# Patent

## Title
Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer

## Abstract

A novel gene 158P3D2 and its encoded protein, and variants thereof, are described wherein 158P3D2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 158P3D2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 158P3D2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 158P3D2 can be used in active or passive immunization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 12/899,433, filed Oct. 6, 2010, now U.S. Pat. No. 8,212,017, which is a continuation of U.S. patent application Ser. No. 10/994,106, filed Nov. 19, 2004, now U.S. Pat. No. 7,811,575, which is a continuation-in-part of U.S. patent application Ser. No. 10/107,532, filed Mar. 25, 2002, now abandoned, which claims priority to U.S. Provisional Patent Application No. 60/283,112, filed Apr. 10, 2001, and U.S. Provisional Patent Application No. 60/286,630, filed Apr. 25, 2001. The contents of the applications listed in this paragraph are fully incorporated by reference herein.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(A), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:












File Name
Date of Creation
Size (bytes)







511582006410Seqlist.txt
Aug. 15, 2012
718,883 bytes









FIELD OF THE INVENTION
The invention described herein relates to genes and their encoded proteins, termed 158P3D2 and variants thereof, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 158P3D2.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 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 urethras. 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 eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 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 (−1.7% per year) while rates for women were still significantly increasing (0.9% 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.
Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have 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 −0.9% 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.
SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 158P3D2, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 158P3D2 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 158P3D2 are provided. The tissue-related profile of 158P3D2 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 158P3D2 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 158P3D2 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 158P3D2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 158P3D2-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 158P3D2 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 158P3D2 genes, mRNAs, or to 158P3D2-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 158P3D2. Recombinant DNA molecules containing 158P3D2 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 158P3D2 gene products are also provided. The invention further provides antibodies that bind to 158P3D2 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.
The invention further provides methods for detecting the presence and status of 158P3D2 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 158P3D2. A typical embodiment of this invention provides methods for monitoring 158P3D2 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 158P3D2 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 158P3D2 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 158P3D2 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 158P3D2. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 158P3D2 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The 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 158P3D2 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 that is immunologically reactive with 158P3D2 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 158P3D2. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 158P3D2 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 158P3D2 production) or a ribozyme effective to lyse 158P3D2 mRNA.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables VIII-XXI and XXII to XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:
i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 5;
ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 6;
iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 7;
iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 8; or
v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 9. [!!!The Figure descriptions need to be revised when using this as a template]



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 158P3D2 SSH sequence of 312 nucleotides.
FIG. 2A) The cDNA and amino acid sequence of 158P3D2 variant 1 clone 158P3D2-BCP-1 (also called “158P3D2 v.1” or “158P3D2 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2B) The cDNA and amino acid sequence of 158P3D2 variant 2A (also called “158P3D2 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 117-827 including the stop codon.
FIG. 2C) The cDNA and amino acid sequence of 158P3D2 variant 2B (also called “158P3D2 v.2”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2249-2794 including the stop codon.
FIG. 2D) The cDNA and amino acid sequence of 158P3D2 variant 3 (also called “158P3D2 v.3”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2E) The cDNA and amino acid sequence of 158P3D2 variant 4 (also called “158P3D2 v.4”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2F) The cDNA and amino acid sequence of 158P3D2 variant 5A (also called “158P3D2 v.5”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1385 including the stop codon.
FIG. 2G) The cDNA and amino acid sequence of 158P3D2 variant 5B (also called “158P3D2 v.5”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1289-1834 including the stop codon.
FIG. 2H) The cDNA and amino acid sequence of 158P3D2 variant 6 (also called “158P3D2 v.6”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2I) The cDNA and amino acid sequence of 158P3D2 variant 7 (also called “158P3D2 v.7”) is shown in FIG. 2I. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2J) The cDNA and amino acid sequence of 158P3D2 variant 8 (also called “158P3D2 v.8”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.
FIG. 2K) The cDNA and amino acid sequence of 158P3D2 variant 14 (also called “158P3D2 v.14”) is shown in FIG. 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-4246 including the stop codon.
FIG. 2L) The cDNA and amino acid sequence of 158P3D2 variant 15 (also called “158P3D2 v.15”) is shown in FIG. 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-3502 including the stop codon.
FIG. 2M) The cDNA and amino acid sequence of 158P3D2 variant 16 (also called “158P3D2 v.16”) is shown in FIG. 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6037 including the stop codon.
FIG. 2N) The cDNA and amino acid sequence of 158P3D2 variant 17 (also called “158P3D2 v.17”) is shown in FIG. 2N. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6175 including the stop codon.
FIG. 2O) The cDNA and amino acid sequence of 158P3D2 variant 18 (also called “158P3D2 v.18”) is shown in FIG. 2O. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2932-4764 including the stop codon.
FIG. 2P) The cDNA and amino acid sequence of 158P3D2 variant 19 (also called “158P3D2 v.19”) is shown in FIG. 2P. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6001 including the stop codon.
FIG. 2Q) The cDNA and amino acid sequence of 158P3D2 variant 20 (also called “158P3D2 v.20”) is shown in FIG. 2Q. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6121 including the stop codon.
FIG. 2R) 158P3D2 v.9 through v.13, SNP variants of 158P3D2 v.1. The 158P3D2 v.9 through v.13 proteins have 1072 amino acids. Variants 158P3D2 v.4 through v.20 are variants with single nucleotide difference from 158P3D2 v.1. 158P3D2 v.10, v.12 and v.13 proteins differ from 158P3D2 v.1 by one amino acid. 158P3D2 v.9 and v.11 proteins code for the same protein as v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above in FIGS. 2A-2Q.
FIG. 3A) The amino acid sequence of 158P3D2 v.1 clone 158P3D2-BCP-1 is shown in FIG. 3A; it has 328 amino acids.
FIG. 3B) The amino acid sequence of 158P3D2 v.2A is shown in FIG. 3B; it has 236 amino acids.
FIG. 3C) The amino acid sequence of 158P3D2 v.2B is shown in FIG. 3C; it has 181 amino acids.
FIG. 3D) The amino acid sequence of 158P3D2 v.3 is shown in FIG. 3D; it has 328 amino acids.
FIG. 3E) The amino acid sequence of 158P3D2 v.4 is shown in FIG. 3E; it has 328 amino acids.
FIG. 3F) The amino acid sequence of 158P3D2 v.5A is shown in FIG. 3F; it has 178 amino acids.
FIG. 3G) The amino acid sequence of 158P3D2 v.5B is shown in FIG. 3G; it has 181 amino acids.
FIG. 3H) The amino acid sequence of 158P3D2 v.10 is shown in FIG. 3H; it has 328 amino acids.
FIG. 3I) The amino acid sequence of 158P3D2 v.11 is shown in FIG. 3I; it has 328 amino acids.
FIG. 3J) The amino acid sequence of 158P3D2 v.12 is shown in FIG. 3J; it has 328 amino acids.
FIG. 3K) The amino acid sequence of 158P3D2 v.13 is shown in FIG. 3K; it has 328 amino acids.
FIG. 3L) The amino acid sequence of 158P3D2 v.14 is shown in FIG. 3L; it has 1393 amino acids.
FIG. 3M) The amino acid sequence of 158P3D2 v.15 is shown in FIG. 3M; it has 1145 amino acids.
FIG. 3N) The amino acid sequence of 158P3D2 v.16 is shown in FIG. 3N; it has 1990 amino acids.
FIG. 3O) The amino acid sequence of 158P3D2 v.17 is shown in FIG. 3O; it has 2036 amino acids.
FIG. 3P) The amino acid sequence of 158P3D2 v.18 is shown in FIG. 3P; it has 610 amino acids.
FIG. 3Q) The amino acid sequence of 158P3D2 v.19 is shown in FIG. 3Q; it has 1978 amino acids.
FIG. 3R) The amino acid sequence of 158P3D2 v.20 is shown in FIG. 3R; it has 2018 amino acids.
As used herein, a reference to 158P3D2 includes all variants thereof, including those shown in FIGS. 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.
FIG. 4. Effect of 158P3D2 RNAi on cell proliferation. SCaBER cells or Cos-1 cells were transfected with Lipofectamine 2000 reagent (LF2K) alone, or with negative control Luc4 oligo (20 nM), positive control Eg5 oligo (20 nM) or 158P3D2.b oligo (20 nM). After 48 hours, the media was replaced and the cells were incubated for 24 hrs, pulsed with 3H-thymidine at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat and counted in scintillation cocktail on a Microbeta trilux counter. Percentage cell proliferation relative to the LF2k control (100%) is shown. The reduction in 158P3D2 levels by the 158P3D2.b siRNA oligo correlated with diminished cell proliferation in the SCaBER cells, but no effect was observed in the 158P3D2-negative cell line Cos-1.
FIG. 5(a)-(i). Hydrophilicity amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 6(a)-(i). Hydropathicity amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 7(a)-(i). Percent accessible residues amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 8(a)-(i). Average flexibility amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 9(a)-(i). Beta-turn amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 10. Exon compositions of transcript variants of 158P3D2. Variant 158P3D2 v.2, v.14 through v.20 are transcript variants. Compared with 158P3D2 v.1; v.2 had six additional exons to the 5′ end, an exon 7 longer than exon 1 of 158P3D2 v.1 and an exon 10 shorter than exon 4 of 158P3D2 v.1. Exons 2, 3, 5, 6 and 7 of 158P3D2 v.1 are the same as exons 8, 9, 11, 12 and 13 of 158P3D2 v.2, respectively. Other variants had different exon compositions as shown above. Numbers in “( )” underneath the box correspond to those of 158P3D2 v.1. Black boxes show the same sequence as 158P3D2 v.1. Length of introns are not proportional.
FIG. 11. Schematic display of protein variants of 158P3D2. Nucleotide variant 158P3D2 v.2 and 158P3D2 v.5 potentially coded for two different proteins, designated as variants 158P3D2 v.2A and 158P3D2 v.2B, 158P3D2 v.5A and 158P3D2 v.5B, respectively. Variant 158P3D2 v.5B shares the same amino acid sequence as variant 158P3D2 v.2B. Variants 158P3D2 v.3 and v.4 were variants with single amino acid variations. Black box shows the same sequence as 158P3D2 v.1. Numbers in “( )” underneath the black boxes correspond to those of 158P3D2 v.1 and those underneath the “brick” boxes correspond to those of v.17. Single amino acid differences are indicated above the box.
FIG. 12. Schematic display of SNP variants of 158P3D2. Variant 158P3D2 v.3 through v.13 are variants with a single nucleotide difference from v.1. Though these alternative SNP alleles were shown separately, they could occur in any transcript variants in any combination (called haplotype). Numbers in “( )” underneath the box correspond to those of 158P3D2 v.1. ‘-’ indicate single nucleotide deletion. Black boxes show the same sequence as 158P3D2 v.1. SNPs are indicated above the box.
FIG. 13. Secondary structure and transmembrane domains prediction for 158P3D2 protein variants.
FIG. 13A (SEQ ID NO:54), FIG. 13B (SEQ ID NO:55), FIG. 13C (SEQ ID NO:56), FIG. 13D (SEQ ID NO:57), FIG. 13E (SEQ ID NO:58), FIG. 13F (SEQ ID NO:59), FIG. 13G (SEQ ID NO:60), FIG. 13H (SEQ ID NO:61), FIG. 13I (SEQ ID NO:62): The secondary structures of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, were predicted using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G., accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein variant in a given secondary structure is also listed.
FIG. 13J, FIG. 13L, FIG. 13N, FIG. 13P, FIG. 13R, FIG. 13T, FIG. 13V, FIG. 13X, and FIG. 13Z: Schematic representation of the probability of existence of transmembrane regions of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIG. 13K, FIG. 13M, FIG. 13O, FIG. 13Q, FIG. 13S, FIG. 13U, FIG. 13W, FIG. 13Y, FIG. 13AA: Schematic representation of the probability of the existence of transmembrane regions of 158P3D2 variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server.
FIG. 14. 158P3D2 Expression in Normal and Cancer Tissue Specimens. First strand cDNA was prepared from a panel of 13 normal tissues (brain, heart, kidney, liver, lung, spleen, skeletal muscle, testis, pancreas, colon, stomach) and pools of 4-7 patients from the following cancer indications: bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, multiple cancer metastasis, cervix, lymphoma as well as from a pool of patient-derived xenografts (prostate cancer, bladder cancer and kidney cancer). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression of 158P3D2 in cancers of the bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, cervix, and lymphoma. Low expression was detected in all normal tissues tested except in normal stomach. Strong expression was also observed in the cancer metastasis pool.
FIG. 15. 158P3D2 Expression in bladder cancer patient specimens. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82) and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of bladder cancer patient specimens tested. Very low expression was detected in normal tissues, but no expression was seen in the cell lines tested.
FIG. 16. 158P3D2 Expression in bladder cancer patient specimens by northern blotting. RNA was extracted from normal bladder, bladder cancer cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and their normal adjacent tissues (NAT). Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal nor NAT tissues.
FIG. 17. 158P3D2 Expression in lung cancer patient specimens. First strand cDNA was prepared from normal lung, cancer cell line A427 and a panel of lung cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) of lung cancer specimens, but not in all 3 normal lung tissues tested.
FIG. 18. 158P3D2 Expression in lung cancer patient specimens by northern blotting. RNA was extracted from normal lung, A427 lung cancer cell line, and a panel of lung cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in normal tissues.
FIG. 19. 158P3D2 Expression in cancer metastasis patient specimens. First strand cDNA was prepared from normal colon, kidney, liver, lung, pancreas, stomach and from a panel of cancer metastasis patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of patient cancer metastasis specimens tested but not in normal tissues.
FIG. 20. 158P3D2 Expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in all 14 cervical cancer patient specimens tested. No expression was detected in normal cervix nor in the cell line tested.
FIG. 21. 158P3D2 Expression in cervical cancer patient specimens by northern blotting. RNA was extracted from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal cervix nor in the cell line.
FIG. 22. 158P3D2 Expression in kidney cancer patient specimens. First strand cDNA was prepared from normal kidney, kidney cancer cell lines (769-P, A-498, CAM-1), and a panel of kidney cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of kidney cancer patient specimens, but not in all 3 normal kidney tissues tested. Low expression was detected in 2 of 3 cell lines tested.
FIG. 23. 158P3D2 Expression in kidney cancer patient specimens by northern blotting. RNA was extracted from normal kidney and a panel of kidney cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.
FIG. 24. 158P3D2 Expression in stomach cancer patient specimens. First strand cDNA was prepared from normal stomach, and a panel of stomach cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of stomach cancer patient specimens. Weak expression was detected in the 2 normal stomach, and only in 1 of the 2 NAT tissues tested.
FIG. 25. 158P3D2 Expression in stomach cancer patient specimens by northern blotting. RNA was extracted from normal stomach and a panel of stomach cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.
FIG. 26. 158P3D2 Expression in colon cancer patient specimens. First strand cDNA was prepared from normal colon, colon cancer cell lines (LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of colon cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of colon cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 3 of 5 cell lines tested.
FIG. 27. 158P3D2 Expression in uterus cancer patient specimens. First strand cDNA was prepared from normal uterus and a panel of uterus cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of uterus cancer patient specimens, but not in normal uterus.
FIG. 28. 158P3D2 Expression in breast cancer patient specimens. First strand cDNA was prepared from normal breast, breast cancer cell lines (MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel of breast cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of breast cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 2 of 5 cell lines tested.
FIG. 29. Serum titer of mice immunized with KLH-peptide encoding amino acids 315-328 of 158P3D2. Serial dilutions of serum taken from immunized mice were incubated on an ELISA plate coated with the 158P3 D2 peptide conjugated to ovalbumin Specific bound antibody was then detected by incubation goat anti-mouse IgG-HRP conjugate and then visualized and quantitated by development with TMB substrate and optical density determination.
FIG. 30. Validation of 158P3D2 siRNA oligo. Cos-1 cells were transfected with 1 μg pcDNA3-158P3D2, which encodes a full-length 158P3D2 protein fusion with a Myc/His tag on the C-terminus, simultaneously with Lipofectamine 2000 reagent (LF2k) alone, or with control CT1 oligo (20 nM), 158P3D2.b oligo (20 nM), or no DNA or oligo (No DNA). After 72 hours, the cells were lysed in 1% Triton buffer, and 50 μg of total soluble cell lysate was analyzed by Western blotting. The upper panel was blotted with anti-Myc (1:1000) to detect the 158P3D2-Myc/His fusion protein and the lower panel was developed with anti-actin. The level of 158P3D2 was diminished by the 158P3D2 siRNA oligo, whereas no change was observed with the control (LF2k) or siRNA oligo CT1. In contrast, no change in the level of actin was noted in the cell lysates, indicating that the loading was equivalent in all lanes.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 158P3D2 Polynucleotides
II.A.) Uses of 158P3D2 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
II.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) 158P3D2-related Proteins
III.A.) Motif-bearing Protein Embodiments
III.B.) Expression of 158P3D2-related Proteins
III.C.) Modifications of 158P3D2-related Proteins
III.D.) Uses of 158P3D2-related Proteins
IV.) 158P3D2 Antibodies
V.) 158P3D2 Cellular Immune Responses
VI.) 158P3D2 Transgenic Animals
VII.) Methods for the Detection of 158P3D2
VIII.) Methods for Monitoring the Status of 158P3D2-related Genes and Their Products
IX.) Identification of Molecules That Interact With 158P3D2
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) 158P3D2 as a Target for Antibody-Based Therapy
X.C.) 158P3D2 as a Target for Cellular Immune Responses
X.C.1 Minigene Vaccines
X.C.2. Combinations of CTL Peptides with Helper Peptides
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
X.D.) Adoptive Immunotherapy
X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
XI.) Diagnostic and Prognostic Embodiments of 158P3D2.
XII.) Inhibition of 158P3D2 Protein Function
XII.A.) Inhibition of 158P3D2 With Intracellular Antibodies
XII.B.) Inhibition of 158P3D2 with Recombinant Proteins
XII.C.) Inhibition of 158P3D2 Transcription or Translation
XII.D.) General Considerations for Therapeutic Strategies
XIII.) Identification, Characterization and Use of Modulators of 109P1D1
XIII.A.) Methods to Identify and Use Modulators
XIII.B.) Gene Expression-related Assays
XIII.C.) Expression Monitoring to Identify Compounds that Modify Gene Expression
XIII.D.) Biological Activity-related Assays
XIII.E.) High Throughput Screening to Identify Modulators
XIII.F.) Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
XIII.G.) Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
XIII.H.) Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
XIII.I.) Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
XIII.J.) Invasiveness into Matrigel to Identify and Characterize Modulators
XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
XIII.L.) In Vitro Assays to Identify and Characterize Modulators
XIII.M.) Binding Assays to Identify and Characterize Modulators
XIII.N.) Competitive Binding to Identify and Characterize Modulators
XIII.O.) Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
XIII.P.) Inhibitory and Antisense Nucleotides
XIII.Q.) Ribozymes
XIII.R.) Use of Modulators in Phenotypic Screening
XIII.S.) Use of Modulators to Affect Peptides of the Invention
XIII.T.) Methods of Identifying Characterizing Cancer-associated Sequences
XIV.) KITS/Articles of Manufacture
I.) 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 C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 158P3D2 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 158P3D2. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 158P3D2-related protein). For example, an analog of a 158P3D2 protein can be specifically bound by an antibody or T cell that specifically binds to 158P3D2.
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-158P3D2 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-158P3D2 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-158P3D2 antibody compositions with polyepitopic specificity.
The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”
A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).
Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).
The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Rr186, Re188, Sm153, Bi212 or 213, P32 and radioactive isotopes of Lu including Lu177. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
The “gene product” is sometimes referred to herein as a protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of FIG. 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of FIG. 2. In another embodiment, the sequences are sequence variants as further described herein.
“High throughput screening” assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).
The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.
The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 158P3D2 genes or that encode polypeptides other than 158P3D2 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 158P3D2 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 158P3D2 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 158P3D2 protein. Alternatively, an isolated protein can be prepared by chemical means.
The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ 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 (i.e., 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 “modulator” or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By “neutralize” is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein. The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.
The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., 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 158P3D2-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 (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. 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 pattern of the primary and secondary anchor residues.
A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in FIG. 2, can also be uracil (U); 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 (U) instead of thymidine (T).
The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.
An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
“Radioisotopes” include, but are not limited to the following (non-limiting exemplary uses are also set forth):
Examples of Medical Isotopes











