI0. 012] 1232 9 ASjGVTLRCP jo.oio 1 1233 29 29 LCSTQLSME j0.010 j[ 1234 13 TLRCPSSWP 0f .010o 1235) [7 Jr LASGVrLRj0. ::V:OfE~p 1237 rable XIII: 1O1P3A11-V3-A24-9mers S9 mlius une Score Seq ID. Numj 71 LFI~HS 6.000 123 jIl QFDACLLQM F2jjjjj 1239 41ACLLQMFAI]1501 1240 1211 FDACLLQMF]I .288 1241 Ir-mr LLIHS 0.501 1242 1 8 Q--I I HSLS 0.140 1243 EJ~j MFISLSG1.50 124 ErableXIV:1O1P3A11-V1-A24-lomers Start Subsequence 11score ISeq. ID Num 37 1(LYLIAVLG 420.000124 220 ISYLLIKT36 0.0001 1248] 491IIVTHS]_O 1249 253 J[FYVPFIGLSM63090[ [265 JrRFSKRRDSPL 4 F00] 1251 VF- IYFILIG;LpL 30. 0 1252 96 1253 297 T Kr~~EIRQRILI 9.600 Jr 1254 I 1 iAFPLCSLYLIJ 7.500 1255 281 IYLLVPPVLNij 7.500 1256 168](QLPFCRSNILJI 7.200 J[ 1257 1213 JrDSLLISFSYL1j 7.200 1258 F16071[APLPVFIKQLII720J 1259 J[EAQFWLAFPL I .20J 1260 0 (VYGLIVIISAJJ 700 1261 8 [YCLHQDV4KL Jr660 1262 24 LPVILAIYL J(6.000 1263 1 [60 ][EPMYIFLCMLT[ 6.000 1 1264 (203 J[LIVIISAIGL 6.000 13265 [93 [TTIQFDACLL [600 1266 101 LLQIFAIHSL[ 6.000 1 1267 6'LAP~S .00 0 1268__ 66~~ -CLGDLH 00 36 ~able XIV: 1O1P3Al1-V1-A24-l0mers [stat~lubaguecej Scre Seq. ID Num 214 IfSLLISFSYLLf 6.000 1270 1 174 ISNILSIHSYCLII 6.000 271 222 IFLJLILKTLiL[ 11.000 1-72 9 [DIRVN~VVYGLJ[ 5.600 Jj 1273 11JSATYFILIGLI[T5.600 1274 [~]NIYLLVPPVL]r 5.600 1275 ]FIFYVPFIGL][ 4.800 1276 34JjLCSLYLIAVL][ -4.800 II 1277 [TI SLHEPMYIFL][ 4.8001 1278 30 ]j LAFPLCSLYL] 4.800 1279 7 JISAILDSLL[4.800 1280 J 120IIGIJDSLLISFI 4.320 1[ 1281 242 IITCVSHVCAVF]I 4.200 1282 IIISAIGLDSLII 4.000 1283__] 149 'VA~kVGAALI 4 1284 W_109 SLSGMESTVLJ 4.000_ 1285____ 26 LISFSLII [92 1 [STTIQFDACLII 4.000 1287 76 [ISTSSMPKLI1 4.00 1288 110 ILSGMESTVLLII 4.000 1[ 1289 79 3.600 1290 6 NGESTFI3.600 1291 16 KQLPFCRSNII 3.600 19 1__ILVPVNI .2 12923 28ICAVFIFYVPFI [3.000 19 1162 1 LPVFIKQLPFIj 3.000 IL 1295 1 156 IHSLHEPMYIFII 3.000 If 1296 3 119 JGLPGLEEAQF 3. 0 00 197 3 IAALMAPLPVFI 3.000 ]f 1298 3 1300 EE EIRQRILRLFI 2.800 If 1299 3 272 [SPLPVILANIII250f 1300 [104[IFAIHSLSGM 1 2.500 1301 3 114 IISVLMFI2.00 1302 23 I[LTREAQAKAFj[240] 1303 67 1 CMLSGIDILIII210 f 10 257 Y[FGLSH1RF 2.100 1305 257 FIGSKVRF 2.-000 J~1306 rT1fMPKMIFWF 1 2.0 1307 88 .00J 1308 r244 3[VSHVCAVFIF3[ 2.000 11 1309 19 1310___ 53 RTHSHEM 1.800 1311 [157 ALMAPLPVFI][.01 1312 [T ][z~vGLIVIjf 1.500 1313 IJIAVLGNLTIII 17500 on 1314 Iable XIV: 1O1P3A11-V1-A24-lorners ItrlSubsequenc] Score jSeq. ID NumJ F:T7TDVKL 1.]L500 .1315 1 F1251 RYVAIC.HPLR F. 00 3 316 -1 21 LLIFSLL 1318 208 IISAIGLDSLLI ]1.500 Lf 1319 F 9 ~KEIRQRILRLI120J 1320 9 ESSATYFILIJI120J 1321 [95 JIQFDACLLQI 1.0 1322 [3 8 VLTLPRVTKI 1.100 J~1323 [~22 IVYGVKTKEI1.0 1324 J GMES~vLA~jf1.050 1325 J [65 J[FLCMLSGIDI f100jf 1326 3 42 IVLGNLTIIYII 1.000 1327 [98 [DACLLQIFAII1.0 1328 129AVFIFYVPFIJ 1.000I 1329 1 78 J[TSSMPKMLAI][I.0 1330 _3 ViTICVSHVCAVFII 1.000oo 1331_7 I~ILAV G II 1.000 1f 1332 IWIAMFDRYVAI1.00 1 1335 3 29]RRDSPLPVIL 0. 960] 1336 3 1150 3[AAVVRGAALMr[ .75 07 1337 3 62][MYIFLCMLSG][ 0.750 1338 j IIPGLEEAQFW:L 0I2 13 IAGCSV 0. 700 0 _3 I~hSFSYLLT 0 160~ 1342 130 CH[LHTL 10. 60 0 JF[ 1343 3 [I275 I LAN~IYhL 0.600o 1344 124 ][DRYVAICHPL]I .50I 1345 75 LISTSSMPKM3[ .55 1346 Fabie XIV:101P3A11-V2-A24-10mer I tart [Subs e__encej Score Ese: ID. New 2 SWPISICWFL 40 1347 21 WIIWLI 6 01 1348 6[IAVLASGV'rL ITOOO349 126 I[CWFLLCSTQL14.01 ]I3 [16 IRCPSSWPISI 1351 119 IrsswPIS CWF] 12. 1352 3]LYLIAVLASGJI1.0501 1353 14 JjTLRCPSSWPI] 1 -000IL 1354 27 IIWFLLCSTQLS jI.

900 I 1355 2_8_1 FLLCSTQLSM L. 1 135 able XIV:10MPA11-V2-A24-lomers

I

Start-Sbsequence jScore ISeq ID. Newl I CSIIYLIAVLAD 0.2101 1357 0LALSVI.1801 1358 11 I SGVTLRCPgSS 0.150[_16 8 LSVLRIO10 1361 24 JSICWFLLCST 0.120 1363 2 SLYLAVLAS 110. 10-1 1364 1 CPSSWPISI F,00 1365 [11GVTLRCPSW01 1 1366 LIAWLAGV 0100 1367_ 13 VTLRCPSSWP 0. 0151 1368 7 JAVILASGVTLR I 0.015 F 1369 16s H PSSWPISICw 10.014 1370 11 LRC~sw~ 0.012[ 1371 125 LSQ HO.012][. 1372 22_11 ISICFLLC 00211 1373 p LLCSTQLSME 0 1 1374 j I [LASGVTLRCP[2 0.010 1375 able XV1131-3A4lmr Start jSubsqec Score ISeq ID_ Numi 2 -1 Q)FDACLLQMF[ 40011 1376 XIVLLQMF=AIHSL][.Oj 1377 10IMFAIHSLSGM]1250j 1378 SDACLMFI[00[ 1379 [1IQFDACLLQMIO.60011 1380 18 ILMFAIHSLSIO. 1 138 1 6 1 1CLLQMFAIHSI10.15 0[ 1382 I FACLLMFA 0.018 1384 9 ][QMFAIHSLSGIO.10I 1385 abeXV: 1O1P3A11-V1-B7-9mers

I

Start Subsequence Score ISeq. ID NumU 1697 LPRSNILI~0 1386 I131 HPLRRATVL 180.000L 18 [T0 EPMYIFLCN 1388 P1i4 I LPRVTKIGV 4000 1389 301EIRQRILRLD4.0 1390 i 3AAVVRGAAL 136.0001 1391 204 IVIISAIGL 2000 1392 151 I[ AVVRGAALM 15.000J 1393 2 11AQFWLAFPL 12.0000 1394__ [Table XV: 1O1P3A11-V1-B7-9mers Subs ecuen ce ore~~nT Tn SbsL eae c1* I 2087 SAGDSLL 112. 0001_ 1395 111] SGMESTVLL 1.00l 1396 12 ATYFILIGL 120Jj 1397 3 FPCLYLI 8 OJ 19 41 AVGNLTI If.ooo 1399 29 WLAFPLCSL If6.000 1i400 254 YVPFIGLSM 1401~i 152 VVG MI S.oo000 402 157I FILIGOjPGL jj4.000 1403 S ][YIVRTEHSL 1400j 1404 102 J[LQIFATHSL ]4001 1405 276 VILAN IYLL 34001 1406 110 IfLSGMESVL3400I 1407 94 TIQFDACLL][Ti[ 1408 93 j[ TTIQFDACL 14 j 1409_7 255 IfVPFIGLSMV 14.000 1 1410 221 IfYLLILKTVL 4011 1411 77 1lSSMPKML4.0 1412 35 T CSYI" 14 13 18 fCLHQDVMKL 4.001[ 1414 21 fLLISFSYLL r4. 000 If 1415 285 7,VP V 11.00! 1416 217] ISFSYLLIL 1f 401 1417 20 I3 ISAIGLDSL 114.000 1418 67 11 CMLSGIDILJ 14. 00011 1419 3 fYLIAVLGNL I14.00011 1420 266 H FSKRRDSPLJ 14. 000 ]1 1421 23 LILKTVLGL 1.00I 1422 17 11NILSHSYCL 1423 [T I~ RGAALMAPL 14.000][ 12 ~i fSLLISFSYL j4.000[ 1425 9 Ef ESATYFIL J40][ 1426 105 If LSGM 1427 PVILAN L[3i ppor 1428 [4 DPGES 2.0 1429 198 8 NVVYGLIVI 1430 PI GVK[ 1431 1196 Af RVNVVYGLIJ 2.01 1432 1~j VV~YG j2.0O 1433 284_H LVPPVLNPI 20][ 1434 7715653 AALMAPLPV io][ 1435 119 LAMFDR 1E. 800 1436 209 IfAIGLDSLLI J1.200 1437 99 IfACLLQIFAI 1 1.200 1438 66 LCML2GIDI 139 able XV:101P3A11-V1-B79mers Stat ubsequence Score Seq. ID Num 79 SSMPKMLAI 1.200 1440 22 GLEEAQFWL 1.200 1441 121 MAFDRYVAI 1.200 1442 IAVLGNLTI Jjf 1443 31 AFPLCSLYL 1.200 1444 76 ISTSSMPKM 1.000 1445 73 DILISTSSM 1.000 1446 305 ILRLFHVAT 1.000 1447 231 LTREAQAXA 1.000 1448 252 IFYVPFICL 0.600 1449 236 QAKAFGTCV 0.600 1450 297 KTKEIRQRI 0.600 1451 168 QLPFCRSNI 0.600 1452 136 ATVLTLPRV 0.600 1453 160 APLPVFIKQ 0.600 1454 VTKIGVAA 0.500 1455 148 1 GVAAVVRGA 0.500 1456 227 TVLGLTREA 150.500 II 13? TVLTLPRVT 0.5001 1458 1 51 IVRTEHSLH j[0.5 0 1459 149 VAAVVRGAA 0.4501 1460 128 AICHPLRIA 0.4501 1461 120 AMAFDRYVA 0.450 1462 272 SPLPVILAN 0.400 1463 216 LISFSYLLI 0.400 1464 274 LPVILANIY 0.400 1465 56 HSLHEP14YI J.400 1466 125 RYVAICHPL 0.400 1467 195 IRVNVVYGL 0.400 1468 RDSPLPVIL 0.400 1469 4_NTIIYI 0.400 1470 158 LMAPLPVFI 0.400 171 81 MPKMLAIFW EO:400 3.472 187 VMKLACDDI 0.400 1473 202 GLIVIISAI 0.400 1474 281 IYLLVPPVL 0.400 1475 161 PLPVFIKQL 0.400 1476 68 MLSGIDILI 0.400 1477 133 LRATVLTL 0.400 1478 139 LTLPRVTKI 0.400][ 1479 61 PMYIFLCML 0.400 1480 LPGLEEAQF 0.400 1481 244 VSHVCAVFI 0.400 1482 SSATYFILI 0.400 1483 DACLLQIFA 110.300q 1484 able XV:101P3A11-V1-B7-9mers [Sta Score Se ID NuI LLL.30 AQAKAFG 0 1485 able XV:1O1P3All-V2-B7-9mer] Start Subsequence Score Secr ID. Numl 20 WPISICWFL 80.000 1486 j 6 AVLASGVTL 60.001 1487 J 16 CPSSWPISI 8.000 1488_ 28 LLCSTQLSM11.000 I 1489 21 PISICWFLL10-4001 1490 26 WFLLCSTQL 0 .400J 1491 8 LASGVTLRC 0.300 1492 5 IAVLASGVT 0.300 1493 4 LIAVLASGV ](o.200[ 1494 13 TLRCPSSWP ]FT1hsj5 1495 22 ISICWFLLC 0.1001 1496 1 SLYLIAVLA 1 .oo 1497 11 GVTLRCPSS 10.100 1 4 9 8 24 ICWFLLCST 110.1001 1499 F 14 LRCPSSWPI 10.040E 1500 9 ASGVTLRCP 1 10.030 1501 10 SGVTLRCPS 0.030 1502 27 FLLCSTQLS 0.020 1503 18 SSWPISICW 0.020 1504 12 VTLRCPSSW 0.020 1505 23 SICWFLLCS 0.020 1506 1 f RCPSSWPIS 10.020 1507 17 PSSWPISIC 1.oil 1508 7 J[ VLASGVTLR IFi11 1509 29 LCSTQLSME 0.010 1510 YLIAVLASG 0.010 1511 SWPISICWF 0.002 1512 2 ](LyLIAVLAS 0.0021 1513 CWFLLCSTO 0.001 1514 able XV:101P3AI1-V3-B7-9mers StartilSubseqence Score Seq ID. NuR 7 LQMFAIHSL 12.0001 ]Isis] 4 I ACLLQMFAI 1.200 1516 3 DACLLQMFA 0.300 1517 1 QFDACLLQM 0.030 Iis ia QMFAIHSLS I 0.020 1519 6 LLQMFAIHS 1F0Ti ii1520 5 CLLQMFAIH 0.010 1521 2 If FDACLLQMF 1522 1911 MFAIHSLSG 110.001 1523 able XVI:11P3A11-V1-B7-lomers Start Subsequence Score Seq. ID Num EPMYIFLCNL 240.000 1524 160 APLPVFIKQL 1240.00OF 1525 274 LPVILANIYL 80.000[ 1526 194 11 DIRVNVVYGL 40.000 1527 141 LPRVTKIGVA[20.000 1528 149 VAAVVRGAAL 12.000 1529 LAFPLCSLYL 12.0001 1530 11 SATYFILIGL[12.000 1531 66 LCMLSGIDIL 12.000 1532 EAQFWLAFPL 12.000 1533 150 AAVVRGAALM 9.000 1534 272 SPLPVILANI 8.000 1535 249 AVFIFYVPFI 6.000 1536 251 FIFYVPFIGLJ[6.000 1537 186 DVMKLACDDI 1 6.000 I1538 216 LISFSYLLIL 4.000 1539 110 LSGMESTVLL 4.000 1540 181 YCLHQDVMKL 4.000 1541 93 TTIQFDACLL 4.000 IF 1542 297 XTKEIRRIL[ 4.000 1 1543 213 DSLLISFSYL[ 4.000 11 1544 iSLHEPMYIFL 4.000 1545 168 QLPFCRSNIL[ 4.000 1546 92 STTIQFDACL 4.000 1547 206 IISAIGLDSL 4.000 1548 203 LIVIISAIGL 4.000 1549 222 LLILKTVLGLI 4.000 1550 1 280 NIYLLVPPVL 4.000 1 1551 76 ISTSSMPKML 4.000 1552 101 ILLQIFAIHSL 4.00071 1553 132 PLRHATVLTL 4.000 1554 174 SNILSHSYCL1 4.000 1555 214 ]SLLISFSYLL 4.000 1556 207 ISAIGLDSLL 4.000 If 1557 109 SLSGMESTVL .000 158 34 LCSLYLIAVH 4.000 1559 157 IALMAPLPVFI 3.600 1560 131 HPLHATVLT 2.000 1561 243 CVSHVCAVFI 2.000 1562 32 FPLCSLYLIA 2.000 1563 29a NYvYGLIVII 1 2.000 1564 292 IfVYGVKTKEI 2.000 1565 4 II DPNGNESSAT 2.000 1566 275 PVILANIYLLI2.000 1567 Mable XVI:11P3A11-V1-B7-l0mers StartSubsequence Score Se. ID Num 302 RQRILRLFHV F 2.000 1568 151 AVVRGAALMA 1.500 1569 13.9 LMAFDRYVA 1.350 1570 120 (AMAFDRYVAI 1.200 1571 40 JIAVLGNTII 1.200 1572 86 __AIFWFNSTTI 1.200 1573 98 DACLLQIFAI 1.200 1574 SAIGLDSLLI 1.200 1575 LISTSSMPK? 1.000 1576 246 HVCAVFIFYV 1.000 1577 284 LVPPVLNPIVJ[ 1.000 1578 196 RVNVVYGLIV][ 1.000 1579 143 IRVTKIGVAAV][ 1.000 IL 1580 179 HSYCLHQDVM 1.000 1581 254 YVPFIGLSMV 1.000 1582 190 LACDDIRVNV f0.900 1583 148q GTVaVVRGAA 07 1584 135 HATVLTLPRV 0.600 1585 28 FWLAFPLCSL 0.600 1s86 155 GAALMAPLPV][ 0.600 1587 167 KQLPFCRSNI[ 0.600 fis88 235 AQAKAFGTCV 0.600 1589 238 IKAFGTCVSHV 0.600 1590 268 KRRDSPLPVI 0.600 1591 279 I ANIYLLVPPV 0.600 1 1592 152 IVVRGAALMAPI 0.500 11 1593 51 IIVRTEHSLHEI 0.500 1594 127 jVAICHPLRHAI 0.450 if 1595 128 AICHPLRHAT 0.450 1596 67 CMLSGIDIL 0.400i 1597 20 LPGLEEAQFW 0.400 1598 124 DRYVAICHPL 0.400 1599 [299 ]1KEIRQRILRLI 0.400 1600 81 [MPMLAIFWFi 0.400 1601 78 TSSMPKnMAI 0.40I 1602 138 VLTLPRVTKI 0.400 1603 9 ESSATYFIL1 0.400 1604 265 RFKRRDS 6 0.400 1605 201 YGLIVIISAI 0.400 1606 2T VIGNLTIIYI 0.400 1607 37 LYLIAVLGNL 0.400 1608 FLCMLSGIDI 0.400 1608 283 LLVPPVLNPf 0.400 1610 162 LPVFIKLPF 0.400 1611 215 LLISFSYLLI 0.400 1612 Iable XVI: 101P3A11-V1-B7-lomers IStJSub equenc[ Score Jjseq. ID Num 0.400 1613 VPPVLNPIVY] 0.-400 1614 1 RGAI-4APLI F0.400 II 1615 1491IFDC2Ii 0.0 1616 163 JIYIFLCMLSGIj 0.400 1 17 14 1~ YFILIGLPGLII 0 F40 1618 21o IPGLEEAQFWL] 0.400 I-1619 220_ SYLLILKTVL 0.40 1620 4.9 1IIYIVRTEHSL 0.400 jj 1621 197JINVYGLIVII1I0.400[ 1622_ 9f IALGNLTI I0.0 f 16 23 [able XVI: 1O1P3A11-V2-B7-IOmners [tIsuisequence I cre FSeq ID. New] 1624 F[6-7IAVL.SGV'rL1.o[ 1625 1ITLRCPSSWPIII] 01 1626 fl IT[cpsswpisic jT 1627 1 ~IFLLCSTQLSMI 1 1628 1 16 11RC1WPSIj0.400 If 1629 JIfSWPISICWFLI 0.400j 1630 [26c 1 cs 1I~. 1 1.631 4m 1 IAVLAS7VI .200 1L....632 f7 11AVLASGVTLR 110. 150 IF 1633 IV VAGVTLRC71 O.looj 1634 1 CSLYLIAVLAI[.Ot 1635 1LIAVLASGVTI0. 100I 1636 24 1f SICWFLLCST 0.T100 31637 12 fIGVTLRCPSSW'[~o] 1638 [ASGVTLRCPS O.079] 1639 SILASGVTLRCP 10..03011 1640 111 IfSGVTLRCPSS 19 ~[SSWPISICWF 1.2] 1642