Isotope
Description of use







Actinium-225
See Thorium-229 (Th-229)


(AC-225)



Actinium-227
Parent of Radium-223 (Ra-223) which is an alpha emitter used to


(AC-227)
treat metastases in the skeleton resulting from cancer (i.e.,



breast and prostate cancers), and cancer radioimmunotherapy


Bismuth-212
See Thorium-228 (Th-228)


(Bi-212)



Bismuth-213
See Thorium-229 (Th-229)


(Bi-213)



Cadmium-109
Cancer detection


(Cd-109)



Cobalt-60
Radiation source for radiotherapy of cancer, for food irradiators, and


(Co-60)
for sterilization of medical supplies


Copper-64
A positron emitter used for cancer therapy and SPECT imaging


(Cu-64)



Copper-67
Beta/gamma emitter used in cancer radioimmunotherapy and


(Cu-67)
diagnostic studies (i.e., breast and colon cancers, and lymphoma)


Dysprosium-166
Cancer radioimmunotherapy


(Dy-166)



Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints


(Er-169)
associated with fingers and toes


Europium-152
Radiation source for food irradiation and for sterilization of medical


(Eu-152)
supplies


Europium-154
Radiation source for food irradiation and for sterilization of medical


(Eu-154)
supplies


Gadolinium-153
Osteoporosis detection and nuclear medical quality assurance devices


(Gd-153)



Gold-198
Implant and intracavity therapy of ovarian, prostate, and brain


(Au-198)
cancers


Holmium-166
Multiple myeloma treatment in targeted skeletal therapy, cancer


(Ho-166)
radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis



treatment


Iodine-125
Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer


(I-125)
treatment, radiolabeling, tumor imaging, mapping of receptors in the



brain, interstitial radiation therapy, brachytherapy for treatment of



prostate cancer, determination of glomerular filtration rate (GFR),



determination of plasma volume, detection of deep vein thrombosis



of the legs


Iodine-131
Thyroid function evaluation, thyroid disease detection, treatment of


(I-131)
thyroid cancer as well as other non-malignant thyroid diseases (i.e.,



Graves disease, goiters, and hyperthyroidism), treatment of leukemia,



lymphoma, and other forms of cancer (e.g., breast cancer) using



radioimmunotherapy


Iridium-192
Brachytherapy, brain and spinal cord tumor treatment, treatment of


(Ir-192)
blocked arteries (i.e., arteriosclerosis and restenosis), and implants for



breast and prostate tumors


Lutetium-177
Cancer radioimmunotherapy and treatment of blocked arteries (i.e.,


(Lu-177)
arteriosclerosis and restenosis)


Molybdenum-99
Parent of Technetium-99m (Tc-99m) which is used for imaging the


(Mo-99)
brain, liver, lungs, heart, and other organs. Currently, Tc-99m is the



most widely used radioisotope used for diagnostic imaging of various



cancers and diseases involving the brain, heart, liver, lungs; also used



in detection of deep vein thrombosis of the legs


Osmium-194
Cancer radioimmunotherapy


(Os-194)



Palladium-103
Prostate cancer treatment


(Pd-103)



Platinum-195m
Studies on biodistribution and metabolism of cisplatin, a


(Pt-195m)
chemotherapeutic drug


Phosphorus-32
Polycythemia rubra vera (blood cell disease) and leukemia treatment,


(P-32)
bone cancer diagnosis/treatment; colon, pancreatic, and liver cancer



treatment; radiolabeling nucleic acids for in vitro research, diagnosis



of superficial tumors, treatment of blocked arteries (i.e.,



arteriosclerosis and restenosis), and intracavity therapy


Phosphorus-33
Leukemia treatment, bone disease diagnosis/treatment, radiolabeling,


(P-33)
and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)


Radium-223
See Actinium-227 (Ac-227)


(Ra-223)



Rhenium-186
Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis


(Re-186)
and treatment of lymphoma and bone, breast, colon, and liver cancers



using radioimmunotherapy


Rhenium-188
Cancer diagnosis and treatment using radioimmunotherapy, bone


(Re-188)
cancer pain relief, treatment of rheumatoid arthritis, and treatment of



prostate cancer


Rhodium-105
Cancer radioimmunotherapy


(Rh-105)



Samarium-145
Ocular cancer treatment


(Sm-145)



Samarium-153
Cancer radioimmunotherapy and bone cancer pain relief


(Sm-153)



Scandium-47
Cancer radioimmunotherapy and bone cancer pain relief


(Sc-47)



Selenium-75
Radiotracer used in brain studies, imaging of adrenal cortex


(Se-75)
by gamma-scintigraphy, lateral locations of steroid secreting tumors,



pancreatic scanning, detection of hyperactive parathyroid glands,



measure rate of bile acid loss from the endogenous pool


Strontium-85
Bone cancer detection and brain scans


(Sr-85)



Strontium-89
Bone cancer pain relief, multiple myeloma treatment, and osteoblastic


(Sr-89)
therapy


Technetium-99m
See Molybdenum-99 (Mo-99)


(Tc-99m)



Thorium-228
Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in


(Th-228)
cancer radioimmunotherapy


Thorium-229
Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213


(Th-229)
(Bi-213) which are alpha emitters used in cancer



radioimmunotherapy


Thulium-170
Gamma source for blood irradiators, energy source for implanted


(Tm-170)
medical devices


Tin-117m
Cancer immunotherapy and bone cancer pain relief


(Sn-117m)



Tungsten-188
Parent for Rhenium-188 (Re-188) which is used for cancer


(W-188)
diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis



treatment, and treatment of blocked arteries (i.e., arteriosclerosis and



restenosis)


Xenon-127
Neuroimaging of brain disorders, high resolution SPECT studies,


(Xe-127)
pulmonary function tests, and cerebral blood flow studies


Ytterbium-175
Cancer radioimmunotherapy


(Yb-175)



Yttrium-90
Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver


(Y-90)
cancer treatment


Yttrium-91
A gamma-emitting label for Yttrium-90 (Y-90) which is used for


(Y-91)
cancer radioimmunotherapy (i.e., lymphoma, breast, colon, kidney,



lung, ovarian, prostate, pancreatic, and inoperable liver cancers)









By “randomized” or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, a library is “fully randomized,” with no sequence preferences or constants at any position. In another embodiment, the library is a “biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
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 158P3D2, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 158P3D2 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, 158P3D2 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “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 (e.g., 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×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° 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. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non-limiting constituents of various supertypes are as follows:

    
    
        A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901, A*0207
        A3: A3, All, A31, A*3301, A*6801, A*0301, A*1101, A*3101
        B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701, B*7801, B*0702, B*5101, B*5602
        B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
        A1: A*0102, A*2604, A*3601, A*4301, A*8001
        A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
        B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08
        B58: B*1516, B*1517, B*5701, B*5702, B58
        B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
    
    


Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).
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” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a 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, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150 or more, e.g., 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, e.g., dendritic cells.
The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 158P3D2 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
The “158P3D2-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 158P3D2 proteins or fragments thereof, as well as fusion proteins of a 158P3D2 protein and a heterologous polypeptide are also included. Such 158P3D2 proteins are collectively referred to as the 158P3D2-related proteins, the proteins of the invention, or 158P3D2. The term “158P3D2-related protein” refers to a polypeptide fragment or a 158P3D2 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.
II.) 158P3D2 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 158P3D2 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 158P3D2-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 158P3D2 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 158P3D2 gene, mRNA, or to a 158P3D2 encoding polynucleotide (collectively, “158P3D2 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 158P3D2 polynucleotide include: a 158P3D2 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 158P3D2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 158P3D2 nucleotides comprise, without limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2, wherein T can also be U;
(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B, from nucleotide residue number 117 through nucleotide residue number 827, including the stop codon, wherein T can also be U;
(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C, from nucleotide residue number 2249 through nucleotide residue number 2794, including the a stop codon, wherein T can also be U;
(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D, from nucleotide residue number 849 through nucleotide residue number 1835, 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 FIG. 2E, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G, from nucleotide residue number 1289 through nucleotide residue number 1834, including the stop codon, wherein T can also be U;
(IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2I, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2J, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;
(XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2K, from nucleotide residue number 65 through nucleotide residue number 4246, including the stop codon, wherein T can also be U;
(XIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2L, from nucleotide residue number 65 through nucleotide residue number 3502, including the stop codon, wherein T can also be U;
(XIV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2M, from nucleotide residue number 65 through nucleotide residue number 6037, including the stop codon, wherein T can also be U;
(XV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2N, from nucleotide residue number 65 through nucleotide residue number 6175, including the stop codon, wherein T can also be U;
(XVI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2O, from nucleotide residue number 2932 through nucleotide residue number 4764, including the stop codon, wherein T can also be U;
(XVII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2P, from nucleotide residue number 65 through nucleotide residue number 6001, including the stop codon, wherein T can also be U;
(XVIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2Q, from nucleotide residue number 65 through nucleotide residue number 6121, including the stop codon, wherein T can also be U;
(XIX) a polynucleotide that encodes a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-Q;
(XX) a polynucleotide that encodes a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-Q;
(XXI) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
(XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXVII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XXVI);
(XXVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XXVII);
(XXIX) a peptide that is encoded by any of (I) to (XXVIII); and;
(XXX) a composition comprising a polynucleotide of any of (I)-(XXVIII) or peptide of (XXIX) together with a pharmaceutical excipient and/or in a human unit dose form;
(XXXI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to modulate a cell expressing 158P3D2;
(XXXII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2;
(XXIII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2, said cell from a cancer of a tissue listed in Table I;
(XXXIV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a cancer;
(XXXV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I; and;
(XXXVI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to identify or characterize a modulator of a cell expressing 158P3D2.
As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325 and 328 or more contiguous amino acids of 158P3D2 variant 1; the maximal lengths relevant for other variants are shown in FIGS. 2A-2Q and 3A-3R respectively.
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 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 158P3D2 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions of a 158P3D2 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 158P3D2 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 158P3D2 sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polynucleotide fragments encoding one or more of the biological motifs contained within a 158P3D2 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 158P3D2 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 158P3D2 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 158P3D2 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
II.A.) Uses of 158P3D2 Polynucleotides
II.A.1. Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 158P3D2 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 158P3D2.” For example, because the 158P3D2 gene maps to this chromosome, polynucleotides that encode different regions of the 158P3D2 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 158P3D2 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 158P3D2 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 158P3D2 was shown to be highly expressed in prostate and other cancers, 158P3D2 polynucleotides are used in methods assessing the status of 158P3D2 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 158P3D2 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 158P3D2 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, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
II.A.2. Antisense Embodiments
Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 158P3D2. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 158P3D2 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, e.g., 158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 158P3D2 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 (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 158P3D2 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
The 158P3D2 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 158P3D2 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 158P3D2 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 158P3D2 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 158P3D2 mRNA. Optionally, 158P3D2 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 158P3D2. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 158P3D2 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet. 12: 510-515 (1996).
II.A.3. Primers and Primer Pairs
Further specific embodiments of these nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 158P3D2 polynucleotide in a sample and as a means for detecting a cell expressing a 158P3D2 protein.
Examples of such probes include polypeptides comprising all or part of the human 158P3D2 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 158P3D2 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 158P3D2 mRNA.
The 158P3D2 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 158P3D2 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 158P3D2 polypeptides; as tools for modulating or inhibiting the expression of the 158P3D2 gene(s) and/or translation of the 158P3D2 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 158P3D2 or 158P3D2 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.
II.A.4. Isolation of 158P3D2-Encoding Nucleic Acid Molecules
The 158P3D2 cDNA sequences described herein enable the isolation of other polynucleotides encoding 158P3D2 gene product(s), as well as the isolation of polynucleotides encoding 158P3D2 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 158P3D2 gene product as well as polynucleotides that encode analogs of 158P3D2-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 158P3D2 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 (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P3D2 gene cDNAs can be identified by probing with a labeled 158P3D2 cDNA or a fragment thereof. For example, in one embodiment, a 158P3D2 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 158P3D2 gene. A 158P3D2 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 158P3D2 DNA probes or primers.
II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 158P3D2 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 158P3D2 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, 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 (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 158P3D2 or a fragment, analog or homolog thereof can be used to generate 158P3D2 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of 158P3D2 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 158P3D2 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 158P3D2 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 158P3D2 and 158P3D2 mutations or analogs.
Recombinant human 158P3D2 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 158P3D2-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 158P3D2 or fragment, analog or homolog thereof, a 158P3D2-related protein is expressed in the 293T cells, and the recombinant 158P3D2 protein is isolated using standard purification methods (e.g., affinity purification using anti-158P3D2 antibodies). In another embodiment, a 158P3D2 coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 158P3D2 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 158P3D2 coding sequence can be used for the generation of a secreted form of recombinant 158P3D2 protein.
As discussed herein, redundancy in the genetic code permits variation in 158P3D2 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 (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/ ̃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 consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
III.) 158P3D2-Related Proteins
Another aspect of the present invention provides 158P3D2-related proteins. Specific embodiments of 158P3D2 proteins comprise a polypeptide having all or part of the amino acid sequence of human 158P3D2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 158P3D2 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 158P3D2 shown in FIG. 2 or FIG. 3.
Embodiments of a 158P3D2 polypeptide include: a 158P3D2 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 158P3D2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 158P3D2 peptides comprise, without limitation:
(I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-Q or FIG. 3A-3R;
(II) a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-Q or 3A-R;
(III) a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-Q or 3A-R;
(IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;
(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
(VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R, in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R, in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
(XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
(XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
(XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
(XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(XIX) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(XXII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide:
i) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 5;
ii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 6;
iii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 7;
iv) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 8; or,
v) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 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 FIG. 9;
(XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
(XXIV) a method of using a peptide of (I)-(XXII), or an antibody or binding region thereof or a composition of (XXIII) in a method to modulate a cell expressing 158P3D2;
(XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2;
(XXVI) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2, said cell from a cancer of a tissue listed in Table I;
(XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a cancer;
(XXVIII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I;
(XXIX) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition and;
(XXIII) in a method to identify or characterize a modulator of a cell expressing 158P3D2.
As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, and 328 or more contiguous amino acids of 158P3D2 variant 1; the maximal lengths relevant for other variants are shown in FIGS. 2A-2Q and 3A-3R.
In general, naturally occurring allelic variants of human 158P3D2 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 158P3D2 protein contain conservative amino acid substitutions within the 158P3D2 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 158P3D2. One class of 158P3D2 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 158P3D2 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) 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 (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 158P3D2 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 158P3D2 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 158P3D2 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 variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
As defined herein, 158P3D2 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 158P3D2 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 158P3D2 variant also specifically binds to a 158P3D2 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 158P3D2 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.
Other classes of 158P3D2-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 158P3D2 protein variants or analogs comprises one or more of the 158P3D2 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 158P3D2 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 158P3D2 protein shown in FIG. 2 or FIG. 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 158P3D2 protein shown in FIG. 2 or FIG. 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 158P3D2 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 158P3D2 protein shown in FIG. 2 or FIG. 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.
158P3D2-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 158P3D2-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 158P3D2 protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; EpimatrixTM and EpimerTM, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).
Motif bearing subsequences of all 158P3D2 variant proteins are set forth and identified in Tables VIII-XXI and XXII-XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 158P3D2 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 158P3D2 motifs discussed above are associated with growth dysregulation and because 158P3D2 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, URL brown.edu/Research/TB-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.
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, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.
A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander 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 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
158P3D2-related proteins are embodied in many forms, preferably in isolated form. A purified 158P3D2 protein molecule will be substantially free of other proteins or molecules that impair the binding of 158P3D2 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 158P3D2-related proteins include purified 158P3D2-related proteins and functional, soluble 158P3D2-related proteins. In one embodiment, a functional, soluble 158P3D2 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 158P3D2 proteins comprising biologically active fragments of a 158P3D2 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 158P3D2 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 158P3D2 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.
158P3D2-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-158P3D2 antibodies or T cells or in identifying cellular factors that bind to 158P3D2. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 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, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.
CTL epitopes can be determined using specific algorithms to identify peptides within a 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(A)-(E); EpimatrixTM and EpimerTM, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 158P3D2 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, All, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 158P3D2 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.
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, e.g., 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 al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 158P3D2 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 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, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)) Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be “applied” to a 158P3D2 protein in accordance with the invention. As used in this context “applied” means that a 158P3D2 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 158P3D2 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 158P3D2-Related Proteins
In an embodiment described in the examples that follow, 158P3D2 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 158P3D2 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or TagS, GenHunter Corporation, Nashville Tenn.). The TagS vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 158P3D2 protein in transfected cells. The secreted HIS-tagged 158P3D2 in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 158P3D2-Related Proteins
Modifications of 158P3D2-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 158P3D2 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 158P3D2 protein. Another type of covalent modification of a 158P3D2 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 158P3D2 comprises linking a 158P3D2 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 158P3D2-related proteins of the present invention can also be modified to form a chimeric molecule comprising 158P3D2 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 158P3D2 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 FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 158P3D2. A chimeric molecule can comprise a fusion of a 158P3D2-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 158P3D2 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 158P3D2-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 158P3D2 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 158P3D2-Related Proteins
The proteins of the invention have a number of different specific uses. As 158P3D2 is highly expressed in prostate and other cancers, 158P3D2-related proteins are used in methods that assess the status of 158P3D2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 158P3D2 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 158P3D2-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 158P3D2-related proteins that contain the amino acid residues of one or more of the biological motifs in a 158P3D2 protein are used to screen for factors that interact with that region of 158P3D2.
158P3D2 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 158P3D2 protein), for identifying agents or cellular factors that bind to 158P3D2 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 158P3D2 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 158P3D2 gene product. Antibodies raised against a 158P3D2 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 158P3D2 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 158P3D2-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 158P3D2 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 158P3D2-expressing cells (e.g., in radioscintigraphic imaging methods). 158P3D2 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 158P3D2 Antibodies
Another aspect of the invention provides antibodies that bind to 158P3D2-related proteins. Preferred antibodies specifically bind to a 158P3D2-related protein and do not bind (or bind weakly) to peptides or proteins that are not 158P3D2-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 158P3D2 can bind 158P3D2-related proteins such as the homologs or analogs thereof.
158P3D2 antibodies of the invention are particularly useful in cancer (see, e.g., 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 158P3D2 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 158P3D2 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 158P3D2 and mutant 158P3D2-related proteins. Such assays can comprise one or more 158P3D2 antibodies capable of recognizing and binding a 158P3D2-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 158P3D2 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 158P3D2 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 158P3D2 expressing cancers such as prostate cancer.
158P3D2 antibodies are also used in methods for purifying a 158P3D2-related protein and for isolating 158P3D2 homologues and related molecules. For example, a method of purifying a 158P3D2-related protein comprises incubating a 158P3D2 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 158P3D2-related protein under conditions that permit the 158P3D2 antibody to bind to the 158P3D2-related protein; washing the solid matrix to eliminate impurities; and eluting the 158P3D2-related protein from the coupled antibody. Other uses of 158P3D2 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 158P3D2 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 158P3D2-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 158P3D2 can also be used, such as a 158P3D2 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 158P3D2-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 158P3D2-related protein or 158P3D2 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 158P3D2 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 158P3D2 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 158P3D2 amino acid sequence are used to identify hydrophilic regions in the 158P3D2 structure. Regions of a 158P3D2 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, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 158P3D2 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 158P3D2 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
158P3D2 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 158P3D2-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 158P3D2 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 158P3D2 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 al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 158P3D2 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., 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. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 158P3D2 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 158P3D2 antibodies with a 158P3D2-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 158P3D2-related proteins, 158P3D2-expressing cells or extracts thereof. A 158P3D2 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 158P3D2 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
V.) 158P3D2 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., 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 November; 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, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern 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.C., 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, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., 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 al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “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 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 158P3D2 Transgenic Animals
Nucleic acids that encode a 158P3D2-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 158P3D2 can be used to clone genomic DNA that encodes 158P3D2. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 158P3D2. 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. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 158P3D2 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 158P3D2 can be used to examine the effect of increased expression of DNA that encodes 158P3D2. 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 158P3D2 can be used to construct a 158P3D2 “knock out” animal that has a defective or altered gene encoding 158P3D2 as a result of homologous recombination between the endogenous gene encoding 158P3D2 and altered genomic DNA encoding 158P3D2 introduced into an embryonic cell of the animal. For example, cDNA that encodes 158P3D2 can be used to clone genomic DNA encoding 158P3D2 in accordance with established techniques. A portion of the genomic DNA encoding 158P3D2 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 158P3D2 polypeptide.
VII.) Methods for the Detection of 158P3D2
Another aspect of the present invention relates to methods for detecting 158P3D2 polynucleotides and 158P3D2-related proteins, as well as methods for identifying a cell that expresses 158P3D2. The expression profile of 158P3D2 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 158P3D2 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 158P3D2 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 158P3D2 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variant 158P3D2 mRNAs, and recombinant DNA or RNA molecules that contain a 158P3D2 polynucleotide. A number of methods for amplifying and/or detecting the presence of 158P3D2 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 158P3D2 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 158P3D2 polynucleotides as sense and antisense primers to amplify 158P3D2 cDNAs therein; and detecting the presence of the amplified 158P3D2 cDNA. Optionally, the sequence of the amplified 158P3D2 cDNA can be determined
In another embodiment, a method of detecting a 158P3D2 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 158P3D2 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 158P3D2 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 158P3D2 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 158P3D2 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 158P3D2-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 detecting the presence of a 158P3D2-related protein in a biological sample comprises first contacting the sample with a 158P3D2 antibody, a 158P3D2-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 158P3D2 antibody; and then detecting the binding of 158P3D2-related protein in the sample.
Methods for identifying a cell that expresses 158P3D2 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 158P3D2 gene comprises detecting the presence of 158P3D2 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 158P3D2, 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 158P3D2 gene comprises detecting the presence of 158P3D2-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 158P3D2-related proteins and cells that express 158P3D2-related proteins.
158P3D2 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 158P3D2 gene expression. For example, 158P3D2 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 158P3D2 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 158P3D2 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 158P3D2-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, e.g., 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 158P3D2 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 158P3D2 in a biological sample of interest can be compared, for example, to the status of 158P3D2 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 158P3D2 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, e.g., Greyer et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 158P3D2 status in a sample.
The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 158P3D2 expressing cells) as well as the level, and biological activity of expressed gene products (such as 158P3D2 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 158P3D2 comprises a change in the location of 158P3D2 and/or 158P3D2 expressing cells and/or an increase in 158P3D2 mRNA and/or protein expression.