[T

1 SLYLIAVLAS 02][ 1643 ISICWFLLCS 0O.020] 1.644 LLSQSE 10 1 1646 [CWFL CST 11 0 03][ 1647 22 [PISICWFLIC]iiT[ 10648 1 27 IWFLLCSTQLS]000] 1650 I 1 LRCPSSWPI7S[f0.0j 1651 3 J LYLIAVLASG]0001 1652 I Iable XVI:3 1OIP3A11-V3-B7-10ners-J F Start Suquencel Score Se D.Nm ACLL OMFAI 4.0I I 1 QFDACLLQM Jio 1655 'i~F-17MFAIHiSLSGM 11. 5o6i I LQM][ IHSLS]I§0 1657 [s ACLLQMFAIH~ .03OE 165 8 6 1 CLQFAHS O.0211 1659 I 2 ][FDACLLMFA 0.01 16620 FD 011 36 able XVII:1O1P3A11-V1-B35-9mers [S ta rt]JISu bsequencel Score Seq. ID Num EPMYIFLCM 140.0001 1,663 274_ LPVILANIY 40.000 1664 3 LPGLEEAQF 113.001 1665 81 IPMLAIFW3000 16 [111 PRHATVL 2000jj 16 7 [69 H LPFCRSNIL 120.0001 1668 [T7~fRSILSSY2000 1669 266 IfFSKRRDSPL 15.0001 1670 [213 11 DSLLI SFSY 110.001 1672 76 1f ISTSSMPCM 1F0.0001 16573 32 1 FPLCLYLI a1 o] 1674 r-10-11 LSGMESTVL- 7.S00[ JL 167 30 LAFPLCSLY] 6.0001[ 1676 105_ FAIEHSLSG- 1677 [~i]JISSYLLIL 1I.ooo[1678 2071 ISAIGLDSL 1 5.000 1679 9 -ESSATYFIL] 16801 CSLYLIAVL] 1I~o]:~z681 I 297 KT ~1IRQRI 1 1-0]L1682 I r_2_8 VPPVLNPIVJ 1 14.0 1683 25 VPFIGLSP-- 11.0] 1684 28 IPPVLNPIVYl 14.000 11 1685 3 [TINGNESSATY 11 4.00 1 1686 3 0 ISAIGLDSLL II3.000 11 168 30 fEIRQR ILRL 3.0001 1688 10]AAVVGAAL IF37.0700 If 1689 [56 HSLHPMYI1j 16901 [121 IfMAFDRYVAIJ1 1691 3 2 5 4 I YVPFI GLSM I 1692 3 [DPNGNESSA 2.000~ 1693 111 IfSGMESTVLL 1[2.000 1 1694 290 IfNPIVYGVKT 1[ 2 00 0I 1695 Table XVII: 1O1PA11-V1-B35-9mers Start Iusflec Scr Tn I 127 211 SPLPVILAN J[2.000ol 1696 244T~ VSHVCAVFI 2000j 1697 11 S SATYF ILI .00O0-1 1698 7791SSMPKMLAI] 12.0001E1 1699 757 I1 SLHEPMYIF j[2.0001J_ 1700 118 Rf LLAMFDRYJ 1701 246 if VCAVFIFY 12.000 1 1702 ".Isl AVVRGAALM 00 011 1703 [7 371 DILISTSSM 1[2.00011 1,704 42 VLGNLTIIY H[2. 000]11 1705 154 1j RGAALMAPL ]2.0001[ 1706 236 jjQAKAFGTCV 1I.800 I 1707 94 TIQFDACLLJ 11.500 1 1708 1821 CLHQDVMKL 0 1.500j11 1709 if IAVLGNLTI 111.20011 1710 87 AVMKLACDDI 1.2001I 1711 22 YLLILKTVL]i o 1712 215 LLISFSYL ]ioo] 1713 214 SLLISFSYL_ 111.R00[ 1714 115 JrS L I4A 1[.oo0][ 1715 -175 JrNILSMSYCL][io][ 16 67 JrCMLSGIDIL 11 738 1 YLIAVLG;NL 1718 .3 TTIQFDACL 1719 7s STSPM 11.0] 1720 2 jAQFWLAFPL 5:10001 JE 1722 243 IfCVSHVCAVF ]iooj 12 [276 IfVILANIYLLI.oI 1724 [204) IVIISAIGL 1r.000 1725 29 IfWLAFPLCSL 111.0001 1726 IfSMPKLo.IAIF 1[.000l 1727 [T15-57- ALMAPLPVF -55 -1728 [223 11 LILKTvLGLT 1[~ol 1729 249 IfAVFIFYVPF 1i.oool[ 17-30 [102 ILIFAIHSL f1.000[F 1731 F1 9 HSYCLHQDV 1.000~ 1732 F-01YIVRTEHSL- 1.000W 1735 [190 IfLACDDIRVN I[MF90 1736 [19-611 RVNVVYGLI 110.80011 1737 [691 LSGIDILIS HJr0.7so 11 1738 14 TiGVAAV Jr .600 1739 AALMAPLPV_ 110.600 11 1740 fable XVII:1O1P3A11-V1-.B35-9mers ~Start jSubsequencej cr Seq. ID Num] [189I KLACDDIRV 0.0 1741 1197 LAMAFDRYV Io&o 1742 231 LTREAQAKA 10.60-01 1743 [209J AIGLDSLLI 0.01 1744 S22 ~fGLEEAQFWL 0.0 f 1745 21 DSPLPVILA oso_ 1746 21 SYLLILKT 0511 1747 78 JrTSSMPKMLA Io.sal] 1748 14T ESTVLLAMA If0.5001 1749 1081 HSLSGMEST If 0.500j 1750 91 NSTTIQFDA 10.5001 1751 I43 jGNLTIIYI J0O01 1752 58~J NVVYGLIVIr nA__ 1753 19 VVYGLIVII 10 .4001DO 1754_ F27__7 LISFSYLLI 0.400 22 GIJIVIISAI 0.400 756 IP2 4__1 0.40011 1757 15-8 1 L14APIPVFI 10.4001 1758 r168]!1 MLSGIDILI 10.4001 1759 "168 1r QLPFCRSNI 10.4001 1760 LCMLSGIDI 110.40011 1761 11 41I AVLGNLTII I1.0 1- 1762 frable XVII:1O1PA11-V2-B35-9mers IstartliSubsequence ScrS-e ID. Num] WPISICWFLJ12.01 1763 I[CPSSWPISI 18001 1764 1 is SSWPISICW 11.0 I 1765 F LLCSTQLSM A12.oo0I0 0 17,6 F67] VLSTL] 1 767001 2~I VTLRCPSSW 0.500 17,68 22 I SICWFLLC 10-500 1 769 18EI LASGVTLRC 11.011770 5 fIAVLASGVT II030! 1771 VT1LIAVLASGV 02011772 1s[RCPSSwpIS I01I0j 1773 1SWPISICWF 0f .10011 1774 1 2 IfWFLLCSTQ 10.100 1775 S7I FLLCS TQLS J110. 10 0 1776 1 IIGVTLRCPSS 1f 0. 100] 1777 _j 3 ESICWFLLCS If101 1 778 I1SGVTLRCPS' 110.100 1779 21 PISICWFLL 0.1001 1780 1 SLYLIAVLA 0.10 78 ~T1 ICWLLCST 0.1001 78 00 00 00 Table XVII: 101P3All-V2-B35-9mer9 Start Susqec Jcor Seq ID. Num [9 ASGVTLRCP 0[.050 1783 17 1PSSWPISIC 0.050T 1784 14 LRCPSSWPI [0.040[ 1785 [13 IfTLRCPSSWP 0F.030[ 1786 _29 IF LCSTQLSME 0j.010] 1787 YLIAVLASG 0~.010] 1788 7 VLASGVTLR if0.0 101 1789 2 YLIAVLAS O.i~ 1790] 11CWFLCST -EO.0 1 79jZ1 Table XVII: 101P3A11-V3-B35-9mers [Starti Subsequence ScorejjSeq ID. NumI 4 ][ACLLQMFAI=04001 1793 3 J[DACLLQMFA 0.300 19 8 J[ QMFAIHSLS 10.1001 79 2 ir FDACLLQMF 10.1001 1796 6 fLLQMFAIHS 00b 1797 1 .06 1798 1 CLLQMFAIH 0.0101 1799 9 MF NAIHSLSG 001 10 fable XVIII: 1O1P3Al1-VI-B35-lomers Start tSubsequence ISCor Seq. ID Numj 81 JIMPKMLAIEFWP 60.00011 10 285 IfVPPVLNPIVY140.0001 1802 YFLCM 20.000 1803 160 APLPVFI KQL]1006 1804 162 1f LPVIKLP 2 .0 1805 274 11 LPVILANIYL 12 0. 000 1806 J I~hLPGLEEAQFW 1[15.000 L_1807 297 ]fKTKEIRQRIL 1_2.000 1.808 179 [HSYCLHQDVMI10.000J 1809 272 ItSPLPVILANII1 8.o00 1810 2141J[LPRVTKIGVA][ 6.000 1811 150 ][AVVRGAALM[ 6.000 1812 [231 ifLTREAQAKAF f6.000~ 1813 3101 LSG.MESTVLL 5~ .000] 1814 [114 IfESTVLLAMAF J5.000 1815 213IDSLLISFSYL 1816 E207iISAIGLDSLL 5.000~ 1817 76__ ISTSMKM 500 1618 I79 ISSMPKMLAIF s.000] 1819 .070F 1821 5.200_ 1 82 jj~able XVIII: lOlP3All-V1-. B3S-10mers I 'tStart Subeuence Scre~Seg. ID Numj 15~6]1AALMAPLPVFI 3. 000 1 1822 I LFPLSLY 113. 0 1823 fDPNGNESSATJ[13.000[ 1824 [300J EIRQRILRLF~I3 oo000 1825 149 3. 0 0 0 1826 194I[DIRVNVVYL[ 0 0 0 1827 248 CAVFIFYVPF If.000 1828 11 ]SATYFILIGLI[3.O000 1829 25~ EAQFWLAFPLI3.o0o0I[ 1830 157]ISLHEPMYIFL 2. 000 1831 J 169 JLPFCRSKILS][ 2.000][ 1832 [2T1 ILDSLLISFI[Moo1 1833_ [6 I1NGNESSATYFJ 2.00011. 1834 [2 1 WLAFPLCSLY IiI 18350 [115LISTSSMPKM f2oI 1836 1 1 3 T1 HPLRIri 1 TI1T2.000] 1837 41IAVLGNLTIIY'1200I 1838 [h ESSATYFILI7 2.000iI 1839

K

3 1 FPLCSLYLIA[2.oolI 1840 71171 VLLAMAFDRYD 2. 000 11 1842 28J[SAIGLDSLLI ie0[ 1843 K~h~~sLGMESv~j1.50 If 1844 [TT]YCLHQDVMKL ioof 1845 19 IQDAL 1846 1 [GLPGLEQF~f150~ 1847 190 I[LACDDIRVNVI[.01 1848 98 ][DACLLQIFAII[1.20011 1849 302 ][RQRILRLFHV II1.200j 1850 40I IAVL3N-LTIII[1.200J 1851 238 if KAFGTCVSHV)[1.200[ 1852 92 ][STTIQFDACL)[1.000] 1854 13]RSNILSHSYCI[1.000[ 1855 168 [QLPFCRSNIL][1.000] 1856 216 LISFSYLLILIF1.000l 1857 J 174 LSSYL1.000 185 214 IfSLLISFSYLLf 1. 0001 1860 206 I ISAIGLDSL f1.00011 1861 34 j(LCSLYLIAVLII1.00011 1862- 242 IfTCVSHVCAVFII fool 1863 203 jILIVIISAIGL 1.000 L- 1864 2 2 2 j L L Q I L K T V L 1 f.0 0 0 E jjj1 8 able XVIII: 1OlP3A1.1-V1-B35-lo0mers StartJ Subsequence Score j e.I u [280 ]NIYLLVPPVL] 1.00011 18-67 [219 FSYLLILKTV 1.000~ 1869 257 IFIGLSMVHRF 1.000 251 FIFYVPFIGLJI1.00011 1870 _108__ HSLSGMESTV][1.000J 1871 J 2361 QAKAFGTCVSJ 0.900j E 1872 j IQFDACLLQI 0f 800J 1873 167 KQLPFCRSNI 0. 800] 1874 1213. MAPDRYVAIC][0.600 1875 144 J[VTKIGVA J0 .600 1876] 155 GAALr4APP 0.60_187 112 GMESTVLLAM f0.60011 1878 1S HATVLTLPRV 0.601 1879[Ziz 69 LSGIDIL 1ST 0.500-1 271 JDSPLPVILAN 0.50o 1881 SMPKMLAIFWIIO.SOOJL 1882 91 flNSTTIQFDAC- 0.5001 1883 268 KRDSPLPVI 0484 119 JLAMAFDRYVAI0.450] 1 8815 201 YGLIVIISAI 10.4001 186 1-98 NVVYGLIVII 10.40011 1887 243 JCVSHVCAVFI 0o.400 1888 283 LLVPPVLNPI 0.400- 188.9 -67 11 CKLSGIDILI 0.400__ 1890 10AIIAFDRYVAI110.4001 1891 11FLCMLSGIDIII0.400D 1892 42 IVLGNLTIIYI 0.400 1893 186 DKLCDI 10.400 I 1894 39 J[LIAVLGNLTI 10.400 I 1895 157 ALMAPLPVFI 1000 1896 22 IVYGVKTKEI 10.4001 1897 13 RVTKIGVAAV F0.400~ 1898 86 AIFWFNSTTI 0. 4001 1899g 19 1RVNVVYGITV 0.-4001 1900-i able XVIII:101P3A1-V2-B35-10mero Star Ssqunc Sco e ID. New 21 WI1CFL20.0001 1901 19 SSWPISICWF 5.000l 1902] 6 IAVLASGVTL [3.0001 1903 J 28 LLCTQLSM 2.000 1904___ 14 TTRCPSSWPi: 1.200 1906 16 ]RCPSWPIS 0.80~j 1907 Table XVIII:101P3AI1-V2-B35-10mers EStartJ Subequence Score seg ID. New -3 IIWLC .0 1908 10n] ASGVTLRCPS][0.500[ 1909 [12 [GVTLRCPSSW [1 0. __00 ___191 1 CSLYLIAVuL][0.50011 1911 [18 ifPSSWPISICW]0. 250 J 1912 [1l YLIAVLASGV, 0.200] 193 26 JCWPFLLCSTQL 11 0.o10[ 1914 SLYLIAVJAS,1 0.100][ 19 11 IAsGVTLRCs 1111001 1918 24ISwISCWFL jo0.100 Ii 19197 9 IAVTLRCVI o.0011 1920 J I-A VTL _P 0 3_ 1921 7[ ALAGLRI .11 1922 15 ]1 LRCPSSWPISI0. 0101 1923 I27 ][WFLLCSTQLs.SLo.loj 1924 jl LLCSTQLSM~EIO.o1o I 1925 3 22]PISI WFLLC][-O.-01 1i926 I TC~WTT QO 0.o1 I1927_ -13 ]vTRucpsswrP][5. po 192 __3__[jLYLALSGT.B 0 0111A 192 971 Iable XVIII:1O1P3A11-V3-B35-10mers] Star][Suse ece:core ISeq ID. Num] 1 ifIQFDACLLQM14.00[_ 1930 4 ]DACLLQIIFAI 1.01 13 7 [LLQMFAIHSL [10 MFAISLSG 0. 193 [6 CLLQMFAIH 0.1001 1934 3 [FDCTLOnFv 0.030 196 LLQMFAR -10 1937 00 Table XXX: Motifs and Post-translational Modifications of lOlP3Al N-glycosylation site Number of matches! 3 t1 7-10 NESS 00 2 44-47 NLTI 3 90-93 NSTT cAMP- and cGMP-dependent protein kinase phosphorylation site 268-271 RRDS Protein kinase C phoephorylation site 266-268 SJUR Casein kinase XI phosphorylation site Number of matches; 3 00 1 56-59 SLHE 2 69-72 SGID 3 110-113 SGME N-myristoylation site Number of matches: 4 1 6-11 GNESSA 2 21-26 GLEEAQ 3 111-116 GMESTV 4 240-245 GTCVSH 0-protein coupled receptors family I signature 112- 128 MESTVLLAMAFDRYVAI 00 00

O

O0

(N

Table XX: Motifs vrg. Name Description Potential Function identity Nucleic acid-binding protein functions as 6-0H2 34% Zinc finger, C2H2 type transcription factor, nuclear location probable Cytochrome b(Ny;tochrome b N 58% terminal)/b6/petB nembrane bound oxidase, generate superoxide domains are one hundred amino acids long and S19% Immunoglobulin domain include a conserved intradomain disulfide bond.

tandem repeats of about 40 residues, each containing a Trp-Asp motif. Function in signal WD40 18% WD domain, G-beta repeat transduction and protein interaction may function in targeting signaling molecules to Z 23% PDZ domain sub-membranous sites short sequence motifs involved in protein-protein -B 28% Leucinc Rich Repeat interactions conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic Akise 23% Protein kinase domain site leckstrin homology involved in intracellular H 16% PH domain signaling or as constituents of the cytoskeleton 3040 amino-acid long found in the extracellular omain of membrane-bound proteins or in EG 34% EGF-like domain secreted proteins Reverse transcriptase (RNA- 3 49% dependent DNA polymerase) Cytoplasmic protein, associates integral lk 25% Ank repeat membranc proteins to the cytoskeleton

NADH-

Ubiquinone/plastoquinone membrane associated. Involved in proton axidored gq 32% (complex various chains translocation across the membrane calcium-binding domain, consists ofal2 residue loop flanked on both sides by a 12 residue alphafhand 24% EF hand helical domain Aspartyl or acid protcases, centered on a catalytic V 79% Retroviral aspartyl protease aspartyl residue extracellular structural proteins involved in formation of connective tissue. The sequence ollagen triple helix repeat (20 consists of the G-X-Y and the polypeptide chains o n 42% copies) forms a triple helix.

Table XX: Motifs Name ar.%DsrpinPotential Function identityDsriin Located in the extraccilular ligand-binding region of receptors and is about 200 amino acid rcsiducs long with two pairs of cysteines involved in n 20% Fibronectin type [if domain disulfide bonds seven hydrophobic transmembrane regions, with the N-terminus located extracellularly while the 7transmembrane receptor -terminus is cytoplasmic. Signal through G 19% (rhodopsin family) roteins 00 00 TABLE XXI: Properties of 1O1P3AII Bioinformatic Variants I and 3 Program URL Outcome ORF ORF Finder http://www.ncbi.nlrmgov/gorf 133-1086 Protein Length Transmembrane region n/a TM Pred HMMTop n/a http:/www.ch.emnbnet.org/ http:/www.enzim.hu/hmnitop/ http:/www.genomc.adjp/SOSuV/ Sosui TMHMM http/www.cbs.dtu.dk/services/TMHM (includes stop) 317 amino acids 7 TMV at an: 27-52, 63-88, 104- 129, 146-165,1396- 224, 239-262,273.

295 7 TM at an: 27.50, 63-86, 99.

121, 146-165,201- 224, 239-262, 275- 294 6 TM, at an: 29-5 1, 63-85, 100- 122, 203-225, 239- 261, 273-295 7 TM, at aa: 29-51, 63-85, 100- 122, 143-165, 202.

224, 236-258, 273- 295 indicates no signal p1 8.7 35.2 kfla Plasma membrane 64% Plasma membrane 56.4% 7 transmembranc receptor (rhodopsin family) Rhodopsin-Iike GPCR supcrtfniily Rhodopsin-like GPCR superfamily G-protcin coupled receptors family I Signal Peptide p1 Molecular weight Localization Signal P p11MW tool p1/MW tool

PSORT

PSORT 11 http://www.cbs.dtu.dklscrvices/SignalP ht//wwepshtos http://www.cxpasy.ch/tools( http://psotwi.xacy.jptoos http://psort.nibb.acjp/ http://www.sangcr.ac.uk/PfarrV http://www.biochem.ucl.ac.uk/ http://www.blocks.fhcrc.org/ http://www.gcnome.adjp/ Motifs Pfarn Prints Blocks Prositc IOIP3AI I var.2 Bioinforrnatic URL Outcome Program ORF ORF finder I 30-348bin including stop Protein length Transmembrane region TM Pred HMMTop Sosui

TMHMM

http//www.ch.cmbnct.orgt http://www.cnzim.hu/hmmtiopt http://www~genome.ad.jp/SOSui/ http://www~cbs.dtu~dktscryices/TMHMM 72aa 2TM helices na28-49, 55-72 N tcrzninus cxtraccllular 2TM helices N terminus extracellular 2TM helices aa28-S0, 52-72 I TM helix, aa27-49 1HA I I var.2 Bioinformatic URL Outcome Program Signal Peptide Signal P hitp:/fwww.cbs.dtu.dkc/scrviccs/SignalP/ no p1 p1/MW tool http://www.expasy.chtools/ p1 4.12 Molecular weight p1/MW tool http://www.expasy.ch/tools/ 7.95kB Localization PSORT http://psort.nibb.ac.jp/ 82% extracellular, 16% peroxisonic PSORT I I http://psort.nibb.ac.jpl 39% cytoplasmic, 17% mitochondrial, 17% nuclear Motifs Pfam http://www.sanger.ac.uk/Pfar/ no Motifs found Prints http://www biocheinucl~ac.uk/ no Motifs found Blocks http://www blocks.fhcrc.org/ Zcin sced storage protein Table XXII 101P3A11 vl. HLA-A1 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 246 H V C A V F I F Y 24 1940 L AF P L C S L Y 21 1941 42 V L G N L T I I Y 21 1942 286 P P V L N P I V Y 20 1943 112 G M E S T V L L A -19 1944 118 L L A M A F D R Y 19 1945 173 R S N I L S H S Y 19 1946 193 D D I R V N V V Y 19 1947 213 D S L L I S F S Y 19 1948 58 L H E P M Y I F L 18 1949 23 L E E A Q F W L A 17 1950 S S A T Y F I L I 16 1951 53 R T E H S L H E P 16 1952 E H S L H E P M Y 16 1953 79 S S M P K M L A I 16 1954 96 Q F D A C L L Q I 16 1955 160 A P L P V F I K Q 16 1956 184 H 0 D V M K L A C 16 1957 2 M V D P N G N E S 15 1958 6 N G N E S S A T Y 15 1959 211 GLDSLLISF 15 1960 274 L P V I L A NIY 15 1961 272 S P L P V I L A N 14 1962 92 S TTIQFDA C 13 1963 122 A F D R Y V A I C 13 1964 139 LTLPRVTKI 13 1965 219 F S Y L L I L K T 13 1966 283 L L V P P V L N P 13 1967 191 A C D D I R V N V 12 1968 192 C D D I R V N V V 12 1969 232 T R E A Q A K A F 12 1970 269 R R D S P L P V I 12 1971 271 D S P L P V I L A 12 1972 12 A T Y F I L I G L 11 1973 22 GLEEAQFWL 11 1974 177 L S H S Y C L H Q 11 1975 217 I S F S Y L L I L 11 1976 Table XXII 101P3A11 v2 HLA Al 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 22 I S I C W F L L C 15 1977 18 S S SWP ISICW W 14 1978 8 L A S G V T LR C 9 1979 23 S I C W F L L C S 8 1980 2 L Y L I A V L A S 7 1981 7 VL A S G V T L R 7 1982 12 V T L R C P S S W 7 1983 28 L L C S T Q L S M 7 1984 Table XXII 101P3A11 v3 HLA Al 9-mers Pos I 1 2 3 4 5 6 7 8 9 score I SEQ ID Table XXII 1OlP3All v3 HLA Al 9-mers Pos 12 3 4 56 78 9 score SEQ ID Q FDA C L LQM 16 1985 Table XXIII lO1P3All vi. HLA-A0201 9-mers POO 1 234 56 78 9 score SEQ ID 288 V L NPI V YGV 30 1986 FI L I GLP GL 29 1987 29 WLA FP L CSL 28 1988 38 Y LI A VL GNL 28 1989 223 LI LK TV L GL 28 1990 67 C ML S GI DIL 26 1991 109 SL SG ME ST V 26 1992 182 C LH Q D VM KL 26 1993 202 G L IVI I SA 1 26 1994 215 LL IS FS Y LL 26 1995 276 V IL A N IYLL 26 1996 158 L MA PLP VFI1 25 1997 221 Y LL ILK T VL 25 1998 277 1L AN I YLL V 25 1999 280 N IY L LV PPV 25 2000 139 LT LP RV TKI1 24 2001 214 SL L ISF S YL 24 2002 s0 Y IV RT E HSL 23 2003 144 V T K IGVA AV 23 2004 189 KLA C D DI RV 23 200S 199 VV YG LI VII1 23 2006 22 GL EE AQ0F WL 22 2007 41 AV L GNL TII1 22 2007 207 1IS AI GL DSL 22 2008 12 AT YF I LI GL 21 2009 61 PM YI FL C ML 21 2010 136 AT VL TL P RV 21 2011 161 P LP V F IKQL 21 2012 175 NIL S HS Y CL 21 2013 208 SA I GL DS LL 21 2014 273 PL PV IL AN 1 21 2015 284 L VPP VL NPI1 21 2016 68 M LSG ID ILI1 20 2017 102 L QI F AI HSL 20 2018 283 LL V PPV L NP 20 2019 300 E I RQ RI LRL 20 2020 305 1IL R LFHV AT 20 2021 1IA V LG NLT1 19 2022 46 L TI IYI V RT 19 2023 93 T T 10FD A CL 19 2024 ill SGM E ST V LL 19 2025 128 A IC HPL R HA 19 2026 133 LR HA T VL TL 19 2027 150 AA VV RG A AL 19 2028 156 AA LM A PL PV 19 2029 157 AL MA PL P VF 19 2030 204 I V I I S A I G L 19 2031 209 A IG LDS LLI1 19 2032 00 00 00 XXIII 1O1P3A11 vi. HLA-A0201 9-merB Poe 12 34 5 67 89 score SEQ ID 217 I S F S Y L L I L 19 2033 220 SY L LI L KTV 19 2034 222 L LIL K T VLG 19 2035 224 -1L KT V LG LT 19 2036 18 G LP G LE EA 18 2037 34 LC SL Y L IAV 18 2038 CS L YL I AVL 18 2039 39 LI AV L G NLT 18 2040 44 GQN LTI I YI V 18 2041 86 A I FW FN STT 18 2042 119 LA MA F DR YV 18 2043 195 1R V NVV Y GL 18 2044 211 G LD SL L ISF 18 2045 216 LI S FS Y LLI 18 2046 247 VC AV F I FYV 18 2047 255 VP F IG LS MV 18 2048 16 1IL10L P G LE 17 2049 64 1IFLC ML SGI1 17 2050 73 DI LI ST S SM 17 2051 94 T1Q0F DA C LL 17 2052 99 AC L L (I FAI1 17 2053 112 GM ES T V LLA 17 2054 121 MA F DRY VAI1 17 2055 168 Q LP FC RSNI1 17 2056 198 NV VY GL IVI1 17 2057 227 T V L L TR EA 17 2058 282 Y LL V PP VLN 17. 2059 32 FP LC SL YLI1 16 2060 57 SL H EP M YIF 16 2061 71 GI DI LI S TS 16 2062 79 S S M PK ML A 1 6 2063 SM P K ML AIF 16 2064 105 -FA I HS LS GM 16 2065 120 AM AF DR Y VA 16 2066 145 T KIG0V A AVV 16 2067 148 GV A AV VR GA 16 2068 187 VM K LA CDDI1 16 2069 231 -L TRE AQ0A KA 16 2070 239 AF GT C VS HV 16 2071 250 VF IF Y VPFI1 16 2072 303 Q RIL RL F HV 16 2073 304 RI L R LF HVA 16 2074 19 GL PG L E EAQ 15 2075 36 S LY LI A VLG 15 2076 43 LG N LT I I YI 15 2077 47 TI IY IV R TE 15 2078 S GI DI LI ST 15 2079 77 S T SS M PK ML 15 2080 132 P LR HAT V LT 15 2081 138 VL TL PR V TK i5 2082 154 R GAA LM A PL 15 2083 191 -AC D DIR V NV 15 2084 192 CD DI RV N VV 1s 2085 205 VI IS A I GLD 15 2086 242 TC VS HV C AV 15 2087 Table XXIII 101P3A11 vl. HLA-A0201 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 252 I F YVPFIG L 15 2088 270 R D S P L P V I L 15 2089 281 I Y L L V P P V L 15 2090 307 R L F H V A T H A 15 2091 17 L I G L P G L E E 14 2092 26 A Q F W L A F P L 14 2093 42 V L G N L T I I Y 14 2094 63 Y I F L C M L S G 14 2095 74 I L I S T S S M P 14 2096 L AIFWFNS ST 14 2097 100 C L LLQI F A I H 14 2098 118 L L A M A F D R Y 14 2099 130 C H P L R H A T V 14 2100 151 A V V R G A A L M 14 2101 169 L P F C R S N I L 14 2102 201 Y G L I VI I S A 14 2103 219 F S Y L L I L K T 14 2104 236 Q A K A F G T C V 14 2105 269 R R D S P L P V I 14 2106 297 K T K E I R Q R I 14 2107 Table XXIII 101P3A11 v2 HLA A0201 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 4 L I A V L A S G V 24 2108 3 Y L I A V L A S G 22 2109 6 A V L A S G V T L 22 2110 1 S L Y L I A V L A 19 2111 7 V L A S G V T L R 19 2112 28 L L C S T Q L S M 19 2113 23 S I C W F L L C S 16 2114 21 P I S I C W F LL 15 2115 27 F L L C S T QL S 15 2116 24 I C W F L L C S T 13 2117 14 L R C P S S W P I 12 2118 W P I S I C W F L 12 2119 26 W F L L C S T Q L 12 2120 .8 LASGVTLRC 11 2121 13 T L R C P S S W P 11 2122 Table XXIII- 101P3A11 v3 HLA A0201 9-mers Poo 1 2 3 4 5 6 7 8 9 score ,SEQ ID 7 L Q M F A I H S L 19 2123 C L L Q M F A I H 14 2124 4 A C CLLQMFAI 13 2125 6 L L Q M F A I H S 13 2126 1 Q F D A C L L Q M 9 2127 8 Q M F A I H S L S 9 2128 Table XXIV: 101P3A1l- Vl HLA-A0203 9-mers- No Results.

Table XXIV: 101OP3A11- V2 HLA-A0203 9-mers- No Results.

Table XXIV: 101P3A11- V3 HLA-A0203 9-mers- No Results.

Table XXV 101P3A11 vl. HLA-A3 9-mers 00 Pos 12 34 5 67 89 score SEQ ID 138 VLT LP R V TK 30 2129 Ct 230 0 T R E AQ AK 27 2130 n-146 K I GVAA VVIR 26 2131 00 151S A VVR G AA LM 24 2132 291 P IVY GV K TK 24 2133 -36 S L YL I AVLG 23 2134 157 AL MA P LP VF 23 2135 ~~48 1I IY IV R TEH 22 2136 ID 51 1IV RT EHS L H 22 2137 143 RVT K IG V AA 22 2138 152 VV R GA AL MA 22 2 13i9 243 CV S HV C A V 22 2140 C1 249 AVF I FY V PF 22 2141 00 117 V LLA MA F DR 21 2142 193 DDI R V NV VY 21 2143 304 RI LR L FH VA 21 2144 305 1L RL FH V AT 21 214S 199 V VY G L IV 1 20 2147 292 1IV Y GV KT KE 20 2148 16 1ILIG LP G LE 19 2149 NLT I IY I VR 19 2150 74 1 LXS TS S MP 19 2151 LI ST SS M PK 19 2152 100 C L LQ1F AI H 19 2153 163 P VFI KQ L PF 19 2154 204 1V II SA I GL 19 2155 222 LL I LKT V LG 19 2156 246 H VC AVF I FY 19 2157 307 R L F HV ATHA 19 2158 41 A V LG0NL TI 1 1 2159 86 A IF WF NS TT 1 2160 206 1 IS AIGL D S 18 2161 221 YLL I LK T VL 18 2162 254 Y VP FI G L S 18i 2163 38 Y LIA VL G NL 17 2164 42 V LG NL T IIY 17 2165 118 L LA M AF DRY 17 2166 132 PL R HAT V LT 17 2167 137 T VLT L PR VT 17 2168 181 Y CL H0D V MK 17 2169 202 C L IV II SA1 17 2170 214 S L L ISFS YL 17 2171 257 1 0L S MV HR 17 2172 262 M VH R FS XRR 17 2173 277 1IL A KIY LLV 17 2174 282 YL LV P PV LN 17 2175 287 PV L NP IV YG 17 2176 289 LN P IV YG VK 17 2177 310 HVA T HA S EP 17 2178 2 M V DP N G NES 16 2179 57 SL HE PM Y IF 16 2180 71 G ID IL IS TS 16 2181 73 DI L IS T SSM 16 2182 116 TV L LA M AFD 16 2183 126 YV AI CH P LR 16 2184 00 00 M ND 0

O-

00

O-

m c Table XXV 101P3A11 vi. HLA-A3 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 145 T K I G V A A VV 16 2185 168 Q L P F C R S N 16 2186 176 L S H S Y C L H 16 2187 196 R V N V V Y G L I 16 2188 198 N V V Y G L I V I 16 2189 211 G L D S L L I S F 16 2190 283 L L V P P V L N P 16 2191 300 E I R Q R I L R L 16 2192 302 R Q R I L R L F H 16 2193 17 L I G L P G L EE s15 2194 47 T I I Y I V R T E 15 2195 103 Q I FP A I H S L S 15 2196 194 D I R V N V V Y G 15 2197 209 A I G L D S L L I 15 2198 224 I L K T V L G L T 15 2199 238 K A FP G T C V S H 15 2200 6 N G N E S S A T Y 14 2201 63 Y I F L C M L S G 14 2202 101 L L Q I F A I H S 14 2203 140 T L P R V T K I G 14 2204 148 G V A A V V R G A 14 2205 165 F I K Q L P F C R 14 2206 189 K L A C D D I R V 14 2207 225 L KTVL G LTR 14 2208 227 TVL G L T R E A 14 2209 256 P F I G L S M V H 14 2210 275 P V I L A N I Y L 14 2211 284 L V P P V L N P I 14 2212 299 K E I RQRIL R 14 2213 Table XXV 101P3A11 v2 HLA A3 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 6 A V L A S G V T L 28 2214 1 S L Y L I A V L A 23 2215 3 Y L I A V L A S G 21 2216 7 V L A S G V T L R 18 2217 13 T L R C P S W P 17 2218 4 L I A V L A S G V 15 2219 11 G V T L R C P SS 15 2220 28 L L C S T Q L S M 15 2221 Table XXV 101P3A11 v3 HLA A3 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID .5C LLMFA IH19 2222 6 L L Q M F A I H S 13 2223 1 Q F D A C L L Q M 10 2224 Table XXVI 1O1P3A11 vl. HLA-A26 9-mers PoS 1 2 3 4 5 6 7 8 9 score SEQ. ID No.

Table XXVI lO1P3AIl vi. HLA-A26 9-mers I Pos 1 2 3 4 5 6 7 8 9 I score I SEQ. ID No.

300 E IR0R I L RL 30 2225 73 D IL IS TS SM 27 2226 249 AV FI FY V PF 27 2227 211 GL D SL LI SF 26 2228 FI LI G LP GL 24 2229 57 SL HE P M Y IF 24 2230 118 L L A MAF D RY 24 2231 223 L I L KT V LGL 24 2232 246 H VC A VFI FY 24 2233 12 A TY FI LI GL 23 2234 38 Y L IA VLG NL 23 2235 115 ST VL L AM AF 23 2236 157 AL MA P LP VF 23 2237 163 P VF I KQL PF 23 2238 182 CL HQ0D V M KL 23 2239 29 W L A P L CSL 22 2240 93 TT IQ FD A CL 22 2241 161 P LPV F I KQL 22 2242 204 1V II SA I GL 22 2243 214 S L LIS FS YL 22 2244 276 VI LA NI Y LL 22 2245 194 D IRV NV V YG 21 2246 243 C VS HVC A VF 21 2247 77 S TSS MP K ML 20 2248 254 YV PF I G LSM 20 2249 275 PV IL A N IYL 20 2250 24 EEAQ0F W L AF 19 2251 42 V LG N L TIIY 19 2252 Y IV RT E HSL 19 2253 151 A VV R GA ALM 19 2254 175 N I LS HS YC L 19 2255 193 DDIR VN V VY 19 2256 215 L LI S F SY LL 19 2257 252 1F YV PF I GL 19 2258 9 ES S AT YF IL 18 2259 22 G L EE A0F WL 18 2260 46 LT II YI V RT 18 2261 E HSL H E PMY 18 2262 E P M YIF LCM 18 2263 89 WF N ST T 1QF 18 2264 94' T IQ FD A CLL 18 2265 186 D V M K L A C D D 18 2266 199 VV Y GL I VI1 18 2267 63 YI F LCM L S G 17 2268 71 G ID IL IS TS 17 2269 S MPK ML A IF 17 2270 97 FDA C LL Q IF 17 2271 105 FA I HSL S GM 17 2272 139 LTL P RV TKI1 17 2273 144 VT KI GV A AV 17 2274 205 VII S AI GL D 17 2275 213 DS L LI SF SY 17 2276 221 YL LI L KT VL 17 2277 257 F I G LS MV HR 17 2278 284 LV P PVL NPI1 17 2279 Table XXVI 1O1P3All vi. HLA-A26 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ. ID No.