158P3D2 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 158P3D2 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 158P3D2 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 158P3D2 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 158P3D2 proteins and/or associations of 158P3D2 proteins with polypeptide binding partners). Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variants, 158P3D2 mRNAs, and recombinant DNA or RNA molecules containing a 158P3D2 polynucleotide.
The expression profile of 158P3D2 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 158P3D2 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 158P3D2 status and diagnosing cancers that express 158P3D2, such as cancers of the tissues listed in Table I. For example, because 158P3D2 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 158P3D2 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 158P3D2 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 158P3D2 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 158P3D2 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 158P3D2 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 158P3D2 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 158P3D2 expressing cells (e.g. those that express 158P3D2 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 158P3D2-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 158P3D2 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., 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 August 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 158P3D2 gene products by determining the status of 158P3D2 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 158P3D2 gene products in a corresponding normal sample. The presence of aberrant 158P3D2 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 158P3D2 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 158P3D2 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 158P3D2 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 158P3D2 mRNA or express it at lower levels.
In a related embodiment, 158P3D2 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 158P3D2 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 158P3D2 expressed in a corresponding normal sample. In one embodiment, the presence of 158P3D2 protein is evaluated, for example, using immunohistochemical methods. 158P3D2 antibodies or binding partners capable of detecting 158P3D2 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 158P3D2 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, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 158P3D2 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 158P3D2 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 158P3D2 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, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No. 5,952,170 issued 17 Jan. 1995).
Additionally, one can examine the methylation status of a 158P3D2 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prey., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of 158P3D2. 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 158P3D2 expression. The presence of RT-PCR amplifiable 158P3D2 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).
A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 158P3D2 mRNA or 158P3D2 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 158P3D2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 158P3D2 in prostate or other tissue is examined, with the presence of 158P3D2 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 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 158P3D2 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by tumor cells, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 158P3D2 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 158P3D2 mRNA or 158P3D2 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 158P3D2 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 158P3D2 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 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., 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 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 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 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and another factor associated with malignancy entails detecting the overexpression of 158P3D2 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 158P3D2 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 158P3D2 and PSA mRNA in prostate tissue is examined, where the coincidence of 158P3D2 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of 158P3D2 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 158P3D2 mRNA include in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques using 158P3D2 polynucleotide probes, RT-PCR analysis using primers specific for 158P3D2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 158P3D2 mRNA expression. Any number of primers capable of amplifying 158P3D2 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 158P3D2 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules that Interact with 158P3D2
The 158P3D2 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 158P3D2, as well as pathways activated by 158P3D2 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, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 158P3D2 protein sequences. In such methods, peptides that bind to 158P3D2 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 158P3D2 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 158P3D2 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.
Alternatively, cell lines that express 158P3D2 are used to identify protein-protein interactions mediated by 158P3D2. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun 1999, 261:646-51). 158P3D2 protein can be immunoprecipitated from 158P3D2-expressing cell lines using anti-158P3D2 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 158P3D2 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 158P3D2 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 158P3D2'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 158P3D2-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 158P3D2 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 158P3D2 function can be identified based on their ability to bind 158P3D2 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 158P3D2 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 158P3D2.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 158P3D2 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 158P3D2 amino acid sequence, allowing the population of molecules and the 158P3D2 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 158P3D2 amino acid sequence, and then separating molecules that do not interact with the 158P3D2 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 158P3D2 amino acid sequence. The identified molecule can be used to modulate a function performed by 158P3D2. In a preferred embodiment, the 158P3D2 amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 158P3D2 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.
Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., “cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.
Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.
Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.
Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.
Accordingly, therapeutic approaches that inhibit the activity of a 158P3D2 protein are useful for patients suffering from a cancer that expresses 158P3D2. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 158P3D2 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 158P3D2 gene or translation of 158P3D2 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 158P3D2-related protein or 158P3D2-related nucleic acid. In view of the expression of 158P3D2, cancer vaccines prevent and/or treat 158P3D2-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 158P3D2-related protein, or a 158P3D2-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 158P3D2 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 158P3D2 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 158P3D2 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 158P3D2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire 158P3D2 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, e.g., 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, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., 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. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
In patients with 158P3D2-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., 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.
X.A.1. Cellular Vaccines
CTL epitopes can be determined using specific algorithms to identify peptides within 158P3D2 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University (URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 158P3D2 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
X.A.2. 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 (e.g. a 158P3D2 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 158P3D2 in a host, by contacting the host with a sufficient amount of at least one 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 158P3D2-related protein or a man-made multiepitopic peptide comprising: administering 158P3D2 immunogen (e.g. a 158P3D2 protein or a peptide fragment thereof, a 158P3D2 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, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 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 158P3D2 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 158P3D2 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, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 158P3D2, in order to generate a response to the target antigen.
X.A.3. Nucleic Acid Vaccines:
Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 158P3D2. Constructs comprising DNA encoding a 158P3D2-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 158P3D2 protein/immunogen. Alternatively, a vaccine comprises a 158P3D2-related protein. Expression of the 158P3D2-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 158P3D2 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. 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. Pat. No. 5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 158P3D2-related protein into the patient (e.g., 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, e.g., U.S. Pat. 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 158P3D2-related nucleic acid molecule. In one embodiment, the full-length human 158P3D2 cDNA is employed. In another embodiment, 158P3D2 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
X.A.4. 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 158P3D2 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 158P3D2 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 158P3D2 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 158P3D2 protein. Yet another embodiment involves engineering the overexpression of a 158P3D2 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 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 158P3D2 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 158P3D2 as a Target for Antibody-Based Therapy
158P3D2 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, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 158P3D2 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 158P3D2-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 158P3D2 are useful to treat 158P3D2-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
158P3D2 antibodies can be introduced into a patient such that the antibody binds to 158P3D2 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 158P3D2, 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 158P3D2 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Sievers et al. Blood 93:11 3678-3684 (Jun. 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. 158P3D2), the cytotoxic agent will exert its known biological effect (i.e. 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 (e.g. an anti-158P3D2 antibody) that binds to a marker (e.g. 158P3D2) 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 158P3D2, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 158P3D2 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
Cancer immunotherapy using anti-158P3D2 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 158P3D2 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., MylotargTM Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).
Although 158P3D2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.
Although 158P3D2 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 158P3D2 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 158P3D2 imaging, or other techniques that reliably indicate the presence and degree of 158P3D2 expression Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
Anti-158P3D2 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-158P3D2 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-158P3D2 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 158P3D2. 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-158P3D2 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 158P3D2 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-158P3D2 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-158P3D2 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-158P3D2 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-158P3D2 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-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
Based on clinical experience with the HerceptinTM mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-158P3D2 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 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
Optionally, patients should be evaluated for the levels of 158P3D2 in a given sample (e.g. the levels of circulating 158P3D2 antigen and/or 158P3D2 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
Anti-idiotypic anti-158P3D2 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 158P3D2-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-158P3D2 antibodies that mimic an epitope on a 158P3D2-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
X.C.) 158P3D2 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, e.g., 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 1-lysine, poly 1-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000)).
Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 158P3D2 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), 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, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) 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, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.
3.) Sufficient supermotif bearing-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 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
4.) 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.
5.) 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.
6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
X.C.1. Minigene Vaccines
A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 158P3D2, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 158P3D2 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) 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 II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, Calif.). 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 (e.g. TGF-β) 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. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr 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 (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. 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, e.g., 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, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO:63), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNVVNS; (SEQ ID NO:64), and Streptococcus 18 kD protein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO:65). 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, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRETM, Epimmune, Inc., San Diego, Calif.) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: xKXVAAWTLKAAx (SEQ ID NO:66), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either d-alanine or 1-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include d-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's 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 peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 158P3D2. 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 158P3D2.
X.D.) Adoptive Immunotherapy
Antigenic 158P3D2-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 (e.g., 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 158P3D2. 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, e.g., 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 158P3D2. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of 158P3D2-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 158P3D2, a vaccine comprising 158P3D2-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg 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 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, 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 μg) 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 5×109 pfu.
For antibodies, a treatment generally involves repeated administration of the anti-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), 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-158P3D2 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 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 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 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body area weekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5×1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
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 liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., 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, e.g., Szoka, et al., Ann Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 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 with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 158P3D2
As disclosed herein, 158P3D2 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, e.g., 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 158P3D2 in normal tissues, and patient specimens”).
158P3D2 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, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto et al., Cancer Detect Prey 2000; 24(1):1-12). Therefore, this disclosure of 158P3D2 polynucleotides and polypeptides (as well as 158P3D2 polynucleotide probes and anti-158P3D2 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 158P3D2 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCR analysis, see, e.g., 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 158P3D2 polynucleotides described herein can be utilized in the same way to detect 158P3D2 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 158P3D2 polypeptides described herein can be utilized to generate antibodies for use in detecting 158P3D2 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 158P3D2 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 158P3D2-expressing cells (lymph node) is found to contain 158P3D2-expressing cells such as the 158P3D2 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 158P3D2 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 158P3D2 or express 158P3D2 at a different level are found to express 158P3D2 or have an increased expression of 158P3D2 (see, e.g., the 158P3D2 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 158P3D2) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
The use of immunohistochemistry to identify the presence of a 158P3D2 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.
The 158P3D2 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.
The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).
Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information concerning the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 158P3D2, the 158P3D2 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 158P3D2 protein and immune responses related thereto very useful. Use of the 158P3D2 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.
Immunohistochemical reagents specific to 158P3D2 are also useful to detect metastases of tumors expressing 158P3D2 when the polypeptide appears in tissues where 158P3D2 is not normally produced.
Thus, 158P3D2 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 158P3D2 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, e.g., 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 158P3D2 in normal tissues, and patient specimens,” where a 158P3D2 polynucleotide fragment is used as a probe to show the expression of 158P3D2 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, e.g., Sawai et al., Fetal Diagn. Ther. 1996 November-December 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 (e.g., a 158P3D2 polynucleotide shown in FIG. 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. 158P3D2 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 158P3D2 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 158P3D2 polypeptide shown in FIG. 3).
As shown herein, the 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 158P3D2 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 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 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 158P3D2 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 158P3D2 gene maps (see the Example entitled “Chromosomal Mapping of 158P3D2” below). Moreover, in addition to their use in diagnostic assays, the 158P3D2-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).
Additionally, 158P3D2-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 158P3D2. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 158P3D2 antigen. Antibodies or other molecules that react with 158P3D2 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 158P3D2 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 158P3D2 to its binding partner or its association with other protein(s) as well as methods for inhibiting 158P3D2 function.
XII.A.) Inhibition of 158P3D2 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 158P3D2 are introduced into 158P3D2 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-158P3D2 antibody is expressed intracellularly, binds to 158P3D2 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).
Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a 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 158P3D2 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 158P3D2 intrabodies in order to achieve the desired targeting. Such 158P3D2 intrabodies are designed to bind specifically to a particular 158P3D2 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 158P3D2 protein are used to prevent 158P3D2 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 158P3D2 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. Pat. No. 5,919,652 issued 6 Jul. 1999).
XII.B.) Inhibition of 158P3D2 with Recombinant Proteins
In another approach, recombinant molecules bind to 158P3D2 and thereby inhibit 158P3D2 function. For example, these recombinant molecules prevent or inhibit 158P3D2 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 158P3D2 specific antibody molecule. In a particular embodiment, the 158P3D2 binding domain of a 158P3D2 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 158P3D2 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 158P3D2, whereby the dimeric fusion protein specifically binds to 158P3D2 and blocks 158P3D2 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 158P3D2 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 158P3D2 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 158P3D2 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 158P3D2 gene comprises contacting the 158P3D2 gene with a 158P3D2 antisense polynucleotide. In another approach, a method of inhibiting 158P3D2 mRNA translation comprises contacting a 158P3D2 mRNA with an antisense polynucleotide. In another approach, a 158P3D2 specific ribozyme is used to cleave a 158P3D2 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 158P3D2 gene, such as 158P3D2 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 158P3D2 gene transcription factor are used to inhibit 158P3D2 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
Other factors that inhibit the transcription of 158P3D2 by interfering with 158P3D2 transcriptional activation are also useful to treat cancers expressing 158P3D2. Similarly, factors that interfere with 158P3D2 processing are useful to treat cancers that express 158P3D2. 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 158P3D2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 158P3D2 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 158P3D2 antisense polynucleotides, ribozymes, factors capable of interfering with 158P3D2 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 (e.g., 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 158P3D2 to a binding partner, etc.
In vivo, the effect of a 158P3D2 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. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).
Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, 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.) Identification, Characterization and Use of Modulators of 158P3D2
XIII.A.) Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
XIII.B.) Gene Expression-related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).
The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokarnik, supra.
A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
XIII.C.) Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.
In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
XIII.D.) Biological Activity-Related Assays
The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
XIII.E.) High Throughput Screening to Identify Modulators
The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
XIII.F.) Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.
XIII.G.) Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.
Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
XIII.H.) Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
XIII.I.) Use of Tumor-Specific Marker Levels to Identify and Characterize Modulators
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
XIII.J.) Invasiveness into Matrigel to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
XIII.L.) In Vitro Assays to Identify and Characterize Modulators
Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
XIII.M.) Binding Assays to Identify and Characterize Modulators
In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or TeflonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.
In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
XIII.N.) Competitive Binding to Identify and Characterize Modulators
In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
XIII.O.) Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
XIII.P.) Inhibitory and Antisense Nucleotides
In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
XIII.Q.) Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
XIII.R.) Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker.
As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
XIII.S.) Use of Modulators to Affect Peptides of the Invention
Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
XIII.T.) Methods of Identifying Characterizing Cancer-Associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
XIV.) Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The 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, e.g., 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 terms “kit” and “article of manufacture” can be used as synonyms.
In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(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. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of 158P3D2 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 158P3D2 and modulating the function of 158P3D2.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene
To isolate genes that are over-expressed in bladder cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues, including invasive transitional cell carcinoma. The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. Included in the driver were also cDNAs derived from 9 other normal tissues. The 158P3D2 cDNA was identified as highly expressed in the bladder cancer tissue pool, with lower expression seen in a restricted set of normal tissues.
The SSH DNA sequence of 312 bp (FIG. 1) shows identity to the fer-1-like 4 (C. elegans) (FER1L4) mRNA. A 158P3D2 cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealing an ORF of 328 amino acids (FIG. 2, FIG. 3).
Amino acid sequence analysis of 158P3D2 reveals 100% identity over 328 amino acid region to dJ477O4.1.1, a novel protein similar to otoferlin and dysferlin, isoform 1 protein (GenBank Accession CAB89410.1).
The 158P3D2 protein has a transmembrane domain of 23 residues between amino acids 292-313 predicted by the SOSUI Signal program.
Materials and Methods
Human Tissues:
The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, Pa.). mRNA for some of the normal tissues were purchased from Clontech, Palo Alto, Calif.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.









DPNCDN (cDNA synthesis primer):



(SEQ ID NO: 67)



5′TTTTGATCAAGCTT303′



 



Adaptor 1:



(SEQ ID NO: 68)



5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′



 



(SEQ ID NO: 69)



3′GGCCCGTCCTAG5′



 



Adaptor 2:



(SEQ ID NO: 70)



5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′



 



(SEQ ID NO: 71)



3′CGGCTCCTAG5′



 



PCR primer 1:



(SEQ ID NO: 72)



5′CTAATACGACTCACTATAGGGC3′



 



Nested primer (NP)1:



(SEQ ID NO: 73)



5′TCGAGCGGCCGCCCGGGCAGGA3′



 



Nested primer (NP)2:



(SEQ ID NO: 74)



5′AGCGTGGTCGCGGCCGAGGA3′






Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.
The gene 158P3D2 sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.
The cDNA derived from of pool of normal bladder tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)+ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.
Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 254 PCR 1 was conducted using the following conditions: 75° C. for 5 mM, 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 mM Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 mM at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 mM After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:75) and 5′ agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:76) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 mM, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 mM After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
To determine expression levels of the 158P3D2 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 158P3D2 SSH sequence and are listed below:









158P3D2.1



(SEQ ID NO: 77)



5′ CATCTATGTGAAGAGCTGGGTGAA 3′



 



158P3D2.2



(SEQ ID NO: 78)



5′ AGGTAGTCAAAGCGGAACACAAAG 3′






Additional primers were also designed to test for expression of the different splice variants and these are listed below:











Primer Name
Sequence







158P3D2 ex. 17-R
GTCCTCCCAGCAACTCCACACA



(SEQ ID NO: 79)


 


158P3D2 ex. 26-F
TGTCCCTTCCACCCAACGTGTGC



(SEQ ID NO: 80)


 


158P3D2 ex. 28-R
TCCTCCATCTCTCCTTCCTCCTCAG



(SEQ ID NO: 81)


 


158P3D2 ex. 9-F
CAGAAACTGGTGGGAGTCAACA



(SEQ ID NO: 82)


 


158P3D2 ex.1-F
ATGGCTCTGACGGTAAGCGTGC



(SEQ ID NO: 83)


 


158P3D2 ex.10-F
ATAGGCACCTTCAGGATGGACC



(SEQ ID NO: 84)


 


158P3D2 ex.10-R
TCCATCCTGAAGGTGCCTATCC



(SEQ ID NO: 85)


 


158P3D2 ex.16-F
CAGAGGAGGAGAAAGAGGAGG



(SEQ ID NO: 86)


 


158P3D2 ex.16-R
TCCTCTTTCTCCTCCTCTGG



(SEQ ID NO: 87)


 


158P3D2 ex.21-F
AGATCCAGAGTCTAATGCTCACG



(SEQ ID NO: 88)


 


158P3D2 ex.21-R
CGTGAGCATTAGACTCTGGATC



(SEQ ID NO: 89)


 


158P3D2 ex.27-F
AAGGTGTGGAGTCTGAGGTC



(SEQ ID NO: 90)


 


158P3D2 ex.27-R
ACCTCAGACTCCACACCTTGC



(SEQ ID NO: 91)


 


158P3D2 ex.34-R
ACTCTGACCAGGAGCTTGATG



(SEQ ID NO: 92)


 


158P3D2 ex.40-F
ACACGGAGGATGTGGTTCTGG



(SEQ ID NO: 93)


 


158P3D2 ex.43-F
TTGAGCTGCTGACTGTGGAGGAG



(SEQ ID NO: 94)


 


158P3D2 ex.43-R
TCCTCCACAGTCAGCAGCTC



(SEQ ID NO: 95)


 


158P3D2 ex.44-R
TGAGTGTCCAAGGTCAGCGAG



(SEQ ID NO: 96)


 


158P3D2 ex.7-F
AGAGAATGAGCTGGAGCTTGAGC



(SEQ ID NO: 97)


 


158P3D2 ex.7-R
TCAAGCTCCAGCTCATTCTCTTC



(SEQ ID NO: 98)


 


AGS-25 long RT PCR-3′
TAACACCAGAAAGTTCCACGTCAG



(SEQ ID NO: 99)


 


AGS-25 long RT PCR-5′
TGACGGTCGCCGTATTTGATC



(SEQ ID NO: 100)


 


AGS-25 short RT PCR-3′
GATTGGCTGCCGAGGCTTGA



(SEQ ID NO: 101)


 


AGS-25 short RT PCR-5′
TGACGGTCGCCGTATTTGATC



(SEQ ID NO: 102)