L AF PLC S LY 16 2280 41 A VLG NL TII1 16 2281 47 TI IY IV R TE 16 2282 53 RT E HS L HEP 16 2283 76 1IST SS M PKM 16 2284 92 -ST T IQF DA C 16 2285 136 ATV L TL P RV 16 2286 148 G VA A VV RGA 16 2287 202 GL IV I ISAI1 16 2288 258 1G LS M VH RF 16 2289 280 NIY L LV P PV 16 2290 31 AF PL C SL YL 15 2291 102 L QI F AIH S L 15 2292 116 T vLL A M AFD 15 2293 128 AI C HPL R HA 15 2294 154 IRG A ALM A PL 15 2295 164 V FI KQ L PF 15i 2296 216 L ISF SY LLI1 15 2297 217 1ISF S YL LIL 15 2298 226 KT V L L T RE 15 2299 273 P LP VI L ANI 15 2300 283 L LV P V L NP 15 2301 287 IPV LN PI V YG 15 2302 288 VL NP IV YG v 15 2303 297 KT KE I RQ RI is 2304 304 R IL RLF H VA 15 2305 2 M VD P NG N ES 14 2306 6 N GNE S'S A TY 14 2307 33 PL C SLY L IA 14 2308 -CS LY LI A VL 14 2309 58 L HE PM Y IFL 14 2310 82 -P KML A IF WF 14 2311 100 CL LQ I F AIH 14 2312 196 RV NVV Y GLI1 14 2313 198 NV V YGL IVI1 14 2314 207 1IS AI0L DS L 14 2315 208 S AIG LD S LL 14 2316 227 TV LG L TR EA 14 2317 231 LT R EAQ0A KA 14 2318 245 SH VC A VF IF 14 2319 291 PI V YGV K TK 1 14 2320 301 1R QR IL R LF 1 14 2321 Table XXVI 101P3A11 V2 HLA A26 -9-mets Pos 12 3 4 56 789 score SEQ ID 6 AV L AS G VTL 20 2322 21 P ISI C WF LL 18 2323 28 LL CS TQ L SM 18 2324 3 Y LI A V LASG 16 2325 19 SW PI SI C WF 16 2326 26 W F LL CS TQL 15 2327 4 LI AV LA S GV 14 2328 7 V L ASGV T LR 14 2329 Table XXVI 10IP3Al1 v2 HLA A26 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 23 S I C W F L L C S 14 2330 11 G V T L R C P S S 12 2331 12 V T L R C P S S W 12 2332 W P I S I C W F L 11 2333 27 F L L C S T Q L S 10 2334 1 S L Y L I A V L A 9 2335 13 T L R C P S S W P 9 2336 Table XXVI 101P3AII v3 HLA A26 9-mers Poo 1 2 3 4 5 6 7 8 9 score SEQ ID 1 Q F D A C L L Q M 20 2337 2 F D A C L L Q M F 18 2338 C L L Q M F A I H 14 2339 7 L Q M F A I H S L 13 2340 6 L L Q M F A I H S 9 2341 Table XXVII 1O1P3All vl. HLA-B0702 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 131 H P L R H A T V L 22 2342 E P M Y I F L C M 21 2343 169 L P F C R S N I L 20 2344 290 N P I V Y G V K T 19 2345 4 D P N G N E S S A 18 2346 L P G L E E A Q F 18 2347 141 L P R V T K I G V 18 2348 285 V P P V L N P I V 17 2349 32 F P L C S L Y L I 16 2350 255 V P F I G L S M V 16 2351 270 R D S P L P V I L 16 2352 150 A A V V R G A A L 15 2353 154 R G A A L M A P L 15 2354 157 A L M A P L P V F 15 2355 252 I F Y V P F I G L 15 2356 300 E I R Q R I L R L 15 2357 9 E S S A T Y F I L 14 2358 29 W L A F P L C S L 14 2359 31 A F P L C S L Y L 14 2360 111 S G M E S T V L L 14 2361 133 L R H A T V L T L 14 2362 160 A P L P V F I K Q 14 2363 223 L I L K T V L G L 14 2364 272 S P L P V I L A N 14 2365 26 A Q F W L A F P L 13 2366 110 L S G M E S T V L 13 2367 125 RY V A I C H P L 13 2368 217 I S F S Y L L I L 13 2369 269 R R D S P L P V I 13 2370 281 I Y L L V P P V L 13 2371 12 A T Y F I L I G L 12 2372 C S L Y L I A V L 12 2373 Table XXVII 1O1P3A11 v1. HLA-B!0702 9-mers Pos 12 34 56 7 89 score SEQ ID 58 LH EP MY I FL. 12 2374 77 ST SS MP K ML 12 2375 143 RV T KIG V AA 12 2376 152 VV R GAA L MA 12 2377 191 AC DD IR V NV 12 2378_____ 195 1R VN VV Y GL 12 27 207 1IS AI GL DSL 12 208 SA IG L DS LL 12 28 221 YL LI LK T VL 12 2382_____ 268 KR RD S PL PV 12 28 305 1ILJRL F HV AT 12 28 F IL IGL P GL 11 28 24 EE A QFW L AF 112386 38 YL IA VL G NL 112387 41 AV LG N L TI 1 2388 78 T SS MP K MLA 112389 79 S S MP KML A 11 2390 81 M PK ML A IFW 112391 93 TT IQ F DA CL 112392 113 M E ST V LL AM 12393 120 A MAF DR Y VA 112394 128 A IC H PL RHA __112395 132 P L RHA T VLT 112396 156 A AL M A P LPV ii___2397 158 L MA PLP V F 11 2398 182 CLH Q D VM KL 112399 204 1 V I I S A I G L 1_____2400 209 A IGL DS L L 11 2401 214 SL L ISF S YL 112402 249 AV F I FYV PF 11 2403 266 FS K RRD S PL 11 2404 276 V I L AN1Y L L 11 2405 286 P PV LN P IVY 11 2406 8 NE S SAT YFI1 10 2407 22 GL EE AQ F WL 10 2408 Y IVR TE H SL 10 2409 61 PMY I FL C ML 10 2410 67 CM LS G I DIL 10 2411 68 MLS GI DI LI1 10 2412 94 -T IQF D A CLL 10 2413 96 QFDA CL LQI1 10 2414 102 LQ I FA I HSL 10 2415 109 S L S G ME STV 10 2416 129 1C HP L R HAT 10 2417 145 TK I GV A AVV 10 2418 161 P LP VF I KQL 10 2419 162 L_ LPVF IK QL P 10 2420 175 NI L SH SY CL 10 2421 199 V VYG L IV I I 10 2422 215 L LIS F S YLL 10 2423 216 L ISF SY LLI1 10 2424 239 AF GT CV S HV 10 2425 243 CV S HVC A VF 10 2426 271 1D SP L PV ILA 10 2427 274 L PV I LANI Y 10 2428 Table XXVII 101P3A11 vl. HLA-B0702 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 275 P V I L A N I Y L 10 _2429 277 I L A N I Y L L V 2430 298 T K E I R Q R I L 10__ 2431 Table XXVII 101P3A11 v2 HLA B0702 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID W P I S I C W F L 21 2432 16 C PSSWPI S I 18 2433 6 A V L A S G V T L 16 2434 21 PI S I C W F L L 12 2435 26 W F L L C S T Q L 11 2436 Table XXVII 101P3A11 v3 HLA B0702 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 7 L Q M F A I H S L 11 2437 1 Q F D A C L L Q M 10 2438 4 A C L L Q M F A I 9 2439 2 F D A C L L Q M F 7 2440 3 D A C L L Q M F A 7 2441 Table XXVIII 101P3A1l vl. HLA-B08 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 300 E IRQRILR L 31 2442 266 F S K R R D S P L 29 2443 150 A AVVRGAA L 24 2444 169 L P F C R S N I L 24 2445 295 G V K T K E I R Q 21 2446 121 M A F D R Y V A I 20 2447 293 V Y G V K T K E I 20 2448 22 G L E E A Q F W L 19 2449 79 S S M P K M L A I 19 2450 161 P L P V F I K Q L 19 2451 187 V M K L A C D D I 18 2452 214 S L L I S F S Y L 18 2453 222 L L I L K T V L G 18 2454 297 K T K E I R Q R I 18 2455 298 T KEIRQRI L 18 2456 131 H P L R H A T V L 17 2457 182 C L H Q D V M K L 17 2458 224 I L K T V L G L T 17 2459 29 W L A F P L C S L 16 2460 38 Y L I A V L G N L 16 2461 57 S L H E P M Y I F 16 2462 81 M P K M L A I F W 16 2463 163 P V F I K Q L P F 16 2464 202 G L I V I I S A I 16 2465 208 S A I G L D S LL 16 2466 Table XXVIII 101P3A11 vl. HLA-B08 9-mers Pos 1 2345678 9 score SEQ ID 215 L L I S F S Y LL 16 2467 221 Y LL I L K T V L 16 2468 234 E A Q A K A F G T 16 2469 276 V I L A N I Y LL 16 2470 305 I L R L F H V A T 16 2471 F I L I G L P G L 15 2472 111 S G M E S T V LL 15 2473 139 L T L P R V T K I 15 2474 165 F I K Q L P F C R 15 2475 223 L I L K T V L G L 15 2476 Table XXVIII 101P3A11 v2 HLA BOB 9-mere Pos 1 2 3 4 5 6 7 8 9 score SEQ ID W P I S I C W F L 16 2477 21 P I S I C W F LL 14 2478 13 T L R C P S S W P 12 2479 16 C P S PSSWPISI 12 2480 6 A V L A S G V T L 11 2481 26 W F L L C S T Q L 11 2482 1 S L Y L I A V L A 10 2483 11 G V T L R C P SS 10 2484 19 SWPISICW F 9 2485 7 V L A S G V T L R 8 2486 27 F L L C S T Q L S 7 2487 Table XXVIII 101P3A11 v3 HLA B08 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 7 L Q M F A I H S L 11 2488 4 A C L L Q M F A I 8 2489 2 F D A C L L Q M F 7 2490 C L L Q M F A I H 6 2491 6 L L Q M F A I H S 6 2492 3 D A C L L Q M F A 5 2493 Table XXIX 101P3A11 vl. HLA-B1510 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 58 L H E P M Y I F L 23 2494 245 S H V C A V F I F 17 2495 270 R D S P L P V I L 16 2496 281 I Y L L V P P V L 16 2497 263 V H R F S K R R D 15 2498 300 E I R Q R I L R L 15 2499 107 I H S L S G M E S 14 2500 207 I S A I G L D S L 14 2501 221 Y L L I L K T V L 14 2502 252 I F Y V P F I G L 14 2503 298 T K E I R Q R I L 14 2504 22 G L E E A Q F W L 13 2505 C S L Y L I A V L 13 2506 E H S L H E P M Y 13 2507 Table XXIX 1O1P3A11 vl. HLA-B1510 9-mlers Poe 12 34 5 67 89 scare SEQ -ID ill SG ME S TV LL 13 2508 195 1R V NV V YGL 13 2509 9 E SS ATY F IL 12 2510 F IL IG L PGL 12 2511 29 WL AF PL C SL 12 2512 67 CM LS G ID IL 12 2513 77 ST SS MP K ML 12 2514 .93 T.TIQ0F D ACL 12 2515 110 LS GM ES T VL 12 2516 131 HP LR H AT VL 12 2517 133 L R HAT V LTL 12 2518 A AVV RG A AL 12 2519 154 R GA AL M APL 12 2520 161 P L PVF IK Q L 12 2521 182 CL HQ D VM KL 12 2522 183 L HQ0DV M KLA 12 2523 204 1V I I SA I GL 12 2524 217 1S FS YL L IL 12 2525 223 L IL K TV L GL 12 2526 276 VI L A NIY LL 12 2527 38 Y LI AV LG NL 11 2528 s0 YI V RT E HSL 11 2529 94 TI Q F DAC LL 11 2530 102 L QIF A IH SL 11 2531 130 CH P LR H AT V 11 2532 134 RHA T VL T LP 11 2533 178 SH S YCL H QD 11 2534 208 SAI G LD S LL 11 2535 258 1IGLS MV H RF 11 2536 Table XXIX 1O1P3A11 v2 HLA B1510 9-mere Poe 12 3 4 56 789 score SEQ ID 6 A VL ASG V TL 13 2537 21 P IS IC WF LL 11 2538 W PIS I C WFL 10 2539 26 W FL L CS TQL 10 2540 19 S wP I SI C wF 7 2541 28 L LC STQL SM 6 2542 Table XXIX 10P3A11 v3 IHLA B1510 9-mere Pos 123456789 score SEQ ID 7LQ MF AI H SL 11 I2543 2FD AC LL Q MF 7 2544 I -Table XXX l1P3A11 vi. HLA-B2705 9-ners_ Pos 123 4 56 7 89 score SEQ ID 195 1IRV NVV Y GL 25 2546 269 RR D S P LPV1 24 2547 133 L RHA TV L TL 23 2548 301 -1R 0R IL RL F 23 2549 306 LR LF HV A TH 23 2550 232 T REA Q A KAF 21 2551 CS L YLI A VL 18 2552 300 E I RQRI L RL 18 2553 7 GNE SS AT YF 17 2554 67 C M LSGCI DIL 17 2555 163 P V F I KQLPF 17 2556 208 S AIG L DS LL 17 2557 211 GL DS LL I SF 17 2558 221 YL LI LK T VL 17 2559 238 KAF G T CV SH 17 2560 270 RD S PL P VIL 17; 2561 281 1IYL L VP PVL 17 2562 296 VK T KEI R QR 17 2563 12 AT YF IL IOGL 16 2564 F I LIGL P GL 16 2565 22 G LEE AQ F WL 16 2566 26 AQ F WLA F PL 16 2567 38 Y LIA VL G NL 16 2568 93 T T IQ FD ACL 16 2569 102 LQI FA I HS L 16 2570 125 RY VA IC H PL 16 2571 131 H PLR HA T VL 16 2572 142 PR VT K I GVA 16 2573 154 RG A ALM A PL 16 2574 182 CL HQ D V MKL 16 2575 202 G L IVI I SAI1 16 2576 204 1V I I SA I GL 16 2577 217 1 S FS YL L IL 16 2578 223 L IL KTV L GL 16 2579 256 PF ICL S M VH 16 2580 258 1G LS MV H RF 16 2581 276 V IL AN I YLL 16* 2582 48 1 1 Y I V R T E H 15 2583 110 LS GM ES T VL 15 2584 115 ST VL LA M AF 15 2585 124 DR YV AI C HP 15 2586 146 KI GV A AV VR 15 2587 157 AL MA PL P VF 1s 2588 169 L PFC RS N IL 15 2589 173 R S NI LS HSY 15 2590 199 V V YGL I VI I 15 2591 207 1S AI GL D SL 15 2592 230 G LT REA Q AK 15 2593 249 A V FI FY V PF 15 2594 252 1I FY V PFI GL 15 2595 275 P VIL A NI YL 2596 291 PIV Y GV K TK 15 2597 299 K E I RQRI LR is 2598 L P GL E E AF 14 2599 -LA FP LC S LY 14 2600 00 00 00 Table XXX lO1P3A11 v1. HLA-B2705 9-mers Pos -1 23 45 678 9 score SEQ ID 31 AF P LCS L YL 14 2601 1IAV L G NLT1 14 2602 41 A V L G N L T I I 14 2603 80 SM PK M L AIF 14 2604 PK ML A IF WF 14 2605 ___100 C L LQ1F A IH 14 2606 138 -V LT L PR VTK 2607 139 LT LP R VT K 1i4 2608 151 A V VR GA ALM 14 2609 161 -PL PV FI K QL 14 2610 175 N I LS HS YCL 14 2611 181 Y CLH Q D VM K 14 2612 193 D D IR V NVVY 14 2613 213 DS L LI SF SY 14 2614 214 SLL I SF S YL 14 2615 215 LL IS FS Y LL 14 2616 261 S MV HRF S KR 14 2617 264 HR FS K RR DS 14 2618 268 1R R DSP L PV 14 2619 294 YG VK T KE IR 14 2620 302 R QR IL R L FH 14 2621 303 QR I LRL F HV 14 2622 6 NG N ES S ATY 13 2623 24 E EAQ F WL AF 13 2624 29 W LAF P LC SL 13 2625 NL TI I YI VR 13 2626 52 V RTE HS L HE 13 2627 57 S L HE PM YIF 13 2628 61 P M Y I F L C M4 L 13 2629 73 DI LI S TS SM 13 2630 L IST SS M PK 13 2631 76 1ST S S MP KM 13 2632 99 ACL L QI FAI1 13 2633 105 F AI HS L SG M 13 2634 ill S GME ST V LL 13 2635 117 V LL A MA FDR 13 2636 127 V AIC HP L RH 13 2637 150 AAV V RG A AL 13 2638 159 M A PL P VF IK 13 2639 165 F I KQL PF CR 13 2640 171 FC RS NI LS H 13 2641 172 CR S NI L SHS 13 2642 188 M KL AC D DIR 13 2643 218 S FS YL L I LK 13 2644 225 L KT VLG L TR 13 2645 243 C VS HVC A VF 13 2646 257 FI G LSM V HR 13 2647 262 M VHR FS K RR 13 2648 YI VR T E HSL 12 2649 58 LH EP MY I FL 12 2650 89 WF N ST TI QF 12 2651 97 F DA CLL Q IF 12 2652 135 H ATV LT L PR 12 2653 153 V RG A AL MAP 12 2654 180 SY CLH Q D VM 12 2655 Table XXX lO1P3AIl v1. HLA-B2705 9-tners Pos 1 234 56 7 89 score SEQ ID 198 NV VY GL IVI1 12 2656 245 SH V CA V FIF 12 2657 266 FS K RR DS PL 12 2658 274 L P VIL A NIY 12 2659 286 P PV L NP I VY 12 2660 289 LN PI VY G VK 12 2661 297 KT KE IRQ0RI1 12 2662 298 TK EI RQ R IL 12 2663 Table XXX 1O1P3A11 v2 HLA B2705 -9-mers Pos 12 3 45 67 89 score SEQ ID 14 L RC P SS.WPI1 20 2664 26 WF L LCS T QL 17 2665 6 A VLA SG V TL 16 2666 7 VLA S GV T LR 15 2667 19 Swp I S I C wF 14 2668 W PI SI C WFL 14 2669 28 L LC ST Q LSM 12 2670 21 PI SI CW F LL 10 2671 Table XXX 101P3A11 v3 lILA B2705 9-mers Pos 12 34 56 7689 score SEQ ID 7 LQ0M F AI HSL 14 2672 C L LQ M PAIH 13 2673 2 FD AC L LQ MF 12 2674 1 QFD A CL L QM 11 2675 4- AC L LQ MFAI1 10 2676 Table XXXI 1O1P3A11 v1. HLzA-B2709 9-mers FOS 1 234 56 7 89 score SEQ ID 295 1R VN VV Y GL 24 2677 269 RR DS PL PVI1 24 2678 133 L RH ATV L TL 22 2679 268 KR RD SP L PV 21 2680 301 1R QR I LR LF 20 2681 232 TR E AQ A KAF 19 2682 303 Q RI LR LF HV 19 2683 125 RYV A IC H PL 16 2684 270 R D SP L PV IL 16 2685 44 G NL TI I Y IV 15 2686 217 1S FS YL L IL 15 2687 12 AT YF I LI GL 14 2688 26 AQ FW LA F PL 14 2689 154 RGA A LM A PL 14 2690 Table XXXI lOlP3A11 V1. HLA-B2709 9-mers Pos 12 34 56 78 9 score SEQ ID 175 N ILS HS Y CL 14 2691 223 L IL K TV LGL 14 2692 258 1G LS MV H RF 14 2693 281 1IY LL VP P VL 14 2694 7 G NE SSA T YF 13 2695 FI L IG LP GL 13 2696 22 G L EE AQF WL 13 2697 67 C MIsS G ID IL 13 2698 131 H PLR H AT VL 13 2699 202 G L IVI I SAI1 13 2700 204 1V I I S A IGL 13 2701 215 L LI SF S YLL 13 2702 252 1IFYV P FI GL 13 2703 264 H R FSK R R DS 13 2704 276 V IL A N IYLL 13 2705 306 LR LF HV A TH 13 2706 31 AF PL CS L YL 12 2707 CS LY LI A VL 12 2708 38 YL I A V LGNL 12 2709 52 VR TE HS L HE 12 2710 61 P MYI FL C ML 12 2711 76 1ISTS S MP KM 12 2712 94 T 10F DA CL L 12 2713 124 DRY V AI C HP 12 2714 136 AT VL TL P RV 2715 139 L TL PRV TKI1 12 2716 150 AA VV R G AAL 12 2717 156 AA L MA PL PV 12 2718 169 LP F CRS N IL 12 2719 182 CL H0D V M KL 12 2720 189 KL AC DD I RV 12 2721 191 AC D DI RV NV 12 2722 196 RV NV V YGLI1 12 2723 211 GL DS LL I SF 12 2724 214 S LLIIS F S YL 12 2725 221 Y LLI LK T VL 12 2726 249 AV F IF YV PF 12 2727 280 NIY L LV P PV 12 2728 288 VL N PIV Y GV 12 2729 297 KT KE I RQRI1 12 2730 300 E IR Q RI LRL 12 2731 32 F PLC S LYLI1 11 2732 1IA VL GN LT1 11 __2733 41 A V L NL TII1 11 __2734 YI V RTE H SL 11 2735 58 L HE PM YI FL 11 2736 64 1IF LC ML SG1 11 2737 77 S TS S MP K ML 11 2738 93 T T IQ(PD A CL 11 __2739 99 A C L LQ1FAI1 11 2740 102 LQ IF AI H SL 11 2741 ill SG ME ST V LL 11 __2742 121 M AF DRY VAI1 11 2743 142 PR VT KI G VA 11 __2744 151 A VV RGA A LM 11 __2745 00 00 0