A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Results show strong expression of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasis pool. Expression of 158P3D2 is also detected in colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and prostate metastases to lymph node, and vital pool 2, but not vital pool 1.
Example 2
Full Length Cloning of 158P3D2
The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 158P3D2. The full-length cDNA clone 158P3D2 v.1 clone 158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were cloned from bladder cancer pool cDNA.
Additional 158P3D2 splice and SNP variants have been identified and these are listed in FIG. 2 and FIG. 3.
Example 3
Chromosomal Mapping of 158P3D2
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
158P3D2 maps to chromosome 8, using 158P3D2 sequence and the NCBI BLAST tool located on the World Wide Web at: (ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4
Expression Analysis of 158P3D2 in Normal Tissues and Patient Specimens
Expression analysis by RT-PCR demonstrated that 158P3D2 is strongly expressed in multiple cancer patient specimens, but unrestricted normal tissues (FIG. 14). First strand cDNA was prepared from a panel of 13 normal tissues (brain, heart, kidney, liver, lung, spleen, skeletal muscle, testis, pancreas, colon, stomach) and pools of 4-7 patients from the following cancer indications: bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, multiple cancer metastasis, cervix, lymphoma as well as from a pool of patient-derived xenografts (prostate cancer, bladder cancer and kidney cancer). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression of 158P3D2 in cancers of the bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, cervix, and lymphoma. Strong expression was also observed in the cancer metastasis pool. Low expression was detected in all normal tissues tested except in normal stomach.
Expression of 158P3D2 in bladder cancer patient specimens and human normal tissues is shown in FIG. 15. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82) and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of bladder cancer patient specimens tested. Very low expression was detected in normal tissues, but no expression was seen in the cell lines tested.
Northern blot analysis of 158P3D2 in bladder specimens is shown in FIG. 16. RNA was extracted from normal bladder, bladder cancer cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and their normal adjacent tissues (NAT). Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal, nor NAT tissues.
FIG. 17 shows 158P3D2 expression in lung cancer patient specimens. First strand cDNA was prepared from normal lung, cancer cell line A427 and a panel of lung cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) of lung cancer specimens, but not in all 3 normal lung tissues tested.
Northern blot analysis of 158P3D2 expression in lung cancer patient specimens is shown in FIG. 18. RNA was extracted from normal lung, A427 lung cancer cell line, and a panel of lung cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in normal tissues.
FIG. 19 shows 158P3D2 expression in cancer metastasis patient specimens. First strand cDNA was prepared from normal colon, kidney, liver, lung, pancreas, stomach and from a panel of cancer metastasis patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of patient cancer metastasis specimens tested but not in normal tissues.
FIG. 20 shows 158P3D2 expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in all 14 cervical cancer patient specimens tested. No expression was detected in normal cervix or in the cell line tested.
Northern blot analysis of 158P3D2 expression in cervical cancer patient specimens is shown in FIG. 21. RNA was extracted from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal cervix nor in the cell line.
FIG. 22 shows 158P3D2 expression in kidney cancer patient specimens. First strand cDNA was prepared from normal kidney, kidney cancer cell lines (769-P, A-498, CAM-1), and a panel of kidney cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of kidney cancer patient specimens, but not in all 3 normal kidney tissues tested. Low expression was detected in 2 of 3 cell lines tested.
FIG. 23 shows 158P3D2 expression in kidney cancer patient specimens by northern blotting. RNA was extracted from normal kidney and a panel of kidney cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.
FIG. 24 shows 158P3D2 expression in stomach cancer patient specimens. First strand cDNA was prepared from normal stomach, and a panel of stomach cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of stomach cancer patient specimens. Weak expression was detected in the 2 normal stomach, and only in 1 of the 2 NAT tissues tested.
FIG. 25 shows 158P3D2 expression in stomach cancer patient specimens by northern blotting. RNA was extracted from normal stomach and a panel of stomach cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.
FIG. 26 shows 158P3D2 expression in colon cancer patient specimens. First strand cDNA was prepared from normal colon, colon cancer cell lines (LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of colon cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×s), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of colon cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 3 of 5 cell lines tested.
FIG. 27 shows 158P3D2 expression in uterus cancer patient specimens. First strand cDNA was prepared from normal uterus and a panel of uterus cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of uterus cancer patient specimens, but not in normal uterus.
FIG. 28 shows 158P3D2 expression in breast cancer patient specimens. First strand cDNA was prepared from normal breast, breast cancer cell lines (MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel of breast cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of breast cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 2 of 5 cell lines tested.
The restricted expression of 158P3D2 in normal tissues and the expression detected in bladder cancer, kidney cancer, colon cancer, lung cancer, pancreas cancer, stomach cancer, ovary cancer, breast cancer, uterus cancer, cervical cancer and lymphoma suggest that 158P3D2 is a potential therapeutic target and a diagnostic marker for the treatment of human cancers.
Example 5
Transcript Variants of 158P3D2
Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.
Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 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, S. O., 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(s1)-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 April; 47(4):654-60; Jia, H. P., 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, K. E., 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 158P3D2 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 158P3D2 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
Using the full-length gene and EST sequences, six transcript variants were identified, designated as 158P3D2 v.2, v.14 through v.18. The boundaries of the exon in the original transcript, 158P3D2 v.1 were shown in Table LI. Exon compositions of the variants are shown in FIG. 10. Each different combination of exons in spatial order, e.g. exon 1 of v.2 and exons 3, 4, 5 and 6 of v.1, is a potential splice variant.
Tables LII(a)-(f) through LV(a)-(f) are set forth on a variant-by-variant bases. Tables LII(a)-(f) show nucleotide sequence of the transcript variants. Tables LIII(a)-(f) show the alignment of the respective transcript variant with nucleic acid sequence of 158P3D2 v.1. Tables LIV(a)-(f) lay out amino acid translation of the transcript variants for the identified reading frame orientation. Tables LV(a)-(f) displays alignments of the amino acid sequence encoded by the splice variant with that of 158P3D2 v.1.
Example 6
Single Nucleotide Polymorphisms of 158P3D2
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 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 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).
SNP are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).
Using the methods described above, twelve SNP were identified in the original transcript, 158P3D2 v.1, at positions 1155 (T/C), 1152 (G/A), 960 (G/T) and 1236 (G/-), 519 (A/G), 440 (T/A), 971 (T/C), 150 (C/G), 1022 (C/A), 1148 (G/A), 1691 (G/T) and 1692 (A/G). The transcripts or proteins with alternative allele were designated as variant 158P3D2 v.3 through v.13, respectively. FIG. 12 shows the schematic alignment of the SNP variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in FIG. 11. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 158P3D2 v.17) that contains the site of the SNP.
Example 7
Production of Recombinant 158P3D2 in Prokaryotic Systems
To express recombinant 158P3D2 and 158P3D2 variants in prokaryotic cells, the full or partial length 158P3D2 and 158P3D2 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 variants are expressed: the full length sequence presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2, variants, or analogs thereof.
A. In Vitro Transcription and Translation Constructs
pCRII:
To generate 158P3D2 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 158P3D2 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 158P3D2 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 158P3D2 at the RNA level. Transcribed 158P3D2 RNA representing the cDNA amino acid coding region of the 158P3D2 gene is used during in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 158P3D2 protein.
B. Bacterial Constructs
pGEX Constructs:
To generate recombinant 158P3D2 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus The GST and 6×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 6×His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScissionTM recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 158P3D2-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli. 
pMAL Constructs:
To generate, in bacteria, recombinant 158P3D2 proteins that are fused to maltose-binding protein (MBP), all or parts of the 158P3D2 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×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 6×His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 158P3D2. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
pET Constructs:
To express 158P3D2 in bacterial cells, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 158P3D2 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-TagTM 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 158P3D2 protein are expressed as amino-terminal fusions to NusA. The cDNA encoding amino acids 155-290 and amino acids 260-328 of 158P3D2 each were cloned into the pET-21b vector. The recombinant proteins can be used to generate rabbit polyclonal antibodies.
C. Yeast Constructs:
pESC Constructs:
To express 158P3D2 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 158P3D2. 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 158P3D2 in the yeast species Saccharomyces pombe, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 158P3D2 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A FlagTM epitope tag allows detection of the recombinant protein with anti-FlagTM antibody.
Example 8
Production of Recombinant 158P3D2 in Higher Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 158P3D2 in eukaryotic cells, the full or partial length 158P3D2 cDNA sequences, or variants thereof, can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 are expressed in these constructs, amino acids 1 to 328, 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 158P3D2 v.1, v.3, v.4, v.10, v.12 and v.13; amino acids 1 to 236, 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 158P3D2 v.2A; amino acids 1 to 181, 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 158P3D2 v.2B or v.5B; amino acids 1 to 178, 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 158P3D2 v.5A; amino acids 1 to 2036, 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 158P3D2 v.17; amino acids 1 to 1990, 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 158P3D2 v.16; amino acids 1 to 1145, 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 158P3D2 v.15; amino acids 1 to 1393, 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 158P3D2 v.14; amino acids 1 to 610, 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 158P3D2 v.18; 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 282P1G3 variants, or analogs thereof.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-158P3D2 polyclonal serum, described herein.
pcDNA4/HisMax Constructs:
To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6×His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. 
pcDNA3.1/MycHis Constructs:
To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. FIG. 19 shows expression of 158P3D2.pcDNA3.1/mychis in transiently transfected 293T cells.
pcDNA3.1/CT-GFP-TOPO Construct:
To express 158P3D2 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 158P3D2 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 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 a 158P3D2 protein.
PAPtag:
A 158P3D2 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 158P3D2 protein while fusing the IgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminus of a 158P3D2 protein. The resulting recombinant 158P3D2 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 158P3D2 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×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. 
pTag5:
A 158P3D2 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 158P3D2 protein with an amino-terminal IgGκ signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 158P3D2 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 158P3D2 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:
A 158P3D2 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 158P3D2 proteins, while fusing the IgGK signal sequence to N-terminus. 158P3D2 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 158P3D2 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 158P3D2 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. 
pSRα Constructs:
To generate mammalian cell lines that express 158P3D2 constitutively, 158P3D2 ORF, or portions thereof, of 158P3D2 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 158P3D2, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAG tag to the carboxyl-terminus of 158P3D2 sequences to allow detection using anti-Flag antibodies. For example, the FLAG sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO:103) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 158P3D2 proteins.
Additional Viral Vectors:
Additional constructs are made for viral-mediated delivery and expression of 158P3D2. High virus titer leading to high level expression of 158P3D2 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 158P3D2 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, 158P3D2 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems:
To control expression of 158P3D2 in mammalian cells, coding sequences of 158P3D2, 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 158P3D2. These vectors are thereafter used to control expression of 158P3D2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 158P3D2 proteins in a baculovirus expression system, 158P3D2 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-158P3D2 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant 158P3D2 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 158P3D2 protein can be detected using anti-158P3D2 or anti-His-tag antibody. 158P3D2 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 158P3D2 which are used for diagnostic and therapeutic purposes.
Example 9
Antigenicity Profiles and Secondary Structure
FIG. 5A-I, FIG. 6A-I, FIG. 7A-I, FIG. 8A-I, and FIG. 9A-I depict graphically five amino acid profiles of 158P3D2 variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18, (A) through (I) respectively, each assessment available by accessing the ProtScale website on the ExPasy molecular biology server.
These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 158P3D2 variant proteins. Each of the above amino acid profiles of 158P3D2 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
Antigenic sequences of the 158P3D2 variant proteins indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-158P3D2 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 158P3D2 protein variants listed in FIGS. 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 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 structures of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from their primary amino acid sequences using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G., accessed from the ExPasy molecular biology server. The analysis indicates that 158P3D2 variant 1 is composed of 32.93% alpha helix, 18.29% extended strand, and 48.78% random coil (FIG. 13A). 158P3D2 variant 2a is composed of 25.58% alpha helix, 18.22% extended strand, and 55.93% random coil (FIG. 13B). 158P3D2 variant 2b is composed of 44.75% alpha helix, 11.60% extended strand, and 43.65% random coil (FIG. 13C). 158P3D2 variant 5a is composed of 9.55% alpha helix, 26.40% extended strand, and 64.04% random coil (FIG. 13D). 158P3D2 variant 14 is composed of 33.88% alpha helix, 13.42% extended strand, and 52.69% random coil (FIG. 13E). 158P3D2 variant 15 is composed of 33.28% alpha helix, 15.11% extended strand, and 51.62% random coil (FIG. 13F). 158P3D2 variant 16 is composed of 32.76% alpha helix, 14.47% extended strand, and 52.76% random coil (FIG. 13G). 158P3D2 variant 17 is composed of 32.86% alpha helix, 14.69% extended strand, and 52.46% random coil (FIG. 13H). 158P3D2 variant 18 is composed of 27.21% alpha helix, 14.75% extended strand, and 58.03% random coil (FIG. 13I).
Analysis for the potential presence of transmembrane domains in the 158P3D2 variant proteins 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. Shown graphically in FIGS. 13L, 13N, 13P, 13R, 13T, 13V, 13X, 13Z are the results of analysis of variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, respectively, using the TMpred program. Shown graphically in FIGS. 13K, 13M, 13O, 13Q, 13S, 13U, 13W, 13Y, 13AA are the results of analysis of variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, respectively using the TMHMM program. Both programs predict the presence of 1 transmembrane domain in variant 1, Both programs predict that variants 2a, 2b, 5a, and 18 lack transmembrane domains and are soluble proteins. The TMpred program predicts that variants 14, 15, 16, and 17 have 2 transmembrane domains of which the more carboxy-terminal transmembrane has a higher probability of existence. The TMHMM program predicts that variants 14 and 15 do not encode transmembrane domains and variants 16 and 17 contain 1 transmembrane domain. Analyses of the variants using other structural prediction programs are summarized in Table VI and Table L.
Example 10
Generation of 158P3D2 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 158P3D2 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 158P3D2 protein variant 1 or other protein variants).
For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 158P3D2 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 11 (“Generation of Monoclonal Antibodies”). For example, in 158P3D2 variant 1, such regions include, but are not limited to, amino acids 1-25, amino acids 37-54, amino acids 60-73, amino acids 187-225, and amino acids 235-271. An extracellular epitope peptide encoding amino acids 315 to 328 is also used to generate antibodies that bind to the extracellular region of 158P3D2 protein. 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 315-328 of 158P3D2 variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 158P3D2 variant proteins, analogs or fusion proteins thereof. For example, the 158P3D2 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.
In one embodiment, amino acids 155-290 of 158P3D2 variant 1 were fused to His using recombinant techniques and the pET21b expression vector. In another embodiment, amino acids 260-328 were cloned into the pET21b expression vector. The proteins are then expressed, purified, and used to immunize rabbits. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 158P3D2 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., 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 TagS and Fc-fusion vectors (see the section entitled “Production of Recombinant 158P3D2 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 1-236 of 158P3D2 variant 2a is cloned into the TagS mammalian secretion vector, and expressed in 293T cells. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified TagS 158P3D2 protein is then used as immunogen.
During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
To test reactivity and specificity of immune serum, such a rabbit serum derived from immunization with the His-fusion of 158P3D2 variant 1 protein, the full-length 158P3D2 variant 1 cDNA was cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 158P3D2 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-158P3D2 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 158P3D2 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 158P3D2-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 158P3D2 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 158P3D2 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-158P3D2 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-158P3D2 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11
Generation of 158P3D2 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 158P3D2 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 158P3D2 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 158P3D2 protein variant sequence, regions predicted to contain functional motifs, and regions of the 158P3D2 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 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 engineered to express high levels of a respective 158P3D2 variant, such as Rat1-158P3D2 variant 1 or 300.19-158P3D2 variant 1 murine Pre-B cells, were used to immunize mice.
To generate mAbs to a 158P3D2 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 158P3D2-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg 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 a 158P3D2 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 1-400 of 158P3D2 of variant 15 is cloned into the TagS mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 158P3D2 variant 15 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 158P3D2 variant.
During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).
In one embodiment for generating 158P3D2 monoclonal antibodies, a peptide encoding amino acids 315-328 of 158P3D2 variant 1 was coupled to KLH and use to immunize mice. Balb C mice were immunized with 10 μg of the KLH-peptide mixed in adjuvant. Mice were subsequently immunized over several weeks with the KLH-peptide. ELISA using the peptide coupled to a different carrier, ovalbumin, determined the titer of serum from the immunized mice (See FIG. 29). Reactivity and specificity of the serum to full length 158P3D2 variant 1 protein was monitored by flow cytometry and Western blotting using recombinant 158P3D2 variant 1-expressing cells and cells endogenously expressing 158P3D2 variant 1 protein. Mice showing the strongest reactivity were rested and given a final injection of antigen and sacrificed for fusion. The lymph nodes of the sacrificed mice were harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells were analyzed by flow cytometry to identify specific-158P3D2 surface binding MAbs. Supernatants were also screened by ELISA, Western blot, immunoprecipitation, and fluorescent microscopy to identify 158P3D2 specific antibody-producing clones.
In other embodiments, 158P3D2 variant specific MAbs are generated by employing immunogens that encode amino acid sequences unique to each variant or created by unique junctions from alternative splicing of exons. For example, a peptide encoding amino acids 1018-1035 of 158P3D2 variant 15 is coupled to KLH and used to immunize mice. In another example, amino acids 1375-1393 of 158P3D2 variant 14 is coupled to KLH and used to immunize mice. Hybridomas resulting from fusion of the B-cells from the mice are screened on cells expressing the respective 158P3D2 variant protein from which the antigen was derived and cross-screened on cells expressing the other variant proteins to identify variant specific MAbs and MAbs that may recognize more than 1 variant.
The binding affinity of 158P3D2 variant specific monoclonal antibodies was determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 158P3D2 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
In addition, equilibrium binding analysis of a dilution series of the MAb was also used to determine affinity defined by the dissociation constant (KD). The KD is determined by non-linear regression of the equilibrium binding data of the concentration series. The KD is defined as the concentration at which half-maximal binding of the MAb to the antigen is attained under equilibrium conditions.
Example 12
HLA Class I and Class II Binding Assays
HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/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 IC50 of a positive control for inhibition by the IC50 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 IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.
Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).
Example 13
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
Computer searches and algorithms for identification of supermotif and/or motif-bearing epitopes
The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 158P3D2 set forth in FIGS. 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 158P3D2 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a1i×a2i×a3i . . . ×ani 