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00 0q Table XXXI 101P3A11 vl. HLA-B2709 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 161 P L P V F I K Q L 11 2746 163 P. V F I K Q L P F 11 2747 172 C R S N I L S H S 11 2748 199 V V Y G L I V I I 11 2749 207 I S A I G L D S L 11 2750 208 S A I G L D S LL 11 2751 209 A I G L D S L L I 11 2752 220 S Y L L I L K T V 11 2753 242 T C V S H V C A V 11 2754 250 V F I F Y V P F I 11 2755 275 P V I L A N I Y L 11 2756 277 I L A N I Y L L V 11 2757 Table XXXI 101P3A11 v2 HLA B2709 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 14 L R C P S S W P I 19 2758 6 A V L A S G V T L 14 2759 W P I S I C W F L 13 2760 26 W F L L C S T Q L 13 2761 21 P I S I C W F LL 10 2762 28 L LCSTQLS M 10 2763 4 L I A V L A S G V 9 2764 16 C P S S W P I S I 9 2765 Table XXXI 101P3A11 v3 HLA B2709 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 4 A C L L 0 M F A I 11 2766 1 Q F D A C L L Q M 10 2767 7 L Q M F A I H S L 10 2768 2 F D A C L L Q M F 8 2769 Table XXXII 101P3A11 vl. HLA-B4402 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 24 E E A Q F W L A F 25 2770 8 N E S S A T Y F I 21 2771 99 A C L L Q I F A I 18 2772 299 K E I R 0 R I L R 18 2773 102 L Q I F A I H S L 17 2774 161 P L P V F I K Q L 17 2775 202 G L I V I I S A I 17 2776 300 E I R Q R I L R L 17 2777 12 A T Y F I L I G L 16 2778 26 A Q F W L A F P L 16 2779 L A F P L C S L Y 16 2780 31 A F P L C S L Y L 16 2781 79 S S M P K M L A I 16 2782 113 M E S T V L L A M 16 2783 150 A A V V R G A A L 16 2784 157 A L M A P L P V F 16 2785 193 D D I R V N V V Y 16 2786 208 S A I G L D S LL 16 2787 249 A VFIFYVP F 16 2788 Table XXXII 1O1P3All vi. HLA-B4402 9-mers Po -1 23 4 56 7 89 sco re SEQ ID 270 RD S P LP VIL 16 2789 276 V I LA N IYILL 16 2790 CSL Y LI A VL 15 2791 41 AV L G NL T II is 2792 59 -HE P MY I FLC 15 2793 77 S TSS M PK ML 15 2794 82 P K ML AI F WF 15 2795 ill S G ME ST VLL 15 2796 115 ST VL LA M AF 15 2797 121 MA FD RY VAI1 15 2798 139 LT L PRV T KI 1s 2799 204 1V II SA I GL 15 2800 232 TR EA QA K AF 15 2801 275 P VIL A NI YL 15 2802 286 PP VL N PI VY 15 2803 301 1IRQ RI L R LF 15 2804 38 Y LIA V L GNL 14 2805 E HSL HE P MY 14 2806 58 L HEP M YI FL 14 2807 67 CM LS GI D IL 14 2808 131 HP L RHA T VL 14 2809 169 LP FC RS N IL 14 2810 209 AI G LD SLLI1 14 2811 215 -LL I SF SY LL 14 2812 217 1ISF SY L LIL 14 2813 281 1IYL LV P PVL 14 2814 284 LV PP VL NPI1 14 2815 9 E SS AT Y FIL 13 2816 S S AT YF IL 1 13 2817 42 V L GNL T IIY 13 2818 43 LG N LTI IYI1 13 2819 68 M LSG ID ILI1 13 2820 SM P KM L AIF 13 2821 89 WF NS T T IQF 13 2822 93 T T IQF D ACL 13 2823 133 LRH A TV L TL 13 2824 158 LM AP LP VFI1 13 2825 163 PV F IK Q LPF 13 2826 199 V V Y G L I V I I 13 2827 211 G LDS L LI SF 13 2828 213 DS L LIS F SY 13 2829 214 SL LI S FS YL 13 2830 223 L I LK TV LGL 13 2831 258 1IGL SM V HRF 13 2832 6 NG NE SS A TY 12 2833 FI L IG L PGL 12 2834 L PG LE E AQF 12 2835 21 PGL E E AQ FW 12 2836 23 LE EA QF W LA 12 2837 YI V RT EH SL 12 2838 81 MP K M LAI FW 12 2839 94 TI QF DA C LL 12 2840 96 F D AC L LQ 1 12 2841 125 R YV A IC H PL __12 2842 175 N IL S HS YCL 12 2843 Table XXXII 101P3A11 vl. HLA-B4402 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 182 C L H Q D V M K L 12 2844 195 I R V N V V Y G L 12 2845 198 N V V Y G L I V I 12 2846 221 Y L L I L K T V L 12 2847 243 C V S H V C A V F 12 2848 245 S H V C A V F I F 12 2849 246 H V C A V F I F Y 12 2850 250 V F I F Y V P F I 12 2851 252 I F Y V P F I G L 12 2852 266 F S K R R D S P L 12 2853 274 L P V I L A N I Y 12 2854 298 T K E I R Q R I L 12 2855 Table XXXII 101P3A11 v2 HLA B4402 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQID 6 .A V L A S G V T L 16 2856 18 S SWPISICW 16 2857 19 S W P I S I C W F 15 2858 W P I S I C W F L 14 2859 12 V T L R C P S S W 13 2860 26 W F L L C S T QL 13 2861 21 P I S I C W F LL 12 2862 14 L R C P S S W P I 11 2863 16, C P S S W P I S I 11 2864 Table XXXII 101P3A11 v3 HLA B4402 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID 4 A CLLQMFAI 15 2865 7 L Q M F A I H S L 15 2866 2 F D A C L L Q M F 11 2867 Table XXXIII 101P3A11 vl. HLA-B5101 9-mers Pos 1 2 3 4 5 6 7 8 9 score SEQ ID I A V L G N L T I 26 2868 32 F P L C S L Y L I 25 2869 121 M A F D R Y V A I 24 2870 131 H P L R H A T V L 23 2871 119 L A M A F D R Y V 22 2872 141 L P R V T K I G V 22 2873 156 A A L M A P L P V 22 2874 43 L G N L T I I Y I 21 2875 255 V P F I G L S M V 21 2876 285 V P P V L N P I V 21 2877 169 L P F C R S N I L 20 2878 236 Q A K A F G T C V 20 2879 139 L T L P R V T K I 19 2880 160 A P L P V F I K Q 18 2881 190 L A C D D I R V N 18 2882 199 V V Y G L I VI I 18 2883 278 L A N I Y L L V P 18 2884 208 S A I G L D S LL 17 2885 284 L V P P V L N P I 17 2886 Table XXXIII lO1P3A11 vi. HLA-B5101 9-mers Pos 1 234 56 7 89 scdre SEQ ID 64 1IF LC ML SG1 16 2887 87 1IF WF N STT1 16 2888 S11 M E S TV LL 16 2889 145 T KIG VA A VV 16 2890 150 AA VV R GA AL 16 2891 198 N VV YG L IV 1 16 2892 272 SPL PV I L AN 16 2893 281 1Y LL VP P VL 16 2894 4 D PN1G NE S SA 15 2895 41 A VL G NLTII1 15 2896 98 DAC LL Q I FA 15 2697 133 LRH A TV L TL 15 2898 223 LIL K TV L GL 152899 280 NI YL L V PPV 15 2900 286 P P VLNP IV Y 15 2901 290 NP IV Y GV KT 15 2902 SS AT YF ILI1 14 2903 66 LC ML S GIDI1 14 2904 LA I F WF NST 14 2905 127_ V AI CH P LRH 14 2906 158 LM A PLP VFI1 14 2907 159 M A PILPV F IK 14 2908 192 CD DI R V NVV 14 2909 201 Y G L I V I I S A 14 2910 210 1IG LD SL LIS 14 2911 216 L I S F S Y L L 1 14 2912 220 -SY L LI L KTV 14 2913 221 YL LI L KT VL 14 2914 238 KA FG TC V SH 14 2915 246 CA VF IF Y VP 14 2916 250 V FIFY V PF 1 14 2917 252 1IFY V PF IGL 14 2918 258 1G LS MV H RF 14 2919 269 RR DS PL PVI1 14 2920 274 L PV I LA NIY 14 i2921 LA FP LC S LY 13 2922 34 L CS LYL I AV 13 2923 56 HS L HEP MYI1 13 2924 68 M L S GID ILI1 13 2925 81 MP K M LAI FW 13 2926 96 QF D ACL LQI1 13 2927 99 AC L LQ1FAI1 13 2928 105 FA IH S LS GM 13 2929 147 1IG V AA V VR 13 2930 149 VA AV VR G AA 13 2931 154 R GAA L MA PL 13 2932 234 E AQA KA F GT 13 2933 244 VS H VC AVFI1 13 2934 293 V YGVK T KEI1 13 2935 297 KT K EI RQRI1 13 2936 6 N GNE S S ATY 12 2937 11 S AT Y FI LIG 12 2938 12 AT Y FI LI GL 12 2939 L PG L EEAQ0F 12 2940 C SL Y LI AVL 12 2941 Table XXXIII 1O1P3A11 v1. MiLA-BS1O1 9-mere Poe 12 34 5 67 89 score SEQIXD 38 YLIXA VL G NL 12 2942 44 G N L T X I Y I V 2943 EP MYIXF L CM 12 2944 79 SS MP KM LAI1 12 2945 ___109 S L SG ME STV 12 2946 ___110 L SG MES T VL 12 2947 155 GAA L M A PL P 12 2948 162 LP V FIXKQL P 12 2949 179 HS YC L HQ DV 12 2950 195 XRV N V VY GL 12 2951 196 R VN VVY GLIX 12 2952 217 XIS FSY L LIL 12 2953 239 AF G TC V SHV 12 2954 240 FGT C VS H VC 12 2955 268 K RR DS P LPV 12 2956 273 PL P VIL ANIX 12 2957 292 XIVY GV K T KE 12 2958 XXXIII 1O1P3A11 v2 HLA B5101 -9-mere Poe 12 34 5 6 789 score SEQ ID 16 C PS S wpIxSI 22 2959 8 L A SGVT L RC 17 2960 WP IS I CW FL 16 2961 1IAV L AS GVT 15 2962 6 AV L ASG V TL 13 2963 14 L R C PSS WPIX 13 2964 4 LXA VL A SG V 11 2965 XXXIII 101P3A11 v3 HLA B5101 9-mere Poe 12 34 5 67 89 score )SEQ ID 3 -D AC L LQ MFA 14 J2966 4 A CLL Q MFAIX 12 2967 7 LQ MF AI H SL 9 J2968 Table XXXIV 1O1P3A11 v1. HLA-A1 Poo 1 23 4 56 7 890 score SEQ) ID 192 C DDIXR VN V VY 27 245 S H VCA V FIXFY 24 2969 41 A VL GN L TIXXY 21 2970 285 V P PVL N PI VY 21 2971 117 VL L A MAF D RY 20 2972 29 W LA F PLC SL Y 18 2973 298 T KE I RQ RIXL 17 2974 22 G LE E AQF W LA 16 2975 23 L E EA QF W LAF 16 2976 53 R T EHS L HE PM 16 2977 54 TE HS L HE PM Y 16 2978 58 L H E P M Y I F L C 16 2979 112 G ME ST VL L AM 16 2980 273 P LP VIXLA NIXY 16 2981 2 MV DP N GN ES S 15 2982 Table XXXIV 101P3A11 vl. HLA-AI 10-mers Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID P N G N E S S A T Y 15 2983 122 A F D R Y V A I C H 2984 172 C R S N I L S H S Y 15 2985 212 L D S L L I S F S Y 15 2986 9 E S S A T Y P I L 13 2987 191 A C D D I R V N VV 13 2988 Table XXXIV 101P3A1l v2 HLA Al Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 22 P I S I C W F L L C 11 2989 2 S L Y L I A V L A S 10 2990 19 S S W P I S I C W F 10 2991 23 I S I C W F L L C S 10 2992 8 V L A S G V T L R C 8 2993 18 PSSWPISICW 8 2994 28 F L L C S T QLS M 8 2995 13 VT L R C P S S W p 7 2996 1 C S L Y L I A V L A 6 2997 ASGVTLRCP S 6 2998 Table XXXIV 101P3A11 v3 HLA Al Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 2 Q F D A C L L Q M F 11 2999 S1I Q F D A C L L Q M 6 3000 9 Q M F A I H S L S G 6 3001 6 C L L Q M F A I H S 5 3002 Table XXXV 101P3A11 vl. HLA-A0201 PoS 1 2 3 4 5 6 7 8 9 0 score SEQ ID 222 L L I L K T V L G L 30 3003 101 L L Q I F A I H S L 29 3004 283 L L V P P V L N P I 27 3005 206 II S A I G L D S L 26 3006 214 S L L I S F S Y L L 25 3007 57 S L H E P M Y I F L 24 3008 63 Y I F L C M L S G I 24 3009 109 S L S G M E S T V L 24 3010 118 L L A M A F D R Y V 24 3011 132 P L R H A T V L T L 24 3012 138 V L T L P R V T K I 24 3013 216 L I S F S Y L L I L 24 3014 39 L I A V L G N L T I 23 3015 42 V L G N L T I I Y I 23 3016 157 A L M A P L P V F I 23 3017 194 D I R V N V V Y G L 23 3018 00 00 00 Table XXXV 101p3A11 vI. HLA-A0201 Pos 12 34 5 67 89 0 score SEQ ID 215 L L I S F S Y L L 1 23 3019 33 P L CS L YL IAV 22 3020 120 A MA FD RY VAI1 22 3021 238 K AF G TC V SHV 22 3022 276 V IL AN IY LL V 22 3023 86 A IF WF NS TTI1 21 3024 140 T LP RV TK IG V 21 3025 203 L I VI IS AI GL 21 3026 14 Y FI L IG L PGL 20 3027 17 L IG LP GL E EA 20 3028 L AF PL CS L YL 20 3029 143 R VT KI GV A AV 20 3030 149 V AA V VRG A AL 168 Q LP FC R SN IL 20 3031 181 Y C LH QD V MKL 20 3032 223 L IL KT VL G LT 20 3033 241 G TC VS HV C AV 20 3034 249 A VFI F YV PFI1 20 3035 251. F I FYV P FIGL 20 3036 272 S PL PV IL ANI1 20 3037 280 N IY LL V PP VL 20 3038 305 1ILR LF HV A TH 20 3039 11 S AT Y FIL I GL 19 3040 16 1ILI G L PGL EE 19 3041 28 FW LA FP L CS L 19 3042 36 S LY LI AV L GN 19 3043 38 Y LI AV L G NLT 19 3044 NL T II YI V RT 19 3045 F L C MLS GIDI1 19 3046 84 M L A IFWF N ST 19 3047 160 A PLP V FI K QL 19 3048 190 L A CD DIR VN V 19 3049 208 S AI GL D SLLI1 19 3050 254 Y VP FI GL S MV 19 3051 277 -1LA N IYL L VP 19 3052 282 Y LL VP P VLN P 19 3053 284 LV P PV LN P IV 19 3054 287 P VL N PIV Y GV 19 3055 34 LC S L YLI A VL 18 3056 37 L Y LI A VL GNL 18 3057 1A V LG NLTI I 18i 3058 43 LG N LT I IYI V 18 3059 67 C M LSG ID ILI1 18 3060 112 G ME ST V LL AM 18 3061 129 1ICH PL R H ATV 18 3062 135 H A TVL TL P RV 18 3063 155 GAA LM AP L PV 183064 158 L M APL PV F IK 18 3065 191 A CD DI RV N VV 18 3066 230 G LT RE AQ A KA 18 i 3067 246 H VC A VF IF YV 18 3068 275 P V IL A NI YLL 18 3069 279 A NI YL LV P PV 18 3070 292 1IV Y GV KT K E 18 3071 299 K E I RQR IL RL 18 3072 Table XXXV 1O1P3A11 vI. HLA-A0201 10-mers Poe 1 23 45 67 8 90 score SEQ ID 49 1IYI V RT E HSL 17 3073 66 L CM LS 0I1D IL 17 3074 68 M LS GI DI L IS 17 3075 LITS TS S MP KM 17 3076 92 ST TI Q FD AC L 17 3077 1IQF DA CL LQI1 17 3078 189 K LA CD DI R VN 17 3079 196 N VV Y GL I VI1 17 3080 201 Y G L I V I I S A 1 17 3081 219 FS Y L LIL K TV 17 3082 228 VLG L TR EAQ()A 17 3083 304 R I LRL F RV AT 17 3084 22 G L E E AQFW LA 16 3085 93 T T IQF D AC LL 16 3086 98 D AC L LQ IFAI1 16 3087 128 A I CHP LR H AT 16 3088 144 V TKI G V A AVV 16 3089 196 R VN V VYG L IV 16 3090 221 Y LLI L KT VL G 16 3091 297 K T KE IR QR IL 16 3092 19 GL P G LE E AQF 15 3093 31 AF P LC SL Y LI 15 3094 127 V AI C HPL R HA 15 3095 146 K I GV AA V VRG 15 3096 174 SN IL S HS Y CL i5 3097 202 GL IV I IS A IG 15 3098 209 A IG L D SLL IS 15 3099 211 G L DSL L IS FS 1s 3100 268 K R RDS PL P VI 15 3101 46 L T IIY IV R TE 14 3102 74 1IL IS T SS MPK 14 3103 108 H SL SG ME S TV 14 3104 110 L SG ME ST V LL 14 3105 S1 G ME S T VL LA 14 3106 207 1ISA IG L DS LL 14 3107 220 S YL LI L KT VL 14 3108 224 1IL KTV LG L TR 14 3109 235 A Q AK A FG TCV 14 3110 243 C VS H VC AVFI1 14 3111 257 F I GLS M V HRF 14 3112 288 V LN PI V YG VK 14 3113 302 RQ0R I LR LF HV 14 3114 Table XXXV 1O1P3A11 V2 -HLA A0201 Pos 1 23 45 67 89 0 score SEQ ID 4 Y LI A VLA S GV 25 3115 6 1IA VLA SG V TL 20 3116 24 S I C W F L L C S T 20 3117 28 F L LCS TQ LS M 20 3118 2 SL Y LI A VL AS 19 3119 14 T LR C PS S Wp1 18 3120 S L I AV LA S GVT 16 3121 29 L LC S TQ LS ME 16 3122 Table XXXV 101P3A11 v2 HLA AO.201 lO-niers Pos 1 23 4 567 8 90 score SEQ ID 8 V LA SG VT L RC 14 3123 3 L Y LI AV L ASG 12 3124 7 AV L AS GV T LR 12 3125 9 L A SGV T L RCP 12 3126 S W PIS IC W FL 12 3127 21 W PI S I CW F LL 12 3128 Table XXXV -1OlP3A11 v3 HLA A0201 Po6 12 3 45 679 score SEQ ID 7 L L Q MF AI HS L I 29 I 3129 Table XXXVI 101P3A11 vl. HLA-A0203 1O-mers Pos 1 23 4 567 8 90 score SEQ ID 142 P RV TK I GV AA 19 3130 148 G V A AVV R GAA 19 3131 113 ME S TV LL A MA 18 3132 228 V LG L TR EAQ0A 18 3133 230 G LT RE A QA KA 18 3134 143 RV TK I GV A AV 17 3135 149 V AA V VRG A AL 17 3136 3 V D PNG NE S SA 10 3137 17 L IG L PG L EEA 10 3138 22 G LE E A QF WLA 10 3139 32 FP L CS LY LI A 10 3140 77 S TS SMP K M LA 10 3141 FN ST T IQ F DA 10 3142 97 FD AC LL Q IF A 10 3143 ill SG ME S TV LL A 10 3144 119 LA MA F DR YV A 10 3145 127 V AI C HPL R HA 10 3146 141 L P RV T KI GVA 10 3147 147 1IGV A AVV R GA 10 3148 151 A VV RG AA L MA 10 3149 182 C LH Q DV M KLA 10 3150 200 V YG LI VI I SA 10 3151 226 K TV L GLT R EA 10 3152 240 F GT CVS H VC A 10 3153 270 R DS PL PV IL A 10 3154 303 Q RI LRL F HV A 10 3155 306 L R LF HV AT HA 10 3156 4 D PN G N ESS AT 9 3157 18 1 G LP G LE E A 9 3158 23 L EE AQFPW L AF 9 3159 33 P LC S LYL I AV 9 3160 78 T S S MPK MLAI1 9 3161 Table XXXVI 101P3A11 vl. HLA-A0203 10-mers Pos 1 23456789 0 score SEQ ID 91 N S T T I Q F D A C 9 3162 98 D A C L L Q I F A I 9 3163 112 G M E S T V L L A M 9 3164 114 E S T V L L A M A F 9 3165 120 A M A F D R Y V A I 9 3166 128 A I C H P L R H A T 9 3167 152 V V R G A A L M A P 9 _3168 183 L H Q D V M K L A C 9 3169 201 Y G L I V I I S A I 9 3170 227 T V L G L T R E A Q 9 3171 229 L G L T R E A Q A K 9 3172 231 L TREAQAKA F 9 3173 241 G T C V S H V C A V 9 3174 271 D S P L P V I L A N 9 3175 304 R I L R L F H V A T 9 3176 307 R L F H V A T H A S 9 3177 Table XXXVI 101P3A11 v2 HLA A0203 Poo 1 2 3 4 5 6 7 8 9 0 score SEQ ID 1 C S L Y L I A V L A 10 3178 2 S L Y L I A V L A S 9 3179 3 L Y L I A V L A S G 8 3180 Table XXXVI 101P3A11 v3 HLA A0203 Poes 1 2 3 4 5 6 7 8 9 0 score SEQ ID 3 F D A C L L Q M F A 10 3181 4 D A C L L Q M F A I 9 3182 A C L L Q M F A I H 8 3183 Table XXXVII 101P3A11 vl. HLA-A3 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 137 T V L T L P R V T K 31 3184 288 V L N P I V Y G V K 28 3185 224 ILKTVLGLTR 27 3186 305 ILRLFHVAT H 27 3187 74 I L I S T S S M P K 26 3188 16 I L I G L P G L EE 23 3189 41 A V L G N L T I I Y 23 3190 151 A V V R G A A L M A 23 3191 259 G L S M V H R F S K 23 3192 19 G L P G L E E A Q F 22 3193 304 R I L R L F H V A T 22 3194 277 I L A N I Y L L V P 21 3195 29 W L A F P L C S L Y 20 3196 116 T V L L A M A F D R 20 3197 117 V L L A M A F D R Y 20 3198 126 Y V A I C H P L R H 20 3199 132 P L R H A T V L T L 20 3200 145 T K I G V A A V V R 20 3201 157 A L M A P L P V F I 20 3202 XXXVII 101P3All vl. HLA-A3 lO-mers Pos 1 23 45 67 8 90 score SEQ ID 196 R VN V V YGL IV 20 3203 36 ISL YL I AV LGN 19 3204 273 PL P VI LA NI Y 19 3205 38 YL I AV LG N LT i8 3206 so -YI VR TE H SLH 18 3207 51 1IV RT EHS L HE 18 3208 109 S LS G M EST VL 18 3209 143 R VT KI GV A AV 18 3210 -189 K LA CD DI R VN 18 3211 -280 N IY LL VP P VL 18 3212 292 1IV YGV KT KEI1 18 3213 -295 G V KTK EI R QR 18 3214 47 TI I YI V RT EH 17 3215 103 IQIF AI HS L SG 17 3216 -152 V VR GA AL MA P 17 3217 -180 S YC L HQD V MK 17 3218 204 1V I IS A IG LD 17 3219 205 V II SA IG L DS 17 3220 221 Y LL IL KT V LG 17 3221 222 L LI L KT V LGL 17 3222 228 V LG L TRE A QA 17 3223 243 C VS H VCA VFI1 17 3224 290 N P IV Y G VKTK 17 3225 39 L IA VL GN LTI1 16 3226 86 A IF W FNS TTI1 16 3227 148 GV AA VV R GA A 16 3228 199 V VY GL I VI IS 16 3229 202 G L I V I I S A I G 16 3230 215 L L I S F S Y L L 1 16 3231 227 T VL GL TR E AQ 16 3232 229 L GL T R EAQ AK 16 3233 230 G LT R EAQ0A KA 16 3234 2 MVD PN GN E SS 15 3235 N L TI I YI VRT 15 3236 48 1I1Y IV RT E HS is 3237 68 M LS G IDI L IS 15 3238 73 D IL IST S S MP i5 3239 100 C LL QI F AI HS 15 3240 106 A IH SL S G MES 15 3241 X46 K I GV AA V VRG 15 3242 176 1ILSH S YC L HQ 15 3243 192 C DD I RV NV V Y15 3244 209 A IG L DSL L IS 15 3245 276 V I LA NIY L LV 15 3246 282 Y LLV P PV L NP 15 3247 300 E I RQ RI L R LF 15 3248 307 RIiF HV A T HAS 15 3249 Table XXXVII 1O1P3A11 v2 -HLA A3 lO-niers Pos 1 23 4 567 8 90 score SEQ ID 7 A VL ASGCV T LR I22 I3250 4 Y LI AV L AS GV 21 3251 2 S L Y LI AV L AS I20 I3252 12 G V TLR C P SSW 17 3253 Table XXXVII 1OlP3All v2 HLA A3 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 28 F L L C S T Q L S M 17 3254 L I A V L A S G V T 14 3255 6 I A V L A S G V T L 14 3256 8 V ASGVTLRC 14 3257 29 L L C S T Q L S M E 14 3258 14 T L R C P S S W P I 13 3259 22 P I S I C W F L L C 12 3260 24 S I C W F L L C S T 11 3261 Table XXXVII 101P3AII v3 HLA A3 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 6 C L L Q M F A I H S 15 3262 A C L L Q M F A I H 13 3263 7 L L Q M F A I H S L 12 3264 S1I Q F D A C L L Q M 9 3265 2 QFDACLLQMF 9 3266 9 QMFAIH SjSG 9 3267 Table XXXVIII 101P3AII vl. HLA-A26 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 300 E I R Q R I L R L F 31 3268 194 D I R V N V V Y G L 29 3269 251 F I F Y V P F I G L 257 F I G L S M V H R F 25 3270 L I S T S S M P K M 24 3271 275 P V I L A N I Y L L 24 3272 19 G L P G L E E A Q F 23 3273 117 V L L A M A F D R Y 23 3274 206 I 1 S A I G L D S L 23 3275 222 L L I L K T V L G L 23 3276 231 L T R E A Q A K A F 23 3277 14 Y F I L I G L P G L 22 3278 41 A V L G N L T I I Y 22 3279 57 S L H E P M Y I F L 22 3280 96 Q F D A C L L QI F 22 3281 216 L I S F S Y L L I L 22 3282 93 T T I Q F D A C L L 21 3283 101 L L Q I F A I H S L 21 3284 104 I F A I H S L S G M 21 3285 297 K T K E I R Q R I L 21 3286 29 W L A F P L C S L Y 20 3287 132 P L R H A T V L T L 20 3288 E P M Y I F L C M L 19 3289 92 S T T I Q F D A C L 19 3290 203 L I V I I S A I G L 19 3291 213 D S L L I S F S Y L 19 3292 273 P L P V I L A N I Y 19 3293 280 N I Y L L V P P V L 19 3294 53 R T E H S L H E P M 18 3295 63 Y I F L C M L S G I 18 3296 73 D I L I S T S S M P 18 3297 109 S L S G M E S T V L 18 3298 00 00

ID

00 Table XXXVIII 1O1P3A1 vl. HLA-A26 POS 1 2 3 4 5 6 7 8 9 0 score SEQ ID 114 E S T V L L A M A F 18 3299 152 V V R G A A L M A P 18 3300 79 S S M P K M L A I F 17 3301 143 R V T K I G V A A V 37 3302 163 P V F I K Q L P F C 17 3303 165 F I K Q L P F C R S 17 3304 168 Q L P F C R S N I L 17 3305 186 D V M K L A C D D I 17 3306 249 A V F I F Y V P F I 17 3307 254 Y V P F I G L S M V 17 3308 46 L T I I Y I V R T E 16 3309 146 K I V A A V V R G 16 3310 199 V V Y G L I V I I S 16 3311 204 I V I I S A I G L D 16 3312 210 I G L D S L L I S F 16 3313 214 S L L I S F S Y L L 16 3314 256 P F I G L S M V H R 16 3315 265 R F S K R R D S P L 16 3316 295 G V K T K E I R Q R 16 3317 17 L I G L P G L E E A 15 3318 81 M P K M L A I F W F 15 3319 115 S T V L L A M A F D 15 3320 156 A A L M A P L P V F 15 3321 160 A P L P V F I K Q L 15 3322 175 N I L S H S Y C L H 15 3323 198 N V V Y G L I VI I 15 3324 211 G L D S L L I S F S 15 3325 215 L L I S F S Y L L I 15 3326 223 L I L K T V L G L T 15 3327 241 G T C V S H V C A V 15 3328 248 C A V F I F Y V P F 15 3329 287 P V L N P I V Y G V 15 3330 299 K E I R Q R I L R L 15 3331 Table XXXVIII 1O1P3A11 v2 HLA A26 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 28 F L L C S T Q L S M 17 3332 19 S S W P I S I C W F 16 3333 24 S I C W F L L C S T 15 3334 29 L L C S T Q L S M E 15 3335 7 A V L A S G V T L R 14 3336 22 P I S I C W F L L C 14 3337 2 S L Y L I A V L A S 13 3338 4 Y L I A V L A S G V 12 3339 12 G V T L R C P S S W 12 3340 L I A V L A S G V T 11 3341 V L A S G V T L R C 11 3342 13 V T L R C P S S W P 11 3343 21 W P I S I C W F LL ii 3344 26 C W F L L C S T Q L 11 3345 6 I A V L A S G V T L 9 3346 S W P I S I C W F L 9 3347 14 T L R C P S S W P I 8 3348 Table XXXVIII 1OlP3Al1 v3 HLA A26 lO-mers Pos 1 23 4 56 78 90 score SEQ ID 2 Q F D AC LL 0 MF 23 3349 7 L LQ MF AI H SL 21 3350 M FA I HSL S GM 21 3351 1 10QF D AC LL QM 16 3352 4 D ACL L QNF A I 11 3353 Table XXXIX 1O1P3A11 vl. HLA-B0702 Poe 1 23 456 7 89 0 score SEQ ID 160 A PL P V FIK QL 23 3354 E PM YI F LC ML 22 3355 274 L PV IL AN I YL 20 3356 4 DP NG N ES S AT 19 3357 131 H PL R HAT V LT 19 3358 141 L P RVT KI G VA 19 3359 162 L PV FI KQ L PF 19 3360 32 F PL CS L YL IA 18 3361 272 S PL P VIL A NI 18 3362 81 M PK ML A IFW F 16 3363 109 S L SG MES T VL 16 3364 132 P LRH AT V LT L 15 3365 265 R FS K RRD S PL 15 3366 34 L CS L YLI A VL 14 3367 110 L SGM E ST V LL 14 3368 153 V RG A AL MA PL 14 3369 206 1I1S AI GL D SL 14 3370 216 LI SFS Y L LI L 14 3371 269 RR DS P L PVI L 14 3372 L AF P LCS L YL 13 3373 149 V AA V VRG A AL 13 3374 157 A LM AP LP VFI1 13 3375 194 D IR V NV V YGL 13 3376 222 LLIIL K TV L GL 13 3377 299 K E IRQR I L RL 13 3378 8 N E SSA T YFPIL 12 3379 L P GLE E AQ FW 12 3380 E AQ F WL AF PL 12 3381 120 A MA FD RY VAI1 12 3382 130 C HP LR HA T VL 12 3383 207 1IS AIG LD SL L 12 3384 220 S YL LI L KT VL 12 3385 280 N IY LL V PP VL 12 3386 286 P PVL N PI V YG 12 3387 9 E SS AT YF ILI1 11 3388 14 Y FI LI GL P GL 11 3389 28 F WL A F PLC SL 11 3390 49 1 Y IV RT E HSL __11 3391 57 S LH E P MYI FL 11 3392 66 LC ML S GI DI L 11 3393 76 1S T SS MP K ML 11 3394 78 TS S MP KM LAI1 11 3395 92 S TT IQ FD AC L 11 3396 Table XXXIX 101P3All v1. HLA-B0702 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 124 DR Y V A I C H P L 11 3397 143 RVTKIG VAAV 11 3398 181 Y CL H Q D VM K L 11 3399 191 ACDDIRVNVV 11 3400 213 D S L L IS F S Y L 11 3401 235 AQAKAF GTCV 11 3402 243 CVSHVCAVFI 11 3403 249 AVFIF Y VPFI 11 3404 251 FIF Y VP F I G L 11 3405 255 VP FIGLSMV H 11 3406 267 SK RRDSPLPV 11 3407 268 KRRDSPLPVI 11 3408 270 R D S P L P V I L A 11 3409 279 ANI Y LL VPPV 11 3410 285 VP P V L N P IVY 11 3411 290 N P I V Y G V K TK 11 3412 297 K TK E I R Q RIL 11 3413 Table XXXIX O101P3All v2 HLA B0702 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID 21 W P IS I C W F L L 20 3414 6 I A V L A S G V T L 13 3415 17 C P S S w P IS I C 12 3416 S W P IS I C W FL 11 3417 26 CW F L L C ST Q L 11 3418 14 TL RCPSSWPI 9 3419 Table XXXIX 101P3A1ll v3 HLA B0702 Pos 1 2 3 4 5 6 7 8 9 0 score SEO ID 7 LL Q M F A I HSL 10 3420 1 IQ F DACLL QM 9 3421 2 QFDACLLQMF 8 3422 3F D A C L L Q M F A 8 3423 4 DACLLQMFAI 7 3424 M F A I HSLSGM 7 3425 A C L L Q M F A I H 4 3426 Table XL- 101P3A1ll V1-HLA B08 10-mers No Results.