where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 Supertype Cross-Reactive Peptides
Protein sequences from 158P3D2 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed 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 158P3D2 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent 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 A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.
Selection of HLA-B7 Supermotif Bearing Epitopes
The 158P3D2 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC50 of ≦500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., 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.
Selection of A1 and A24 Motif-Bearing Epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 158P3D2 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14
Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening:
The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/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 monocytes are purified by plating 10×106 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. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the Detacha-Bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml Detacha-Bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 μg/ml β2-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 1×105 cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant 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.
Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×106 cells/ml and irradiated at  ̃4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 ml 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 501 U/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 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ 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 51Cr Release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) 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 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl 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 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample−background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.
In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.
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, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 Supermotif-Bearing Peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce 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 158P3D2. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
Evaluation of A*03/A11 Immunogenicity
HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.
Evaluation of B7 Immunogenicity
Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2- and A3-supermotif-bearing peptides.
Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology
Example 15
Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.
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, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.
Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of 5000 nM 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, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).
In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.
Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.
Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.
Analoging at Secondary Anchor Residues
Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 158P3D2-expressing tumors.
Other Analoging Strategies
Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-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 α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.
Example 16
Identification and Confirmation of 158P3D2-Derived Sequences with HLA-DR Binding Motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-Supermotif-Bearing Epitopes.
To identify 158P3D2-derived, HLA class II HTL epitopes, a 158P3D2 antigen is analyzed for the 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 (15 amino acids total).
Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., 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, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
The 158P3D2-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and 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. 158P3D2-derived peptides found to bind common HLA-DR alleles are of particular interest.
Selection of DR3 Motif Peptides
Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.
To efficiently identify peptides that bind DR3, target 158P3D2 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. 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 17
Immunogenicity of 158P3D2-Derived HTL Epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 158P3D2-expressing tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage
This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].
Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., tota1=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, All, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, 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 A1 and A24 motifs. On average, A1 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 A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
Immunogenicity studies in humans (e.g., 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.
With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.
Example 19
CTL Recognition Of Endogenously Processed Antigens after Priming
This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 158P3D2 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 158P3D2 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
Example 20
Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 158P3D2-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 158P3D2-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-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.
Cell Lines:
Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
In Vitro CTL Activation:
One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
Assay for Cytotoxic Activity:
Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E):target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: R1/50,000)−(1/500,000)1×106=18 LU.
The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.
Example 21
Selection of CTL and HTL Epitopes for Inclusion in a 158P3D2-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 (i.e., 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 158P3D2 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 158P3D2. For example, if it has been observed that patients who spontaneously clear 158P3D2-expressing cells generate an immune response to at least three (3) epitopes from 158P3D2 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an IC50 of 500 nM or less for an HLA class I molecule, or for class II, an IC50 of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.
In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.
When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as 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 within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of 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 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 158P3D2.
Example 22
Construction of “Minigene” Multi-Epitope DNA Plasmids
This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 158P3D2, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 158P3D2 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° 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 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 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 23
The Plasmid Construct and the Degree to which it Induces Immunogenicity
The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).
Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.
For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.
It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.
To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., 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 (e.g., Barnett 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, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson 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 μg of a DNA minigene encoding the immunogenic peptides including at least one
HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”
Example 24
Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 158P3D2 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 158P3D2-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 μg, generally 100-5,000 μg, 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 158P3D2-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 25
Polyepitopic Vaccine Compositions Derived from Native 158P3D2 Sequences
A native 158P3D2 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 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 158P3D2 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of 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 peptide sequences that are actually present in native 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.
Example 26
Polyepitopic Vaccine Compositions from Multiple Antigens
The 158P3D2 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 158P3D2 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 158P3D2 as well as tumor-associated antigens that are often expressed with a target cancer associated with 158P3D2 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27
Use of Peptides to Evaluate an Immune Response
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 158P3D2. 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, 158P3D2 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 158P3D2 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 β2-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, β2-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, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 158P3D2 epitope, and thus the status of exposure to 158P3D2, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 158P3D2-associated disease or who have been vaccinated with a 158P3D2 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 158P3D2 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 (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) 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 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release-spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.
The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 158P3D2 or a 158P3D2 vaccine.
Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 158P3D2 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 10 U/ml IL-2. Two days later, 1 μCi 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
Example 29
Induction of Specific CTL Response in Humans
A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:
A total of about 27 individuals are enrolled and divided into 3 groups:
Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 158P3D2
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 158P3D2. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 158P3D2, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.
There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 158P3D2.
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 158P3D2-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) 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 5×109 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 158P3D2 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells (DC)
Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 158P3D2 protein from which the epitopes in the vaccine are derived.
For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as 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, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as ProgenipoietinTM are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×106 DC, then the patient will be injected with a total of 2.5×108 peptide-loaded PBMC. The percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex Vivo Activation of CTL/HTL Responses
Alternatively, ex vivo CTL or HTL responses to 158P3D2 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, i.e., tumor cells.
Example 33
An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 158P3D2. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 158P3D2 to isolate peptides corresponding to 158P3D2 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
Example 34
Complementary Polynucleotides
Sequences complementary to the 158P3D2-encoding sequences or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 158P3D2. 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, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 158P3D2. 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 158P3D2-encoding transcript.
Example 35
Purification of Naturally-Occurring or Recombinant 158P3D2 Using 158P3D2-Specific Antibodies
Naturally occurring or recombinant 158P3D2 is substantially purified by immunoaffinity chromatography using antibodies specific for 158P3D2. An immunoaffinity column is constructed by covalently coupling anti-158P3D2 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 158P3D2 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 158P3D2 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/158P3D2 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.
Example 36
Identification of Molecules which Interact with 158P3D2
158P3D2, or biologically active fragments thereof, are labeled with 121 l Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 158P3D2, washed, and any wells with labeled 158P3D2 complex are assayed. Data obtained using different concentrations of 158P3D2 are used to calculate values for the number, affinity, and association of 158P3D2 with the candidate molecules.
Example 37
In Vivo Assay for 158P3D2 Tumor Growth Promotion In Vivo Assay of 3T3 Cell Growth by Recombinant Expression of 158P3D2
To address the determination of 158P3D2 to accelerate the growth of non-tumorigenic cells in an in vivo mouse model, non-transformed 3T3 cells are prepared by infection with either a virus containing an empty vector control (Neo gene alone) or with a vector containing the 158P3D2 full-length gene. 3T3 cells are selected for survival in G-418, and expression of 158P3D2 confirmed by Northern blot analysis. To assess the growth of these cells, 1×106 158P3D2 expressing 3T3 cells or 1×106 Neo control are mixed with Matrigel®, then injected intratibially or subcutaneously in SCID mice and allowed to grow for 30 days. The growth of these cells is assessed on day 30 by visual inspection and by necropsy. The 158P3D2 expressing 3T3 cells show a potent effect in comparison to the 3T3-Neo cells, indicating that the 158P3D2 protein enhanced the growth of the cells in Matrigel®. 158P3D2 promotes the growth of non-tumorigenic cells and provides a growth advantage in vivo that mimics the role of this protein in human malignancies.
Example 38
158P3D2 Monoclonal Antibody-Mediated Inhibition of Bladder, Lung, Colon and Breast and other Tumors In Vivo
The significant expression of 158P3D2 in cancer tissues, together with its restrictive expression in normal tissues makes 158P3D2 a good target for antibody therapy. Similarly, 158P3D2 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-158P3D2 MAbs in human bladder cancer xenograft mouse models is evaluated by using recombinant cell lines such as J82-158P3D2 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): p. 16-23), as well as human bladder xenograft models (SCaBER).
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft model. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality (see below), as appreciated in the art. Anti-158P3D2 MAbs inhibit formation of bladder xenografts. Anti-158P3D2 MAbs retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice. MAb effects on tumor growth in mouse models support the utility of anti-158P3D2 MAbs in the treatment of local and advanced stages of bladder cancer (see, e.g., Saffran, D., 2001, et al., PNAS 10:1073-1078).
Administration of the anti-158P3D2 MAbs leads to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. Therefore, 158P3D2 is an attractive target for immunotherapy, and anti-158P3D2 MAbs have therapeutic potential for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 158P3D2 monoclonal antibodies are effective to inhibit the growth of human bladder tumor xenografts grown in SCID mice; accordingly, a combination of such efficacious MAbs is also effective.
MAb-Toxin Conjugates:
Another embodiment of MAb therapy is through the use of toxin conjugation of MAbs for targeted delivery of cytotoxic agents to cells expressing the protein target. Major advances have been made in the clinical application of MAb toxin conjugates with the development of Mylotarg for acute myeloid leukemia (Bross, P. F., et al., 2001, Clin. Cancer Res. 7:1490-1496). Mylotarg is a humanized MAb directed to CD33 which is conjugated to a highly potent DNA-alkylating agent (calichemicin) via an acid labile hydrazone bond (Hamann, P. R., et al., 2002, Bioconjug. Chem. 13:40-46; ibid., 13:47-58). Additional toxins for MAb conjugation in development include maytansinoid, doxorubicin, taxoids and the potent synthetic dolastatin 10 analogs auristatin E and monomethylauristatin E (Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Ross, S., et al., 2002, Cancer Res. 62:2546-2553; Francisco, J. A., et el., 2003, Blood 102 :1458-1465; Mao, W., et al., 2004, Cancer Res. 64:781-788). Such applications have potential to deliver a cytotoxic agent to cells expressing the protein target of the MAb. Internalization of the target protein upon MAb binding is important for toxin delivery, and the mechanism spares the non-targeted tissues from the potentially harmful effects of the cytotoxic agent.
158P3D2 MAbs conjugated to toxins are used to induce cell killing in vitro using established protocols for cytotoxicity assays and clonogenic assays (Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Mao, W., et al., 2004, Cancer Res. 64:781-788). Toxin conjugated anti-158P3D2 MAbs induce cytotoxicity of cells expressing endogenous 158P3D2 (SCaBER cells) and recombinant 158P3D2 (PC3-158P3D2, 3T3-158P3D2, Rat-1-158P3D2 and B300.19-158P3D2). This methodology allows confirmation that the toxin conjugated MAb is functional against cells expressing the 158P3D2 protein on their surface versus those that do not express the target.
The MAb toxin conjugates are tested for their ability to inhibit tumor growth in vivo. Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft model, a mouse lung cancer xenograft model, or mouse colon or breast cancer xenograft model. Administration of the anti-158P3D2 MAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 158P3D2 is an attractive target for immunotherapy and demonstrate the therapeutic potential of toxin-conjugated anti-158P3D2 MAbs for the treatment of local and metastatic cancer. This example demonstrates that toxin-conjugated 158P3D2 monoclonal antibodies are effective to inhibit the growth of human bladder, lung, breast and colon tumor xenografts grown in SCID mice; accordingly, a combination of such efficacious MAbs is also effective. The methodology allows the targeted delivery of a cytotoxin using a plasma stable linker in a MAb-toxin conjugate. Such a mechanism of action reduces the potential harmful effects of the toxin on non-targeted tissues.
Tumor Inhibition Using Multiple Unconjugated or Toxin-Conjugated 158P3D2 Mabs
Materials and Methods
158P3D2 Monoclonal Antibodies:
Monoclonal antibodies were raised against 158P3D2 as described in the Example entitled “Generation of 158P3D2 Monoclonal Antibodies (MAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 158P3D2. Epitope mapping data for the anti-158P3D2 MAbs, as determined by ELISA and Western analysis, recognize epitopes on the 158P3D2 protein Immunohistochemical analysis of bladder 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° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). 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 SCaBER or J82-158P3D2 tumor xenografts.
The MAbs to 158P3D2 are conjugated to various different toxins (listed above) using any of a variety of methods described elsewhere in the art (Hamann, P. R., et al., 2002, Bioconjug. Chem. 13:40-46; ibid., 13:47-58; Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Ojima, I., et al. 2002, J. Med. Chem. 45:5620-5623; Dubowchik, G. M., et al., 2002, Bioconjug. Chem. 13:855-869; King, H. D., 2002, J. Med. Chem. 45:4336-4343; Ross, S., et al., 2002, Cancer Res. 62:2546-2553; Francisco, J. A., et el., 2003, Blood 102 :1458-1465; Mao, W., et al., 2004, Cancer Res. 64:781-788).
Cell Lines
The bladder carcinoma cell lines, J82 and SCaBER, as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in media supplemented with L-glutamine and 10% FBS. J82-158P3D2 and 3T3-158P3D2 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc. Natl. Acad. Sci. USA, 1999, 96(25):14523.
Xenograft Mouse Models
Subcutaneous (s.c.) tumors are generated by injection of 1×106 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 protein not expressed in human cells. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as: Length×Width×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 (5×105) mixed with Matrigel® are injected into the bladder wall in a 10-μl volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure BTA levels. For prostate orthopotic models, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. Tumor cells, e.g. SCaBER cells (5×105) mixed with Matrigel® are injected into the bladder in a 10-μl volume (Yoshida Y et al, Anticancer Res. 1998, 18:327; Ahn et al, Tumor Biol. 2001, 22:146). The mice are segregated into groups for the appropriate treatments, with anti-158P3D2 or control MAbs being injected i.p.
Anti-158P3D2 MAbs Inhibit Growth of 158P3D2-Expressing Xenograft-Cancer Tumors
The effect of anti-158P3D2 MAbs on tumor formation is tested on the growth and progression of bladder cancer xenografts using SCaBER and J82-158P3D2 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse bladder, and prostate, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem., 1994. 56(1): p. 4-8). 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, or lung, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 μg of anti-158P3D2 MAb, 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-cytokeratin 20 for bladder cancer models (Lin S et al, Cancer Detect Prey. 2001; 25:202).
Mice bearing established orthotopic tumors are administered 1000 μg injections of either anti-158P3D2 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.
Anti-158P3D2 antibodies inhibit the formation of tumors, retard the growth of already established tumors, and prolong the survival of treated mice. Moreover, anti-158P3D2 MAbs demonstrate a dramatic inhibitory effect on the spread of local bladder tumors to distal sites, even in the presence of a large tumor burden. Thus, anti-158P3D2 MAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies in Humans
Anti-158P3D2 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-158P3D2 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 158P3D2 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-158P3D2 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-158P3D2 mAb specifically binds to carcinoma cells. Thus, anti-158P3D2 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 158P3D2. Shedding or release of an extracellular domain of 158P3D2 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 158P3D2 by anti-158P3D2 antibodies in serum and/or urine samples from suspect patients.
Anti-158P3D2 antibodies that specifically bind 158P3D2 are used in therapeutic applications for the treatment of cancers that express 158P3D2. Anti-158P3D2 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-158P3D2 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “158P3D2 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-158P3D2 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.
Example 40
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-158P3D2 Antibodies In Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 158P3D2, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 158P3D2 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-158P3D2 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-158P3D2 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-158P3D2 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).
Monotherapy:
In connection with the use of the anti-158P3D2 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.
Imaging Agent:
Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-158P3D2 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 158P3D2. In connection with the use of the anti-158P3D2 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (111In)-158P3D2 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 158P3D2 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified.
Dose and Route of Administration
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-158P3D2 antibodies can be administered with doses in the range of 5 to 400 mg/m2, with the lower doses used, e.g., in connection with safety studies. The affinity of anti-158P3D2 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-158P3D2 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-158P3D2 antibodies can be lower, perhaps in the range of 50 to 300 mg/m2, and still remain efficacious. Dosing in mg/m2, as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.
Three distinct delivery approaches are useful for delivery of anti-158P3D2 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-158P3D2 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-158P3D2 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 158P3D2 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, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-158P3D2 antibodies are found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-158P3D2 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-158P3D2 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-158P3D2 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-158P3D2 antibody with dosage of antibody escalating from approximately about 25 mg/m2 to about 275 mg/m2 over the course of the treatment in accordance with the following schedule:


