Table XL- 101P3A11 V2-HLA B08 10-mers No Results.

Table XL- 101P3A11 V3-HLA B08 10-mers No Results.

Table XLI 101P3Al1 vI HLA B1510 10-mers No results.

Table XLI 101P3A11 v2 HLA B1510 10-mers No results.

Table XLI 101P3All v3 HLA B1510 10-mers No results.

Table XLII 101P3A11 vi HLA B2705 10-mers No results.

Table XLII 101P3A1ll v2 HLA B2705 10-mers No results.

I1' IL. I u L'a u JLi.'z i i ijLlI Table XLII 101P3A11 v3 HLA B2705 10-mers No results.

Table XLIII 101P3AII vl HLA B2709 10-mers No results.

Table XLIII 101P3A11 v2 HLA B2709 10-mers No results.

Table XLIII 101P3A11 v3 HLA B2709 10-mers No results.

Table XLIV 101P3A11 vl. HLA-B4402 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ. ID No.

299 K E I R Q R ILR L 29 3427 23 L E E A 0 F W L A F 23 3428 160 A P L P V F I K Q L 23 3429 8 N E S S A T Y F I L 22 3430 54 T E H S L H E P M Y 20 3431 275 P V I L A N I Y LL 18 3432 41 A V L G N L T I I Y 17 3433 LAF P L C S L Y L 16 3434 34 L C S L Y L I A V 16 3435 79 S S M P K M L A I F 16 3436 88 FWFNSTTIQF T 1 0 P 16 3437 156 A A L M A P L P V F 16 3438 222 L L I L K T V L G L 16 3439 14 Y F I L I G L P G L 15 3440 31 A F P L C S L Y L I 15 3441 66 L C M L S G I D I L 15 3442 93 T T I Q F D A C L L 15 3443 114 E S T V L L A M A F 15 3444 174 S N I L S H S Y C L 15 3445 208 S A I G L D S L L I 15 3446 231 L T R E A Q A K A F 15 3447 300 E I R R I L R L F 15 3448 9 E S S A T Y F I L I 14 3449 49Y IYI V R T E H S L 14 3450 E P M Y I F L C M L 14 3451 S M P K M L A I FPW 14 3452 86 A I F W F N S T T I 14 3453 I Q F D A C L L Q I 14 3454 98 D A C L L Q I F A I 14 3455 101 L L Q I F A I H S L 14 3456 109 S L S G M E S T V L 14 3457 120 A M A F D R Y V A I 14 3458 130 C H P L R H A T V L 14 3459 157 ALMAPLPVFI 14 3460 167 K Q L P F C R S N I 14 3461 201 YGLIVIISAI 14 3462 210 I G L D S L L I S F 14 3463 220 S Y L L I L K T V L 14 3464 249 AVFIFYVPFI 14 3465 251 F I F Y V P F I G L 14 3466 272 S P L P V I L A N I 14 3467 280 N I Y L L V P P V L 14 3468 285 V P P V L N P I V Y 14 3469 00 00 00 Table XLIV lOlP3All v2 HLA 84402 Poe 1 23 45 67 8 90 score SEQ ID 19 S S WP I S I CW F 17 3470 21 W P I S I C W F L L 14 3471 26 C W PL LCS T QL 14 3472 6 1A V LA S GV TL 13 3473 12 G V TLR C P SSW 12 3474 18 P S S WPI SIC w 12 3475 S WP I S IC WFL 12 3476 16 R CPS S WP ISI1 11 3477 14 T LR CPS S WPI1 8 3478 XLIV I01P3A11 v3 HLA B4402 Poe 1 2 34 56 7 890 score SEQ ID 7 L LQM F AI HS L 14 3479 2 QFDA C LL Q MF 12 3480 4 D AC L LQM FAI1 11 3481 1 1IQ FDA CL L QM 6 3482 A CLLQ()MF A IH 6 3483 Table XLV 1O1P3A11 vi HLA B5101 lO-mers No Results Table XLV 1O1P3A1l v2 MLA B5101 lO-mers No Results Table XLV 10lP3All V3 HLA B5101 10-mere No results.

Table XLVI 101P3All vl. DRB-0101 Pos 12 34 56 7 890 1 234 5 score SEQ ID 201 Y GLIV I IS A IGL D SL 36 3484 69 L S G ID I LI S TS SMPK 34 3485 63 YI FL CM LS GI D IL IS 33 3486 104 1IFAI HS LS GM E S TVL 32 3487 46 LT II YI V RTE HS L HE 31 3488 194 DI RV NV VY G LIV II S 31 3489 278 L A NI YLL VP PVL NP 1 31 3490 98 D AC LL QI FAI HS L SG 30 3491 107 1H SL SG ME ST VL L AM 30. 3492 241 GT C VSH VCA V FI F YV 30 3493 11 SA TY F ILI GL PG L EE 29 3494 290 NP IV YG V KTK E I RQR 29 3495 12 AT Y FI L IG LP G LEEA 28 3496 251 F IFY V PF IGL SM V HR 27 3497 14.1 LP RVT K IG VA AV V RG 26 3498 184 HOQDV M KLA CD DI R VN 26 3499 218 SF S YLL IL KT VL G LT 26 3500 17 LI GLP G LE E AQF W LA 25 3501 EAQ F WLA F P LCS L YL 25 3502 37 LY LI A VLG NL T I IY1 25 3503 71 GI D ILIS T SS M PK ML 25 3504 112 G M ES TV LLA MA F DRY 25 3505 149 VA AV VR GA AL MA PL P 25 3506 163 PV FI KQ L PF C R SNIL 25 3507 198 NV VY G LIV I I S AI GL 25 3508 212 LD S LL IS FSY L LI LK 25 3509 219 F SYL LI L KTV L GL TR 25 3510 14 Y FI L IGL PG L EE AQF 24 3511 31' A FP LC S LY LIA V LGN 24 3512 1IAV LG NL TI I YI VRT 24 3513 78 T SS M PKM LA I F W F S 24 3514 86 AI F WF NS T TI0F DA C 24 3515 138 VL TL P RVT K IGV A AV 24 3516 152 VV R GAA L MA PL PVFI1 24 3517 162 L PVF I KQL PF CR SNI1 24 3518 197 V NV VY G L IVII S A IG 24 3519 203 L IV I I S AI GL DS LLI1 24 3520 209 AI G LDS L LIS F SY LL 24 3521 249 A VFI FYV P FI GL SM V 24 3522 252 1FY V PF IG LS MV H RF 24 3523 84 N L A I F W F N S T T I Q F D 23 3524 102 L QI FA IH SL SG M EST 23 3525 166 1IKQL PF CR S NIL SH S 23 3526 204 1V I I SA IG LD SL L IS 23 3527 222 LL IL KT VL G L TRE AQ 23 3528 279 A NI YL L VP PV LN PIV 23 3529 28 FW L A FPLC SL YL I AV 22 3530 36 SLY L IA VL G NLT II Y 22 3531 62 M YIF LC M LS G IDILI1 22 3532 66 LCM L SGI DI LI ST SS 22 3533 81 M PKM LA I F W NS TTI1 22 3534 146 KI GV A AV VRG AA L MA 22 3535 Table XLVI 1lP3A11 vI. DRB-0101 Poe 1 234 56 78 9 012 34 5 score SEQ ID 147 1G VA AV V RGA A L MAP 22 3536 155 GAA L MA PL PV F IKQ L 22 3537 206 1I1SA IG LD SL L ISF S 22 3538 244 vS H VC AV F IF YV PF1 22 3539 271 DS P LP VIL AN I YL LV 22 3540 275 P VIL AN IY LL V P PVL 22 3541 282 YLL V PP VL NP IV Y GV 22 3542 C SL YLI A VL G NLTII 1 21 3S43 SGI DI L IS TS SM P KM 21 3544 153 VR GA AL M APL P VF IK 21 3545 300 EIR Q RI LR L FHV A TH 21 3546 101 L LQI F AIH S LS GM ES 20 3547 136 A TV LT L PRV T KI GVA 20 3548 142 PRV TK I GVA A VV R GA 20 3549 192 CD DI R VN VVY GL IVI1 20 3550 200 V Y GL IV I I SA IG L DS 20 3S51 263 V HR F SKR RD SP LPVI1 20 3552 272 SP LP VIL AN I YL LV P 20 3553 29 WLA F PL C S LY LI AVL 19 3554 59 HE P MY IF LC ML SG ID' 19 3555 EPM YI F LC M LSG IDI1 19 3556 61 P MYI FL CM LS G ID IL 19 3557 99 AC L LQI F AIH S LS GM 19 3558 216 LI S FSYL L IL K TV LG 19 3559 220 SY LL IL K TVL G LT RE 19 3560 229 L GL T RE AQA KAF G TC 19 3561 233 RE AQ AK AF GT CV S HV 19 3562 247 VC A VFI FY VP FI GL S 19 3563 298 T KE IRQ RI L RL F HVA 19 3564 4 DP N GN E SS AT YF ILI 18 3565 FI L IG LP G LE E A FW 18 3566 26 AQ0F WL AFP LC SL Y L 1__18 3567 43 LG NL TI IY IV R TEH S 18 3568 47 T IIY IV R TE H SL HEP 18 3569 79 S SMP K MTA IF W F NST 18 3570 LA IF WF NS TT I QF DA 18 3571 F NST TI QF D AC L LQI 18 3572 94 TI QF DAC L LQ01F A IH 18 3573 116 T VLL AM A FD RY V AIC 18 3574 120 AM A FD R YV AI CH PLR is 3575 128 AI C HP LRH AT V LT LP 18 3576 130 C H PL RHA TV LT L PRV 18 3577 148- GVA AV V RGA A LM A PL 18 3578 150 AA VV R G AAL MA P LPV 18 3579 217 1ISF S YL LIL KT V LGL 18 3580 228 V LG L TR E AQ AK A FT 18 3581 250 V FI F YV PF IGCLS M VH 18 3582 254 Y VPF I GLS M VHR F SK 18 3583 265 vpPPV LNP I VY GV K TK 18 3584 287 PV L N PIV YG V KT KEI1 18 3585 304 R-IL R LF HV A TH AS EP 18 3586 13 TY FI LIG LP G LE E AQ 17 '3587 Table XLVI 1O1P3Al1 v1. DRB-0101 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 23 L E E A Q F W L A F P L C S L 17 3588 34 L C S L Y L I A V L G N L T I 17 3589 73 D I L I S T S S M P K M L A I 17 3590 96 Q F D A C L L Q I F A I H S L 17 3591 114 E S T V L L A M A F D R Y V A 17 3592 118 L L A M A F D R Y V A I C H P 17 3593 123 F D R Y V A I C H P L R H A T 17 3594 124 D R Y V A I C H P L R H A T V 17 3595 133 L R H A T V L T L P R V T K I 17 3596 140 T L P R V T K I G V A A V V R 17 3597 180 S YC L H Q D V M K L A C D D 17 3598 196 R V N V V Y G L I V I I S A I 17 3599 199 V V Y G L I V I I S A I G L D 17 3600 207 I S A I G L D S L L I S F S Y 17 3601 214 S L L I S F S Y L L I L K T V 17 3602 224 I L K T V L G L T R E A Q A K 17 3603 226 K T V L G L T R E A Q A K A F 17 3604 248 C A V F I F Y V P F I G L S M 17 3605 255 V P F I G L S M V H R F S K R 17 3606 273 P L P V I L A N I Y L L V P P 17 3607 281 I Y L L V P P V L N P I V Y G 17 3608 Table XLVI 1OP3A1 v2 DRB 0101 PoS 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 29 S I C W F L L C S T Q L S M E 34 3609 7 S L Y L I A V L A S G V T L R 32 3610 2 A F P L C S L Y L I A V L A S 24 3611 4 P L C S L Y L I A V L A S G V 24 3612 17 G V T L R C P S S W P I S I C 24 3613 L C S L Y L I A V L A S G V T 23 3614 8 L Y L I A V L A S G V T L R C 22 3615 23 P S S W P I S I C W F L L C S 21 3616 9 Y L I A V L A S G V T L R C P 18 3617 14 L A S G V T L R C P S S W P I 17 3618 A S G V T L R C P S S W P I S 16 3619 Table XLVI 101P3All v3 DRB 0101 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID M F A I H S L S G M E S T V L 32 3620 9 D A C L L Q M F A I H S L S G 30 3621 13 L Q M F A I H S L S G M E S T 23 3622 12 L L Q M F A I H S L S G M E S 20 3623 1 F N S T T I Q F D A C L L Q M 18 3624 T I Q F D A C L L Q M F A I H 18 3625 7 Q F D A C L L Q M F A I H S L 17 3626 A C L L Q M F A I H S L S G M 17 3627 2 N S T T I Q F D A C L L Q M F 16 3628 6 I Q F D A C L L Q M F A I H S 16 3629 Table XLVII 1O1P3All vl. DRB-0301 Poo 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 17 L I G L P G L E E A Q F W L A 26 3630 Table XLVII 1OlP3A11 vi. DRB-0301 Poe 12 34 5 67 89 0 1234 5 score SEQ ID 207 1SA I GL DS LL IS FS y 23 3631 92 ST T IQ FDA CL L QI FA 22 3632 118 LL A M AF D RY V AIC HP 22 3633 39 LI AV LG NL T II YI VR 21 3634 180 S YCL HQ DV M KL AC DD 21 363S 212 LD S LL ISF SY LL I LK 21 3636 220 SY LL I LKT V LG LT RE 21 3637 273 PL PVI L AN IY LL V PP 21 3638 27 F WL AF P LCS L YL IA 20 3639 115 ST VL LA M AFD RY VAI1 20 3640 130 C HP LRHA T VL T L PRV 20 3641 135 HAT V LT LP R V*TK IGV 20 3642 187 V MKL AC DD I RV NVV Y 20 3643 201 YG LI V IIS AI GL D SL 20 3644 271 DS PL P VI LA N IY LLV 20 3645 298 TKE I RQ RI L RLF H VA 20 3646 12 AT Y FI LIG LP G LE EA 19 3647 E HS L HEP MY I FL C ML 19 3648 107 1H S LSGM E S TVL LA M 19 3649 166 1IKQL P FCR S NIL SH S 19 3650 192 CD DI R V NV VYGL IVI1 19 3651 204 1 V I I S A I G L D S L L I S 3652 214 S LL I SF S YL LIL KTV 1__9 3653 225 LK TVL G LT R E AQA KA 1__9 3654 228 V LGL TR E AQ AK AF GT 19 3655 249 AV FI FYV P FI G LS MV 19 3656 255 V PFI GL SM VH R F SKR 19 3657 278 LA NIY L LV PP V LN P1 19 3658 37 L YL I A VLGCN LT IIY1 18 3659 94 T I Q FD AC L LQ1F AIH 18 3660 99 ACL L QI F AI HS L SGM 18 3661 126 YV AIC HP LR H AT V LT 18 3662 159 M AP L PVF IKQ()L PF CR 18 3663 188 MK L ACD DI RV N VV YG 18 3664 218 SF S YLL IL K TVL G LT 18 3665 226 KT VL GL TR E AOA K AF 18 3666 282 YL L VPP VL NP IV Y GV 18 3667 289 L NPI VY GV KT KE IROQ 18 3668 19 GL P GLE E AQF WL AF P 17 3669 NL TI IY I VRT E HS LH 17 3670 146 KI G VAA V VR G AA LMA 17 3671 160 AP LP V FIK QL P FC RS 17 3672 257 FI GL SM VH RF SK R RD 17 3673 260 L SMV H RFS KR R DS PL 17 3674 138 VL TL PR VT KI GV A AV 16 3675 263 VHR F SK RR DS PL PVI1 16 3676 295 V KT K E I RORI L RLF 16 3677 47 T I I YIV RT E HS L HE 1i 3678 52 VRT EH SLH E PM YI FL 15 3679 173 RS NI LS HS Y CL HQ DV 15 3680 190 L ACD D IR V NV VYGL 1 15 3681 213 DSL L IS FS YL LI L KT 15 3682 00 00 00 Table XLVII lO1P3All vi. DRB-0301 Pos 12 34 56 78a9 01 234 5 score SEQ ID 219 FS YL LI LK T VLG L TR 15 3683 272 S PL P VILA N IYL L VP is 3684 280 1IYL LV PP V LNP I V 15i 3685 13 TYF I LI GL P G LE E A 14 3686 36 S LY LIAV L GN L TI IY 14 3687 F LC MLS GID I LI ST S 14 3688 141 LP R VT KI GV AA V V R 14 3689 274 LP VI L ANI YL LV P PV 14 3690 302 R QR ILRL F HV AT H AS 14 3691 14 YFI L IGL PG LE E AQ F 13 3692 48 1I1Y IV R TE HS L HEPM 13 3693 72 1D I LIS TS SM P KM LA 13 3694 81 MPK M LAI FW F NS TT 1 13 3695 110 L S G MEST V LLA M AFD 13 3696 114 E ST VL LA M AF DR YVA 13 3697 136 AT V LTL PR VT K IG VA 13 3698 196 RV NV V YGL IV II SA 1 13 3699 203 LI V IIS AI GL D SLL 1 13 3700 221 YL LI L KT V LGLT R EA 13 3701 222 LL IL KT VL GL T R E A 13 3702 265 RF S KR RD SP LP V ILA 13 3703 281 1Y L LVP P VLN PI V yG 13 3704 303 QR I LR LF HV AT H ASE 13 3705 FI LI GL P GLE EA QF W 12 3706 L PG L EE AQ FWLA F PL 12 3707 31 A FP LC S L Y LI AVL GN 12 3708 34 LC S LY LI AVL GNFLTI1 12 3709 43 L G N L T I r Y I V R T E H S 12 3710 49 1Y IV RT EH S LHE PM Y 12 3711 59 HE P MYI F LC ML S GID 12 3712 63 YI F LCM LS GI DI LI S 12 3713 71 G IDI L IS T SS MP KML 12 3714 73 DI LI STS SM PK M LAI1 12 3715 98 DA C LL QI F AI HS LSG 12 3716 104 1IF AI HS LS G ME STVL 12 3717 108 HSL SG M ES TV L LA MA 12 3718 149 VA AV V RGA AL MA P LP 12 3719 150 A AVV RG A AL MAP L PV 12 3720 154 RG0A ALM A P LPV FI KQ 12 3721 155 G AA L MAPL PV FI KQ0L 12 3722 156 AA LM AP L PVF I KQL P 12 3723 163 PV FI K QL P FC RS NIL 12 3724 185 Q DV MKLA C DD I RV NV 12 3725 200 VYG L IV I IS AI GLD S 12 3726 202 G LI V IIS AI GL D SLL 12 3727 209 AXG L DSL L IS FS YL L 12 3728 211 G LDS LL IS FS YL L IL 12 3729 285 VP PV LN PI V YGV K TK 12 3730 293 VY GV K TKE IR Q RILR 12 3731 Table XLVII 101P3A1I v2 DRB 0301 Poe 12 34 56 78 9 0 123 45 1Score ISEQ ID Table XLVII 101P3A11 v2 DRE 0301 Pos 1 2 3 4 5 6 7 8 9 0 1 2.3 4 5 score SEQ ID L I A V L A S G V T L R C P S 14 3732 L C S L Y L I A V L A S G V T 13 3733 2 A F P L C S L Y L I A V L A S 12 3734 7 S L Y L I A V L A S G V T L R 12 3735 9 Y L I A V L A S G V T L RC p 12 3736 11 IAVLASGVTLRCPSS 12 3737 A S G V T L R C P S S W P I S 12 3738 17 G V T L R C P S S W P I S I C 12 3739 S W P I S I C W F L L C S T Q 12 3740 3 F P L C S L Y L I A V L A S G 11 3741 24 S S W P I S I C W F L L C S T 11 3742 27 P I S I C W F L L C S T Q L S 11 3743 8 L Y L I A V L A S G V T L R C 10 3744 22 C P S S W P I S I C W F L L C 10 3745 29 S I C W F L L C S T Q L S M E 9 3746 23 P S S W P I S I C W F L L C S 8 3747 13 V L A S G V T L R C P S S W P 7 3748 Table XLVII 101P3All v3 DRB 0301 Poo 1 2 3 4 5 6.7 8 9 0 1 2 3 4 5 score SEQ ID 3 S T T I Q F D A C L L Q M F A 22 3749 A C L L Q M F A I H S L S G M 19 3750 T I Q F D A C L L Q M F A I H 18 3751 9 D A C L L Q M P A I H S L S G 12 3752 M F A I H S L S G M E S T V L 12 3753 12 LLQMFAIHSLSGMES 11 3754 1 F N S T T I Q F D A C L L Q M 10 3755 2 N S T T I Q F D A C L L Q M F 10 3756 Table XLVIII 101P3All vl. DR1-0410 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 37 L Y L I A V L G N L T I I Y I 26 3757 46 L T I I Y I V R T E H S L H E 26 3758 69 L S G I D I L I S T S S M P K 26 3759 84 M L A I F W F N S T T I Q F D 26 3760 135 H A T V L T L P R V T K I G V 26 3761 146 K I G V A A V V R G A A L M A 26 3762 225 L K T V L G L T R E A Q A K A 26 3763 228 V L G L T R E A Q A K A F G T 26 3764 257 F I G L S M V H R F S K R R D 26 3765 282 Y L L V P P V L N P I V Y G V 26 3766 290 N P I V Y G V K T K E I R Q R 26 3767 302 R Q R I L R L F H V A T H A S 26 3768 12 ATYPILrGLPGLEEA 22 3769 E A Q F W L A F P L C S L Y L 22 3770 26 A Q F W L A F P L C S L Y L I 22 3771 C S L Y L I A V L G N L TI I 22 3772 L A I F W F N S T T I Q F D A 22 3773 123 F D R Y V A I C H P L R H A T 22 3774 198 N V V Y G L I V I I S A I G L 22 3775 Table XLVIII l1P3Al1 vi. DRl-0410 Pos 1 234 56 7 89 012 3 45 score SEQ ID 216 L ISF S YL L IL KT VLG 22 3776 218 S FSY L LIL KT V L GLT 22 3777 251 FI F Y VPFI GL SM V HR 22 3778 279 A NIY L LVP P VLN PI V 22 3779 L PGL EE AOQFW L AF PL 20 3780 31 AF PL CS LY L IA VL GN 20 3781 34 LC SL Y LIA VL G NLTI1 20 3782 36 SL YLI AV L GN LT I IY 20 3763 1IAV L GNLT II YI V RT 20 3764 43 L G N L T I I Y I V R T E H S 20 3785 NL TII Y IV R TEH S LH 20 3786 49 1Y IV R TE HSL HE P MY 20 3787 59 HEP MY I FL C MLS G ID 20 3788 63 YI FL C MLS GI DI LI S 20 3789 66 LC ML S GID IL I S TSS 20 3790 72 1D IL I STS S MP KM LA 3791 81 M P KM LA I FWFNS T T 1 20 3792 82 P KML AI F WF N STT IQ __20 3793 92 ST TIQ FD A C LLQ I FA 20 3794 98 D ACL LQI F AI HS L SG 20 3795 99 A C LLQ 1F AI HS L S M 20 3796 .101 L LQI FA I HS LSG ME S 20 3797 104 1F AI H SL S GME S TVL 20 3798 107 1HS L SG M EST VL L AM 20 3799 116 TV LL AM AF D RYV A IC 20 3800 118 L L AM A FDR YV AI C HP 20 3801 126 YV AI C HPL RH AT V LT 20 3802 130 CHP L R H ATVL T LP RV 20 3803 138 V LT LP RV T KI GV AAV 20 3804 141 L PR V T KI GV A AVV R 20 3805 156 AA L MA P LPVF I KQ LP 20 3806 163 PV FI KQ LP FC RS N IL 20 3807 166 1IK 0LP FCR SN I LS HS 20 3808 180 SY C L HQDVM K LA C DD 20 3809 184 HQ DV MK LA C D DIR VN 20 3810 187 VM KL AC DD IR V NV VY 20 3811 194 DI RV NV V YGL IV I IS 20 3812 197 V NVV YG L IV IIS A IG 20 3813 200 VYG LI V I ISA I GL DS 20 3814 201 Y G LIVI IS AI GL D SL 20 381S 203 L IVII S AI GL DS LLI1 20 3816 204 1V I IS AIG LD SL LI S 20 3817 207 1S AI G LD SLL I SF SY 20 3818 209 A IGL DS LL I SFS Y LL 20 3819 212 LD SL LI S FSY L LI LK 20 3820 213 DSL LI SF S YL LI L KT 20 3821 219 FS Y LL IL K TV LG LTR 20 3822 241 GT CV S HVC AV F IF YV 20 3823 244 VS HV C AVF I FYV PFI1 20 3824 247 VC AV F IF YVP FI G LS 20 3825 249 AV FI FYV P FI GL S MV 20 3826 252 IF Y VP FI G LSM V H RF 20 3827 Table XLVIII lO1P3AIl vl. DRl-0410 iS-mers Pos 12 34 5 67 890 12 345S score SEQ ID 273 PL PV I LA NI YLL V PP 20 3828 278 LAN I YL LV PP VL NPI1 20 3829 286 PP VL NP IV YG VK T KE 20 3830 19 GL P GL EE AQF WL A FP 18 3831 28 FWL A FP LC SL YL I AV 18 3832 SGI D IL IS TS S M PKM 18 3833 1 QF D AC L LQ1FA I HS 18 3834 100 CLL QI FA I HS LS G ME 18 3835 108 HS LS G M EST V LL AMA 18 3836 117 V L LA MA F DRY V A ICH 18 3837 127 V AI CH P LR H ATV LTL 18 3838 165 FI KQ L PFCR S N IL SH 18 3839 177 L S HSY CL HQ0DV M KLA 18 3840 188 M K L AC DDI RV NV V YG 18 3841 206 1 1 S A I G L 0) S L L I S F S 18 3842 234 E A 0A KA FG TCV S HVC 18 3843 238 K AFGT C VS H VCA VFI1 18 3844 272 SpL P VI L ANI Y LLV P 18 3845 294 YG V KT KE I RQ RI LR L 18 3846 295 GV KT KEI R QR I LR LF 18 3847 11 S AT YF IL I GLPG L EE 16 3848 29 WL AFP L CS LY LI A VL 16 3849 E PMY I FLC M LSG IDI1 16 3850 62 M YIF LC ML SG ID ILI1 16 3851 86 A IFW F NS T TIQF D AC 16 3852 102 LQI F AI HS LS G ME ST 16 3853 178 S HSY CL HQ DV MK LA C 16 3854 237 AKA F GT CV S HV CA VF 16 3855 250 VF IF YV P FI GL S MVH 16 3856 254 Y VP FI GL S M VHR FSK 16 3857 14 YF IL IGL P GL EE AQ F 14 3858 is F I LI GL P GL EE AQFW 14 3859 17 LI GL PG L EEAQ0F W LA 14 3860 39 LI AV LGN L TI I YI VR 14 3861 48 1I1Y I VRTE HS L HE PM 14 3862 E HSLH E PM YI FL C ML 14 38653 61 PM YI FL C ML S GI DIL 14 3864 FL C MLS GI D ILI S TS 14 3865 71 GI DI LI S TSS M PK ML 14 3866 73 DIL I ST SS MPK M LAI1 14 3867 110 LS GM E ST V L LA MAFD 14 3868 114 E ST VL L A MAF DR YVA 14 3869 136 AT VL TL PRV T KI G VA 14 3870 144 V T K IG VA A VV R AAL 14 3871 149 VAA V VR GA AL M AP LP 14 3872 150 AA VVR GA A LM AP L PV 14 3873 155 G AA L MAP LP V FI KQL 14 3874 159 MA PL PVF IKXQ LP F CR 14 3875 174 SNI L S HSY C LHQ D VM 14 3876 185 QDV M KL AC DD IR V NV 14 3877 192 C DD IR VNVV Y GL IVI1 14 3878 196 RVN V VY GL IV II SAI1 14 3879 Table XLVIII 1O1P3A11 vl. DR1-0410 Poe 12 34 5 678 90 12 34 5 score SEQ ID 214 SL LI S FS YLL IL KT V 14 3880 221 YLL IL KT V L GLT R EA 14 3881 222 LL I LK T V LGLT RE AQ 14 3882 226 K TV LGL T RE AQA K AF 14 3883 260 LS M VHR FS KR R DS PL 14 3884 271 DS PL PV IL A NI YLL V 14 3885 274 L PVI LA N IY LL V PPV 14 3886 275 PV IL A NIY L LV P PVL 14 3887 281 1Y LL VP P VL NPI V YG 14 3888 285 VPP VL N PI VY GV K TK 14 3889 303 R I LRLF HV A TH A SE 14 3890 2 MVD P NGN ES S AT YFI1 12 3891 3 V DPNG N ES SA TY F IL 12 3892 PN GN ES S AT YF I LIG 12 3893 6 NG NE SS A TY FIL I GL 12 3894 9 ES S AT YFI L IGL P GL 12 3895 51 1V RT EHS LH EP M YI F 12 3896 58 LH EP M YIF LCM L SGI1 12 3897 67 CM LS GI DI LI ST S SM 12 3898 68 M LSG I DI LIS TS S MP 12 3899 SM PK ML AI FW F NS TT 12 3900 83 KM L AI FW F NST T IQ F 12 3901 88 F WF NS T T10F DA C LL 12 3902 91 N S T TI0F D AC L LQ1F 12 3903 93 T TIQ FD A CL LQI FAI1 12 3904 96 F D ACL LQ IF AI H SL 12 3905 ill SG M ES T VL L AM AF DR 12 3906 122 A FDR YV AI CH PL R HA 12 3907 129 1ICH PL RHA T VL T LPR 12 3908 132 PL RH A TVL T LP RV TK 12 3909 133 LRH A TV LTL P RV TKI1 12 3910 145 T KI GV AA V VR GA ALM 12 3911 147 1G V A AVV RGA A L MAP 12 3912 151 AV VR G AA L MA PL PVF 12 3913 158 LM A PLP V FI KQL P FC 12 3914 160 AP L P VFI K L P FCR S 12 3915 170 PF CR S NI LS HS Y CLH 12 3916 171 FC RS N I LS HS Y CL H 12 3917 172 CR S NIL SH SY C L HQD 12 3918 176 1ILS HS Y CL HQ1)V M KL 12 3919 189 KL AC DD IR V NVV Y GL 12 3920 193 D DIR V NVV Y GL IVII1 12 3921 199 VV YG L IVI IS A IG LD 12 3922 210 1IG LD S LLI S FSY LL1 12 3923 211 G L DSL LIS FS YL L IL 12 3924 217 1S FS YLL I LK T VL GL 12 3925 224 1L KT V L GL T R EAQAK 12 3926 231 LT RE AQ AK AF G TC VS 12 3927 233 RE AQA K AF GT CV S HV 12 3928 2S6 PFI G LS MV HR FS K RR 12 3929 261 SM VH RF SK R RD S PLP 12 3930 265 RF SK R RD SPL P VI LA 12 3931 Table XLVIII lOlP3A11 vl. DR1-0410 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 268 K R R D S P L P V I L A N I Y 12 3932 270 R D S P L P V I L A N I Y L L 12 3933 277 I L A N I Y L L V P P V L N P 12 3934 287 P V L N P I V Y G V K T K E I 12 3935 299 K E I R Q R I L R L F H V A T 12 3936 300 E I R Q R I L R L F H V A T H 12 3937 Table XLVIII 101P3AII v2 DRB 0401 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 29 S I C W F L L C S T Q L S M E 28 3938 8 L Y L I A V L A S G V T L R C 26 3939 2 A F P L C S L Y L I A V L A S 20 3940 L C S L Y L I A V L A S G V T 20 3941 7 S L Y L I A V L A S G V T L R 20 3942 17 G V T L R C P S S W P I S I C 20 3943 27 P I S I C W F L L C S T Q L S 20 3944 6 C S L Y L I A V L A S G V T L 16 3945 23 P S S W P I S I C W F L L C S 16 3946 11 I A V L A S G V T L R C P S S 14 3947 Table XLVIII 101P3All v3 DRB 0401 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 3 S T T IQ F D A C L L Q M F A 20 3948 A C L L Q M F A I H S L S G M 20 3949 12 L L Q M F A I H S L S G M E S 20 3950 M F A I H S L S G M E S T V L 20 3951 6 I Q F D A C L L Q M F A I H S 18 3952 11 C L L Q M F A I H S L S G M E 18 3953 13 L Q M F A I H S L S G M E S T 16 3954 9 D A C L L Q M F A I H S L S G 14 3955 2 N S T T I Q F D A C L L Q M F 12 3956 4 T T I Q F D A C L L Q M F A I 12 3957 7 Q F D A C L L Q M F A I H S L 12 3958 T I Q F D A C L L Q M F A I H 10 3959 Table XLIX 101P3AII vl. DRBI-I101 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID 146 K I G V A A V V R G A A L M A 28 3960 123 F D R Y V A I C H P L R H A T 25 3961 218 S F S Y L L I L K T V L G L T 25 3962 198 N V V Y G L I V I I S A I G L 24 3963 11 S A T Y F I L I G L P G L E E 23 3964 256 P F I G L S M V H R F S K R R 23 3965 N L T I I Y I V R T E H S L H 22 3966 E P M Y I F L C M L S G I D I 22 3967 159 M A P L P V F I K Q L P F C R 22 3968 238 K A F G T C V S H V C A V F I 22 3969 L I S T S S M P K M L A I F W 21 3970 135 H A T V L T L P R V T K I G V 20 3971 138 V L T L P R V T K I G V A A V 20 3972 163 P V F I K Q L P F C R S N I L 20 3973 00 00 00 Table XLIX 1O1P3All VI. DR~l-1101 15-mere Pos 1 23 456 7 890 12 34 5 score SEQ ID 200 V YGL IV I ISA I GL DS 20 3974 225 LKT V L G LTRE A QA KA 20 3975 257 FI G LS MVH R FS KR RD 20 3976 291 p IV YGV K TKE IR QRI1 20 3977 302 R Q RIL RL FHV A TH AS 20 3978 66 LC M LSG ID I LI ST SS 19 3979 101 L LQ I FAI HS L SG MES 19 3980 197 V NVVY G LIV I IS A IG 19 3981 219 FS YLL I L KTV LG L TR 19 3982 248 C AV FIF Y VP FIG L SM 19 3983 275 P VIL A NI YL LV P PVL 19 3984 46 LT II Y IV RTE H S LHE 18 3985 69 L SGI DI LI S T SS MPK 18 3986 81 MP KM L AIF WF NS TTI1 18 3987 98 DAC L LQI FA I HS L SG 18 3988 104 1F AI HS LS G ME S TVL 18 3989 209 AIG L DSL L IS FS Y LL 18 3990 250 V FI F YVPF I GL S MVH 18 3991 62 MYI F LC MLTSG ID IL 1 17 3992 216 L IS F SY LL ILK T VLG 17 3993 260 L S MVHR FS KR R DS PL 17 3994 279 A NIY LL VP PV L N PIV 17 3995 289 LN PI V YGV KT K EI RQ 17 3996 12 AT YF IL IG LP G LE EA 16 3997 EA QF WL AF P LC S LYL 16 3998 43 LGN LT IIY IV RT E HS 16 399.9 254 Y V P I GLS MV H RF SK 16 4000 48 11Y I VR TE HS L HE PM 15 4001 100 CL LQ IF AI HS LS G ME 15 4002 117 VLL A MA FD RY V AIC H 15 4003 144 VT KI GV AA VV RG A AL 15 4004 180 S YC L H DV M KLA C DD 15 4005 228 V LG L TR E AQAK A FGT 15 4006 261 SMV H RF S KR R DS PL 15i 4007 262 MV HR FS KR RD SP L PV 15 4008 278 LAN IY LL V PP VL N PI 15 4009 286 P PVL N P IV YG V K TKE 15 4010 115 ST VL LA MA FD R YVAI1 14 4011 126 Y VA ICH PLR H AT V LT 14 4012 127 V AIC H PLR H ATV L TL 14 4013 141 L P RVTK I GVA AV V RG 14 4014 171 FC RS NI LS HS YC L HQ 14 4015 181 Y C L HQD VM K L AC DD1 14 4016 194 DI RV NV VY GL IV II S 14 4017 230 G L TR E AQAK AFG TC V 14 4018 271 DS PL PV IL A NI YL LV 14 4019 299 KE I RQR IL RL FH V AT 14 4020 SS AT YF IL I GL PG LE j 13 4021 LP GL EE A0F W LA F PL J13 4022 33 P LCS LY LI A VLG N LT 1 3 4023 36 S LYL I A V LGNL T IIY J13 4024 37 LYL I AV LG NL T II Y 1 13 4025 Table XLIX 1OlP3All vi. DRBl1-1101 Pos 1 234 56 7 890 1 234 5 score SEQ ID 59 -H E P M Y I F L C M L S G 1 D) 13 4026 107 1H SL SG ME ST VL L AM 13 4027 152 V VR GA ALMA P LP VFI1 13 4028 1 -56 AA LM AP LP V FIK QL P 13 4029 207 1IS AIGL DS L LI S FSY 13 4030 237 AKA F GT C VS HV C AVF 13 4031 249 AV FIF YV P FI G LS MV 13 4032 268 K RRD SPL P VI LA N IY 13 4033 280 N IYLL VP P VL N PI VY 13 4034 282 Y LLV P PVL N PI VY CV 13 4035- Table XLIX 1Q1P3A11 v2 DRB 1101 Pos 12 34 56 7 090 1 234 5 score SCO ID LC SL YL IA VL A SG VT 18 4036 6 CS LYL I AV LA S GV TL 18 4037 29 S IC WFL LC S TQ LS ME 17 4038 13 VL ASG V TL RC PS SW p 15 4039 8 LY L I AVLAS G V T LRC 14 4040 4 P LCSL Y LI AV L AS GV 13 4041 12 AV L ASG V TL RCP S SW 13 4042 2 A FPL CS LY LI AV L AS 12 4043 7 SLY LI A VL AS G VT LR 12 4044 14 LA SG VT L RCP SS WPI1 12 4045 11 1A V LAS GV TL RC P SS 10 4046 23 P SS WPI SI C WF LL CS 10 4047 is AS GV T LRC PS S WP IS 8 4048 Table XLIX 1O1P3A11 v3 DRB 1101 Poe 12 34 56 78 90 1 234 5 score SEOQID 12 L LQ MF AI H SL SG MES 19 4049 9 DA C LL Q MF AI HS LSG 18 4050 MF AI HS L S GMES T VL 18 4051 11 CL LQ MF AI H SL SG ME is 4052 6 1IQ F D AC LLQMF AI HS_ 12 4053 TI QF D AC LL Q MF AIH 10 4054 13 LQ MF A XHS LS G ME ST 10 4055 Table LIV. Nucleotide sequence 1 61 121 181 241 301 361 421 481 541 601 661 721 is 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 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661

TGCGCTCCAC

TGGCCTGGCT

CTGGGATTAC

CCAAAACCTT

TGTTGTCAAT

AACACAAACC

CAAGCCTGGC

GGTCTCGAAC

AGGCGTGAGC

GCCCTGGCAA

TACTTTAGAT

TCTCTTGAAA

GATTATAGTA GGTCTTAAAG CCCTAACCCC AGGATACATC AAGTCTACTC TGATGGGAAA AGATOCAGGA CTGTGTTACA CAGTAATAGA ATCTGCTTGT TGTCCAGGAA GTTGTATATA

CAGGATGACT

TTTCTCTCCT

ACCCTGGATT

GAAATCTGGT

TACACTTTTG

TGACCATTGT

TGGCCATGCT

GTCTTTT1CCT

TTATCCATGC

TTGTGGCCAT

AGATTGGACT

TCAAGTrGGTT

ATATTATGAA

TCCTCTCAGT

GGGCTGTTTT

CCCACCTCTG

GGCTGGGTGG

CACCTGTAGT

TCTGTATGTT

ACAGAAGATG

TTGTGCTG3TC

AGTACTGAAC

TATGGTGGTC

GGAGGCTGTA

CTTGCCCCCA

CCTAAAGTGG

GGAGAGACAG

GAGGGTGTGT

TATTTGTCCA

TGTGGACACT

GTACTGGTTG

ACGTGTCTCT

GTCTACGTGA

GGTAAGAATG

TGTGTGGAAT

GTATGTCCAC

TAGTTGTAAG

TATTTTCGTG

GTGTATGTGA

TCTGTATGTA

ATAATTTCCA

CCTGGGTGAT

TTTCACAGGC

ACCTCTTCCT

TCCTGCCTCA

CCAAACATGT

GTGTGATGGC

GAGCCCGCAA

ACAGAGCAAG

TCTCCTAATC

in the 5' region close to 1O1P3A11 gene.

TAACTTT TGC ATTTTFAATA GAGGCAGGGT TTCACCATGT CCCTGACCTT GCGATCTGCC CACCTCGGCC TCCCAAAGTG CACTGTACCT GGCGGGGCTT ATTGTTTTTT AAAAAGATTr TTCTGATrI-r CTGGGCCTGG AGCAGGACCT GGAGGGATGG GTTTCTATCA GGAAAGTTTG AGAAATGGTA TTCAGGCCTA TCTrCATCCCA GACTGAGCCC CTGCTCCCTA TCTTAAATTA TCAGCTGTAG ACTGAGCCTC TAAATCTGAA CCCAGACCCA AGAAGAGCTG GTCAATGTGG ACCATTCTGA GCAATCCTGC P.GGCTAAGAG CAGTGCCCTG GGCAGCAACA TCAGCTCTGA TGTTTTATGA GTGGGTCTTC ACACACTGAG ATTCATGGGA GCAGCACTGG GGCCTTGGAG GGTCAGGGTA AGGCTCAAGA AGGAGAATCA GAGCAGAGAG AGACTAGGGT TCAGAATTAC rGTTACTGTC ACCACTCCAA TGCCTTTTCC TCATTAGTCC FCTAAATGAT GTTTCTACTT TTCCCTTTCT ACTTTCCTAG AAGCCCCAGC TCTTG.GTCCC TATCATAGCC ACTTCAAATG rACTTCCTTT TGGTGGGTAT CCCTGGCCTG GGGCCTACCA CCACTGTGTT TTATGTATGC CrGGCCACC CTGGGTAACC CGTG;TGGAGA GGCGACTGCA TGAGCCCATG TACCTCTTCC

TAGTCCTGTT

CTGAGCCACA

TTGTATGCAG

CCACGCAGCA

GCTGGCTTT C

CCTCATCATT

TTCCACTATT

GATGGGCATC

TCTGTCAGCC

TTGCCACCCA

ATCTGCCCTG

GTCCTACTGC

GCTGTCCTGT

CATGGGTGTG

GGAGCTGTCC

TGCTGTTCTG

TCCCACCTCC

CAACCCCCTT

CTCACAAGGT

GGAATATTAG

TTCTTCCAGC

TTATGACCCT

TTGCCT'rCTC

AAGATCACAC

TGTGCCCATG

TTACCCAGCC

CAACAAGACA

TGGCAGATTT

TAAGCTQCC

TGCTTT-TCAG

TGTCCTGGTG

GATTGCGCAT

GAATGCTGGT

CTGCCTCTAC

GTGTGGAATG

TGTGCATAAT

TCGGTGAAAT

CATATGTCTA

CACGAATGGG

TCTTGGAATG

AAGTTGAGTG

AGCTCAGTCT

CAGACCACAC

ACGTTTCCTG

ATAGCAAGTC

ATAAAAGTCC

GCGTGCCTGT

GG'rGGAGGTT

ACTCTGTGTC

GACCTAGTCC

CAGGAGATCG

GTGGAGTCAG

TTGCGCCATG

TCTCCTCTAT

AGTTCAACAT

CTGTCCTGCT

CTTCTG3TGCT ACCAGGGT TTGTATTCTT CAAACACATA CTGTCACACA ACTGACACCA GGGTCAATGT GACTCTCTCT TCATTGGCTT TCTCGGAGfJG CAGCACTCAA GTCTTCTATG TACCCCTCAT CTCCTCCATG TGGTTATGGC GTCTATGGAG CCAAGACCAA GGCAAGTGAG ACACCTTAGT GATCCTATTG AATGCCTTGG AATTTAAGTA GATCATGTAT GTCTGGACAT CCTGGAGAAT CTTCTCTCTC AGCTAGAAAA CTCATGGTTC ATTCCAGTTT TTGGTGAATT TGCATGGACT ATAATCAGGG GTTAATGAAG AGAGGCAGCT CACATGCAAT GTGAAATCTG CCCATTTGCA ATGTATTCTQ GTTGTGGGTG TGTGCGTATA TGTGAGAGAG AGTGTGGTAG CTATGTCCTG TTGTATTTCT GTGGTATCTG GTCTGTATCT GCATIGGTGGG CTTTTCTTCC TATTTGTACT TGTAGTATTG GGATGCCTGT ATTTGAGATG TAAAACCATT GTACATCTGA ATTCTGTGTG GTGTATATGT TTTAAGGCAA GACAGCATCT GTATTTCTGA

CACCATGCCC

TTGCCTGGCC

GGCCATGGCT

GACAGGGTGT

CTTCCCACTG

CTCCTTCTGT

GGTTTATGGA

CTCATATATC

GGCTTTCAAC

TGGGCTCTCG

TAATACCTAC

AGAGATCTGT

GTCTCGCTTC

TGATTAAAGT

TCTGTCTCCA

GACTGCACTA

TACATCTAGT

TGAAGTATGA

ATAAACGTTA

GTATTTGGGG

OTTGAAGTT

TCTGTATGGC

TGAATGTGTG

AGGGTGCACA

GCACATGTAT

TTAGTTGGTA

CAGTACCTTT

ATGTGAATGT

ATCTTTCAGC

TTGTIGCGGTA

CATATTGTTG

ACTTTCTTTG

GCATGGATTG

TCTGGCTGTG

GTTTCTCGGC

GGTGCTTCCT

GGATTTCAAG

GGCCTCCCTA

TTCCCTTTTT

AAAATACAAC

CTGAGGCAGG

CACTGCACTC

AGCCTTGGTT

CAGTCAATCT

AAGATGGCCA

CAGATGTTCC

TTTGACCGCT

ACTGTGGCCA

CCCTTCATCC

CT'GCACCAAG

CTCTTCATCA

CTCATCCT

ACCTGCATCT

GTGGTGCATA

TTGCTGCTAC

TCAAGGGTCC

TACTACTACT

ATCAAACCTA

GGAATGTGTC

GTCCCTCTGC

TTTGACATG

TTTTAATGTr

TTGCAAATAC

AATAGTAACT

CTOTATGCAA

TCTATATGAC

GGTGTGTrTA

CATGGAATAC

GTICATGAG

TATGATATGT

ATGTOTATCT

GGTGCATGAA

GTGTTTGGGT

TATGTGTTAT

GTACTGATGC

TGTGTTGGGT

ATGTGTGGTG

AGCAGCAGAA

CTGGGGTCAG

TGCTCCACCT

GGCTTTTGTT

AACCCTGCCC

GGCTTATCTO

AGAAACGCTT

CAGCCTGGTG

GTAGGGAGTT

CCCTTCTQTT

GAGGAGGGAG

ACATGACTCr

GTCAGAATGA

CTTCTTCATC

CCTCTGCTAT

ATGGTATCCT

TTGGTTCCCC

AGTCCCAGCT

GCAGTGAGCC

ATTGAAGAAG

AAGATGCCCA

AAGGAAACAC

CTGAACACAA

CCGAGGCACT

CACCTCTCCC

ATCTCTACTA

AGTTGGGAGG

GAGATCATGC

AAAAAAAAAA AAAAAAAAAA CCTCTGGGAA AGCAAGGGTG GAGGGGAAGC 3721 3781 3841 3901 53961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981

GCCGCATGGA

TCACCTTGTC

GAAGTAGAAG

ATGGAGGCAG

AGGCACCAGA

TTGCCATGAT

CACAGTTGTG

TATTTATAGA

TRGTTCTCTA

TGTCATTGGT

TGTCCTCAGC

CTGGGGACCT

AACTCCCTTA AGGCAGGAAG CTGAAAAAAC TCATGTCTCA CTGTCCTTCC ACATGTCTCA TCCCTTTGGT ATTTTTTAAA GTCTTTGCCA CAGAGATGGC TCCAGGGTTC

AACTGTTGTC

CTATTTAACA

AAACTGATCA

TGTAGrrTTT

ACTCTCTAAA

CTTACGTGGC

CAGAAGTTCA

TGGTTATAAG

ACCAGTAATT

TAGCTTGATT

GTAAAAAATA

ACTTATTTCA

ATGTTCTCTG

TGGTGTTGGG

GTAATCCAAG

TrATCCAGAC

GGAAAAGGAG

AAATGAGAGA

GTGG3AGTGTC

AGAGACTTGG

TACTCACTTT

TGATACCAAG

TTGATGTGGT

TAGCGTGTCC

AGGGGAGACC

AAATACGAGG

ACTATTTTTG

GGTGGGGAGG

GCCAGAGAGT

ACAGGGACCA

GATGGACCCA

AGGGAGGACA

TCTGCAAACT

ATAAAGACCG

CCCTATCTAT

TGTAGCATTC ACCTCATTAT TTGTTACTCC ATATTGGATG TGTCTAAGTT AATGAGGTTA TGTCAGGCTG CGTGCTCTGT CTGAGTTAGA ATGGTCTGAT TGTGTTCTGA ATTTAA.AACT AACTAAAAAC CATGTTGTTC TATTTAGTTT TACATTCAAA CCCTTAAGGG AGAAACCAGA GTTAAAGAAA CCACGTTCTC GGACGGCAGA GGCACTGTCC GAGCCTGGGA GACAAAAAAA GTGAATTCCA CGCTTAGCAA GAAAGAACAC TTCAGGATGG TCCTTTGCCA TTCTCCTGTT TCACGAAGAC TACACTAATG CCTTCACATT GGGAGTTGGC TCCCTTTCCC CATTGCCTCT GCTGAGAAGC AGTCCAGAAC GGACAGCATT CTGCCCCACC AATAAATAAC CCTGACCAGG caqtgcagcc tgccaqacct GATGTAGCCC TAGGGCTTTG GGACCTAAAC AGTGTCCCCC

AAATGGGCTG

TGAGTTTGAT

AGTTTCTTCT

ATGAGGATCC

CCTCCCCATA

CACAGTGGGC

CTGCAGGAAG

GAGOTCCAAG

ACACTTAACC

AAACCTGAGA

AGCTTTTTTC

CAGCAGCCCA TCAGGGGAGG ACTCTAGAGA CCCCCAGGCA TATCCTCCCA GGGCACGGAA TAGTGAGGGG TACCTGCTGA TGTACCCTTT AAGCAGAAGG AGGGAGAGGG TGAGGCAGAG GOAGTAGOCO GAGAcaqaqa qa'ctatattt Note: The three high score predictions of promoters were bold and underlined. The lower case sequence indicates the beginning part of the transcript of 1O1P3A11 gene.

Table LV: Promoters and their positions predicted by Neural Network Promoter Prediction computer program.

Start 665 2477 3139 3420 4092 4953 End 75 715 2527 3189 3470 4142 5003 Score 0.91 0.95 0.91 0.82 0.96 0.99 0.97 Promoter Sequence

TTTGCATTTTTAATAGAGAGGGTTTCACCATGTTGGCCTGTC

CAGGAAGTTGTATATAAGGAGAATCAGAGCAGAGAGAGATAGGG-ICAG

TCAGTGTGCGTATATGTGAGAGAGAGGGTGCACACATGGJATACGTACTG

TGACATGACTCTAAGATGCCCAGTTTCTCGGCCTGGGGTCAGCCTGGGTG

GCAAAGAAAGCTGTTCCTTTCAAAAA

AACTG-ATCAGTAAAAAATAAGGGGAGACCAACTAAAAACCATGTTGTCT

AGGCAGAGAATAAATAAcCCTGACCAGGGAGGTCCAAJGAGTAGGCGGA Table LVI: Alignment of five homologous s, upstream genomic regulatory regions of the human lOlP3Ali and PSA genes.

Query: 5' upstream regulatory region of the PSA gene Subject: Putative S' upstream regulatory region of the lOIP3A1l gene.

Nucleic acid sequences predicted to be binding sites for the indicated transcription factors are bolded, underlined, or italicized.

Query: SbJct: Query: Sbj ct Query: Sbjct: 2.

Query: Sbjct: Query: Sbj ct: Query: Sbjct 3.

Query: Sbj ct Query: Sbjct.

3864 3598 3924 3538 3984 3478 4670 3496 4730 3556 4790 3615 NF-l SP-l NV-i ccaggctggagtgcagtggcgcagtctcggctcactgcaacctctgcc tcccaggttcaa ccgcgatcggctaccgcccgactcctgggta gtgattctcctgcctcagcctcctgagt tgctgggattacaggcatgcagcaccatgccc agctaatttttgtatttttagtagagatgggg 4015 ggctaattgttgtatttttagtagagatgggg 3447 cctgtagtcccagctagttgggaggctgaggcaggagaaacgct tgagcccgcaaggtgg SPI NF-E NF-i NP-i OR OR aggttgcaa tgagccgagat tgcgccac tgcactccagcctzgggtgacagtagtgagactc aggttgcagtgagccgagatcatgccac tgcactccagcct-ggtgacagagcaagactc tgtctcaaaaaaaaaaaa 4807 tgtgtzcaaaaaaaaaaaa 3632 3923 3539 3983 3479 4729 3555 4789 3614 Query: Sbjct: 4.

Query: Sbjct: Query: Sbjct: OR NF-l SPi 142 tgagactgagtctcgctctgtgcccaggctggaatgcagtggtgcaaccttggctcactg 201 3621 tgacacagagtcttgctctgtcaccaggctggagtgqcagtggcatgatctcggctcactg 3562 202 caagctccgcctcctgggttcacgccattctcctgcctcagcctcctgagtagctgggac 261 3561 caacctccaccttgcgggctcaagcgtttctcctgcctcagcctcccaactagctgggac 3502 262 tacaggcacccgccaccacgcctggctaannnnnnngtatttttagtagagatgggg 318 3501 tacaggcacgcgccatcacacccggctaa--ttgttgtatttttagtagagatgggg 3447 300 atttttagtagagatggggtttcactgtgttagccaggatggtctcagtctcctgacctc 359 31 atttttaatagaggcagggtttcaccatgttggcctggctggtctcgaaccctgacctt SPl NP-i LP-Al CP2 360 gtacgcactgcccaggcggtaaggggecggc 419 91 gcgatctgcccacctcggcctcccaaagtgctgggattacaggcgtgagccactgtacct 150 00 Query: 420 ggc 422 00 5 Sbjct: 151 99c 153 NF-1 Query: 4506 gcagaat~ttagcgatccaactgagtaaggga 4565 111111II IIITIIIIIIIIiIiijigi ii I II iiiI 111111 III Sbjct: 153 gcagaalSccccttaccgattgagcagggaa 94 Query: 4566 caggtaggtggcactacaagtaattttt 4620 Sbjct: 93 cgagtaggtggcacagcaagtaactctt 39 Table LVII >1O1P3A11 v.1 aa 1-318: for 9-mers, lO-mers, MMVDPNGNES SATYFILIOL PGLBEAQFWL AFPLCSLYLI AVLGNLTIIY PMYIFLCMLS GIDILISTSS MPKMLAIFWF NSTTIQFDAC LLQZFAIHSL MAFDRYVAIC HPLRHiATVLT LPRVTKIGVA AVVRGAAL4A PLPVFIKQLP YCLHQDVMKL ACDDIRVNVV YGLIVIISAI GLDSLLISFS YLLILKTVLG GTCVSHVCAV Fl FYVPFIGL SMVHRFSKRR DSPLPVILAN IYLLVPPVLN IRQRILRLFH VATHASEP >1O1PA11 v.2 9-mera, aa 36-72 SLYLIAVLASGVTLRCPSSWPIS

ICWFLLCSTQLSME

1O-mers, aa 35-72 CSLYLIAVLASGVTLRCPSSWPIS

ICWFLLCSTQLSME

15-mers, aa 30-72 LAFPLCSLYLIAVLASGVTLRCPSSWPIS

ICWFLLCSTQLSME

>101P3A11 v.3

IVRTEHSLHE

SGMESTVLLA

FCRSNILSHS

LTREAQAKAF

PIVYGVKTKE

9-mere; aa 96-112

QFDACLLQNFAIHSLSG

aa 95-113 IQFDACLLQNFAIHSLSO3M iS-mers, aa 90-118

FNSTTIQFDACLLQMFAIHSLSGMESTVL

Claims (37)

1. A composition comprising: 00 a substance that a) modulates the status of a protein of Figure 2 (SEQ ID NOS: or b) a molecule that is modulated by a protein of Figure 2, whereby the status of a cell that expresses a protein of Figure 2 is modulated.

2. A composition of claim 1, further comprising a physiologically acceptable carrier. 00 3. A pharmaceutical composition that comprises the composition of claim 1 in a human unit dose form.

4. A composition of claim 1 wherein the substance comprises an antibody or fragment thereof that specifically binds to a protein that is related to a protein of Figure 2. An antibody or fragment thereof of claim 4, which is monoclonal.

6. An antibody of claim 4, which is a human antibody, a humanized antibody or a chimeric antibody.

7. A non-human transgenic animal that produces an antibody of claim 4.

8. A hybridoma that produces an antibody of claim

9. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses a protein of Figure 2 (SEQ ID NOS: said method comprising: providing the cytotoxic agent or the diagnostic agent conjugated to an antibody or fragment thereof of claim 4; and, exposing the cell to the antibody-agent or fragment-agent conjugate. A composition of claim I wherein the substance comprises a polynucleotide that encodes an antibody or fragment thereof, either of which immunospecifically bind to a protein of Figure 2.

11. A composition of claim I wherein the substance comprises a protein related to a protein of Figure 2.

12. A protein of claim 11 that is at least 90% homologous to an entire amino acid sequence shown in Figure 2 (SEQ ID NOS: 00 O O (N

13. A composition of claim I wherein the substance comprises: c a) a peptide of eight, nine, ten, or eleven contiguous amino acids of a protein of Figure 2; 00 b) a peptide of Tables V to XVIII (SEQ ID NOS:_ c) a peptide of Tables XXII to XLVII (SEQ ID NOS: or, d) a peptide of Tables XLVIII to LI (SEQ ID NOS:

14. A composition of claim 1 wherein the substance comprises a CTL polypeptide or an analog 0 thereof, from the amino acid sequence of a protein of Figure 2 (SEQ ID NOS: 00 15. A composition of claim 14 further limited by a proviso that the epitope is not an entire 0 amino acid sequence of Figure 2 (SEQ ID NOS:).

16. A composition of claim 14 wherein the substance comprises a CTL polypeptide set forth in Tables V to XVIII (SEQ ID NOS:

17. A composition of claim 16 further limited by a proviso that the polypeptide is not an entire amino acid sequence of a protein of Figure 2 (SEQ ID NOS:

18. A composition of claim I wherein the substance comprises an antibody polypeptide epitope from an amino acid sequence of Figure 2 (SEQ ID NOS:

19. A composition of claim 18 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: A composition of claim 18 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of Figure 2 (SEQ ID NOS: in any whole number increment up to the end of said peptide, wherein the epitope comprises an amino acid position selected from: a) an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure b) an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; c) an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; d) an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; e) an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9; f) a combination of at least two of a) through e); g) a combination of at least three of a) through e); a combination of at least four of a) through or a combination of five of a) through e).

21. A composition of claim 20 further limited by a proviso that the epitope is not an entire S amino acid sequence of Figure 2 (SEQ ID NOS:

22. A polynucleotide that encodes a protein of claim 11.

23. A polynucleotide of claim 22 that comprises a nucleic acid molecule set forth in Figure 2.

24. A polynucleotide of claim 22 further limited by a proviso that the encoded protein is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: A polynucleotide of claim 22 wherein T is substituted with U.

26. A composition of claim 1 wherein the substance comprises a polynucleotide that comprises a coding sequence of a nucleic acid sequence of Figure 2 (SEQ ID NOS:

27. A polynucleotide of claim 22 that further comprises an additional nucleotide sequence that encodes an additional protein of claim 11.

28. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 22.

29. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 27.

31. A composition of claim 1 wherein the substance comprises a) a ribozyme that cleaves a polynucleotide having a 101P3A11 coding sequence, or b) a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier.

32. A composition of claim 1 wherein the substance comprises human T cells, wherein said T cells specifically recognize a 101P3AI I peptide subsequence in the context ofa particular HLA molecule.

33. A method of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising: 00 O administering to the cells the composition of claim 1.

34. A method of claim 33 of inhibiting growth of cancer cells that express a protein of Figure 2, 00 the method comprising steps of: administering to said cells an antibody or fragment thereof, either of which specifically bind to a O 101P3A1 -related protein. A method of claim 33 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: 00 administering to said cells a 101P3AI 1-relalcd protein. O C 36. A method of claim 33 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: administering to said cells a polynucleotide comprising a coding sequence for a 101P3AI 1-related protein or comprising a polynucleotide complementary to a coding sequence for a 101P3A1 l-related protein.

37. A method of claim 33 of inhibiting growth of cancer cells that express a protein of Figure 2, the method comprising steps of: administering to said cells a ribozyme that cleaves a polynucleotide that encodes a protein of Figure 2.

38. A method of claim 33 of inhibiting growth of cancer cells that express a protein of Figure 2 and a particular HLA molecule, the method comprising steps of: administering human T cells to said cancer cells, wherein said T cells specifically recognize a S peptide subsequence of a protein of Figure 2 while the subsequence is in the context of the particular HLA molecule.

39. A method of claim 33, the method comprising steps of: administering a vector that delivers a nucleotide that encodes a single chain monoclonal antibody, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express a protein of Figure 2. A method of generating a mammalian immune response directed to a protein of Figure 2, the method comprising: exposing cells of the mammal's immune system to a portion of a) a 101P3Al 1-related protein and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to said protein. 00 O O C 41. A method of generating an immune response of claim 40, said method comprising: F1 providing a 101P3A 1 -related protein that comprises at least one T cell or at least one B cell epitope; and, 00 contacting the epitope with a mammalian immune system T cell or B cell respectively, whereby the S T cell or B cell is activated. (0 42. A method of claim 41 wherein the immune system cell is a B cell, whereby the induced B M) cell generates antibodies that specifically bind to the 101P3A 1 -related protein. 00 N

43. A method of claim 41 wherein the immune system cell is aT cell that is a cytotoxic T cell S (CTL), whereby the activated CTL kills an autologous cell that expresses the 101P3A 11-related protein.

44. A method of claim 41 wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producing activity ofa B cell. A method for detecting, in a sample, the presence of a 101P3AI 1-related protein or a 101P3A1 l-related polynucleotide, comprising steps of: contacting the sample with a substance of claim I that specifically binds to the 101P3A1 l-related protein or to the 101P3A ll-related polynucleotide, respectively; and, determining that there is a complex of the substance with the 101P3AI I-related protein or the substance with the 101P3A1 l-related polynucleotide, respectively.

46. A method of claim 45 for detecting the presence of a 101P3Al1 -related protein in a sample comprising steps of: contacting the sample with an antibody or fragment thereof either of which specifically bind to the 101P3AI 1-related protein; and, determining that there is a complex of the antibody or fragment thereof and the 101P3AI 1-related protein.

47. A method of claim 45 further comprising a step of: taking the sample from a patient who has or who is suspected of having cancer.

48. A method of claim 45 for detecting the presence of a protein of Figure 2 mRNA in a sample comprising: producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using 101P3A11 polynucleotides as sense and antisense primers, wherein the 101P3AI polynucleotides used as the sense and antisense primers serve to amplify a 101P3AI cDNA; and, 00 0 detecting the presence of the amplified 101P3A 11 cDNA.

49. A method of claim 45 for monitoring one or more 101P3AI 1 gene products in a biological 00 sample from a patient who has or who is suspected of having cancer, the method comprising: Sdetermining the status of one or more 101 P3A 1 gene products expressed by cells in a tissue sample from an individual; comparing the status so determined to the status of one or more 101P3A11 gene products in a IND corresponding normal sample; and, identifying the presence of one or more aberrant gene products of 101P3AI in the sample relative to the normal sample. 00 The method of claim 49 further comprising a step of determining if there are one or more elevated gene products of a 101P3A1 I mRNA or a 101P3A I protein, whereby the presence of one or more elevated gene products in the test sample relative to the normal tissue sample indicates the presence or status of a cancer.

51. A method of claim 50 wherein the cancer occurs in a tissue set forth in Table I.