Day 0
Day 7
Day 14
Day 21
Day 28
Day 35






















mAb Dose
25
75
125
175
225
275



mg/m2
mg/m2
mg/m2
mg/m2
mg/m2
mg/m2


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: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. 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-158P3D2 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42
Human Clinical Trial
Monotherapy with Human Anti-158P3D2 Antibody
Anti-158P3D2 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-158P3D2 antibodies.
Example 43
Human Clinical Trial
Diagnostic Imaging with Anti-158P3D2 Antibody
Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-158P3D2 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44
158P3D2 Functional Assays
158P3D2 protein, and variants thereof, is a member of a family of related proteins, the ferlins. This family of membrane proteins is characterized by the presence of intracellular C2 domains, so named by their homology to a conserved protein kinase C (PKC) motif. The canonical C2 domain is a 130 amino acid long Ca2+ dependent membrane targeting module that is found in proteins involved in signal transduction or membrane trafficking (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998)). The function of the C2 domain amongst the >100 proteins identified to date varies between these proteins, however a common feature is that the C2 domain has been shown to bind to phospholipids, particularly phosphatidylserine and phosphatidylcholine. In some cases, the C2 domain may not bind to Ca2+ or to phospholipids but rather to other proteins (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998)). 158P3D2, and variants thereof, are Ca2+ binding proteins with the capacity to bind to both phospholipids and to proteins. The different variants of 158P3D2, which express different numbers of C2 domains, have different functions with respect to the unique combinations of expressed C2 regions.
Dysferlin is a member of this family of C2 containing proteins that has a function in muscle membrane repair. Human mutation of dysferlin leads to specific autosomal recessive muscular dystrophies (limb-girdle MD type 2B and Miyoshi myopathy) (reviewed in Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213). Dysferlin is localized in the plasma membrane of cells where it interacts with annexin A1 and A2, and is also found in vesicles. Membrane disruption (for example in muscle) causes an increase of localized Ca2+ at the wound site and an accumulation of vesicles containing dysferlin. The dysferlin protein facilitates both docking and fusion of the vesicles with the plasma membrane through interaction with the annexins and/or other membrane-associated proteins. Fusion between the repair vesicles and the plasma membrane seals the wound (Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213).
158P3D2 protein, and variants thereof, functions in a similar fashion as dysferlin by inducing repair of cellular plasma membranes following their disruption. Given the high rate of cell division and stress conditions such as hypoxia and reduced nutrient supply during tumor formation, membrane repair becomes a critical component of tumor survival. Expression of 158P3D2, and variants thereof, on tumors provides an advantage for such cells to grow under stressful conditions such as hypoxia or nutrient deprivation.
The C2 domain of the lipid phosphatase/tumor suppressor PTEN is regulated by threonine phosphorylation (Raftopolou, M., et al., 2004, Science, 303, 1179-81). This phosphorylation event inhibits cell migration independent of the lipid phosphatase activity, which may relate to the tumor suppressive activity of PTEN. However, given the regulation of C2-induced function by phosphorylation, the status of that phosphorylation event alters the migratory capacity of the cell. 158P3D2 protein, and variants thereof, reside in the plasma membrane of tumor cells as C2-containing regulators of cell migration due to alterations in the phosphorylation status of 158P3D2. Upon phosphorylation of 158P3D2, the C2 domains influence the migratory capacity of 158P3D2-positive tumor cells, conferring an advantage for them to migrate to distal sites to seek secondary growth (metastasis). 158P3D2, and variants thereof, also bind to signal transduction proteins, providing important signaling cascades for tumor cells that confer a growth advantage and increased capacity for cell migration and adhesion. Such advantages are key elements for increased survival and metastasis for bladder, lung, colon and breast cancer cells.
Enhanced proliferation and entry into S-phase of tumor cells relative to normal cells is a hallmark of the cancer cell phenotype. To address the effect of expression of 158P3D2 on the proliferation rate of normal cells, two rodent cell lines (3T3 and Rat-1) are infected with virus containing the 158P3D2 gene and stable cells expressing 158P3D2 antigen are derived, as well as empty vector control cells expressing the selection marker neomycin (Neo). The cells are grown overnight in 0.5% FBS and then compared to cells treated with 10% FBS. The cells are evaluated for proliferation at 18-96 hr post-treatment by a 3H-thymidine incorporation assay and for cell cycle analysis by a BrdU incorporation/propidium iodide staining assay. Rat-1 cells expressing the 158P3D2 antigen grow effectively in low serum concentrations (0.1%) compared to the Rat-1-Neo cells. Similar data are obtained for the 3T3 cells expressing 158P3D2 versus Neo only. To assess cell proliferation by another methodology, the cells are stained with BrdU and propidium iodide. Briefly, cells are labeled with 10 μM BrdU, washed, trypsinized and fixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC (Pharmigen) is added to the cells, the cells are washed and then incubated with 10 μg/ml propidium iodide for 20 min prior to washing and analysis for fluorescence at 488 nm. An increase in labeling of cells in S-phase (DNA synthesis phase of the cell cycle) in 3T3 cells that express the 158P3D2 protein is observed relative to control cells. This confirms the results of those measured by 3H-thymidine incorporation. Accordingly, 158P3D2 expressing cells have increased potential for growth as tumor cells in vivo during stress, including nutrient deprivation, hypoxia or reduced osmolarity.
Example 45
158P3D2 RNA Interference (RNAi)
RNA interference (RNAi) technology is implemented to a variety of cell assays relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNA (siRNA) have the correct composition to activate the RNAi pathway targeting for degradation, specifically some mRNAs. See, Elbashir S. M., et al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411(6836):494-8 (2001). Thus, RNAi technology is used successfully in mammalian cells to silence targeted genes.
Loss of cell proliferation control is a hallmark of cancerous cells; thus, assessing the role of 158P3D2 in cell survival/proliferation assays is relevant. Accordingly, RNAi was used to investigate the function of the 158P3D2 antigen. To generate siRNA for 158P3D2, algorithms were used that predict oligonucleotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of the 158P3D2 protein when introduced into cells. Accordingly, one targeted sequence for the 158P3D2 siRNA is: 5′ CCTCGGCAGCCAATCAGCTAT 3′ (SEQ ID NO:104)(oligo 158P3D2.b). In accordance with this Example, 158P3D2 siRNA compositions are used that comprise siRNA (double stranded, short interfering RNA) that correspond to the nucleic acid ORF sequence of the 158P3D2 protein or subsequences thereof. Thus, siRNA subsequences are used in this manner are generally 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 or more than 35 contiguous RNA nucleotides in length. These siRNA sequences are complementary and non-complementary to at least a portion of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In preferred embodiments, these siRNA achieve knockdown of 158P3D2 antigen in cells expressing the protein and have functional effects as described below.
The selected siRNA (158P3D2.b oligo) was tested in numerous cell lines in the thymidine incorporation/proliferation assay (measures 3H-Thy uptake and incorporation into DNA). Moreover, this 158P3D2.b oligo achieved knockdown of 158P3D2 antigen in cells expressing the protein and had functional effects as described below using the following protocols.
Mammalian siRNA Transfections:
The day before siRNA transfection, the different cell lines were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at 2×103 cells/well in 80 μl (96 well plate format) for the survival/MTS assay. In parallel with the 158P3D2 specific siRNA oligo, the following sequences were included in every experiment as controls: a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA (targeted sequence: 5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO:105); and, c) Eg5 specific siRNA (targeted sequence: 5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO:106). SiRNAs were used at 10 nM and 1 μg/ml Lipofectamine 2000 final concentration.
The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 uM μM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 μg/ml (10-fold concentrated) for the total number transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted 10-fold concentrated siRNA and incubated at RT for 20-30′′ (5-fold concentrated transfection solution). 20 μls of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37° C. for 96 hours before analysis.
3H-Thymidine Incorporation Assay:
The proliferation assay is a 3H-thymidine incorporation method for determining the proliferation of viable cells by uptake and incorporation of label into DNA.
The procedure was as follows: Cells growing in log phase are trypsinized, washed, counted and plated in 96-well plates at 1000-4000 cells/well in 10% FBS. After 4-8 hrs, the media is replaced. The cells are incubated for 24-72 hrs, pulsed with 3H-Thy at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat and counted in scintillation cocktail on a Microbeta trilux or other counter.
To address the validation of the 158P3D2 siRNA in reducing the expression of 158P3D2 protein in cells, Cos-1 cells were transfected with pcDNA.3 vector expressing a Myc/His-tagged version of 158P3D2 alone (LF2k) or together with siRNA for either CT1 (bacterial negative control) or 158P3D2 oligo (158P3D2.b). For additional control, a mock transfection was also included (No DNA). Western blot analysis using an antibody to the Myc tag on 158P3D2 protein showed that the 158P3D2 siRNA significantly reduced the expression level of 158P3D2 protein (FIG. 30). These data show that the specific 158P3D2.b siRNA will have utility to probe the function of 158P3D2 protein in cells.
In order to address the function of 158P3D2 in cells, 158P3D2 was silenced by transfecting the endogenously expressing 158P3D2 cell lines (SCaBER, a bladder cancer cell line) with the 158P3D2 specific siRNA (158P3D2.b) along with negative siRNA controls (Luc4, targeted sequence not represented in the human genome) and a positive siRNA control (targeting Eg5) (See FIG. 4). SCaBER cells were shown to express 158P3D2 by Northern blot of total cellular RNA. The results indicated that when these cells were treated with siRNA specifically targeting the 158P3D2 mRNA, the resulting “158P3D2 deficient cells” showed diminished cell proliferation as measured by this assay (see oligo 158P3D2.b treated cells). This effect is likely caused by an active induction of apoptosis. The reduced viability is measured by the decreased uptake of labeled thymidine.
As control, Cos-1 cells, a cell line with no detectable expression of 158P3D2 mRNA or protein (by Western blot), was also treated with the panel of siRNAs (including oligo 158P3D2.b) and no phenotype was observed (FIG. 4). This result reflects the fact that the specific protein knockdown in the SCaBER cells is not a function of general toxicity, since the Cos-1 cells did not respond to the 158P3D2.b oligo. The differential response of the two cell lines to the Eg5 control is a reflection of differences in levels of cell transfection and responsiveness of the cell lines to oligo treatment (FIG. 4).
Together, these data indicate that 158P3D2, and variants thereof, play important roles in the proliferation of cancer cells and that the lack of 158P3D2 clearly decreases the survival potential of these cells. It is to be noted that 158P3D2 is constitutively expressed in many tumor cell lines. 158P3D2 serves a role in malignancy; it expression is a primary indicator of disease, where such disease is often characterized by high rates of uncontrolled cell proliferation and diminished apoptosis. Correlating cellular phenotype with gene knockdown following RNAi treatments is important, and allows one to draw valid conclusions and rule out toxicity or other non-specific effects of these reagents. To this end, assays to measure the levels of expression of both protein and mRNA for the target after RNAi treatments are important, including Western blotting, FACS staining with antibody, immunoprecipitation, Northern blotting or RT-PCR (Taqman or standard methods). Any phenotypic effect of the siRNAs in these assays should be correlated with the protein and/or mRNA knockdown levels in the same cell lines. Knockdown of 158P3D2 is achieved using the 158P3D2.b oligo as measured by Western blotting and RT-PCR analysis.
Another method to analyze 158P3D2 related cell proliferation is performing clonogenic assays. In these assays, a defined number of cells are plated onto the appropriate matrix and the number of colonies formed after a period of growth following siRNA treatment is counted.
In 158P3D2 cancer target validation, complementing the cell survival/proliferation analysis with apoptosis and cell cycle profiling studies are considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that commits the cell to die. A method to observe fragmented DNA in cells is the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELISA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of the cells and can detect DNA degradation in cells that do not proliferate in vitro (i.e. freshly isolated tumor cells).
The most important effector molecules for triggering apoptotic cell death are caspases. Caspases are proteases that when activated cleave numerous substrates at the carboxy-terminal site of an aspartate residue mediating very early stages of apoptosis upon activation. All caspases are synthesized as pro-enzymes and activation involves cleavage at aspartate residues. In particular, caspase 3 seems to play a central role in the initiation of cellular events of apoptosis. Assays for determination of caspase 3 activation detect early events of apoptosis. Following RNAi treatments, Western blot detection of active caspase 3 presence or proteolytic cleavage of products (i.e. PARP) found in apoptotic cells further support an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation, membrane blebbing, apoptotic bodies help to further support cell death as apoptotic. Since not all the gene targets that regulate cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain the profile of DNA content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to the leaking out to the cytoplasm (sub-G1 population). In addition, the use of DNA stains (i.e., propidium iodide) also differentiates between the different phases of the cell cycle in the cell population due to the presence of different quantities of DNA in G0/G1, S and G2/M. In these studies the subpopulations can be quantified.
For the 158P3D2 gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in identifying assays to screen for mAbs that are active anti-tumor therapeutics. Further, siRNA are administered as therapeutics to cancer patients for reducing the malignant growth of several cancer types, including those listed in Table 1. When 158P3D2 (and variants) plays a role in cell survival, cell proliferation, tumorigenesis, or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46
Homology Comparison of 158P3D2 to Known Sequences
The 158P3D2 v.17 protein has 2036 amino acids with a calculated molecular weight of 227.6 kDa and a pI of 5.64. 158P3D2 is predicted to be a predominantly cytoplasmic protein with plasma membrane association (PSORT-II). 158P3D2 contains a single transmembrane region from amino acids 2000-2022 with high probability that the amino-terminus resides outside, consistent with the topology of a type I transmembrane protein. Based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel, TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993), 158P3D2 contains a primary transmembrane region from amino acids 2003-2020 (contiguous amino acids with values greater than 0 on the plot have high probability of being transmembrane regions) with an orientation in which the amino terminus resides inside and the carboxyl terminus outside (type II). Another transmembrane algorithm indicated that 158P3D2 contains a transmembrane domain from amino acids 2003-2022, with the N-terminus oriented intracellularly consistent with a type II topology. The transmembrane prediction algorithms are accessed through the ExPasy molecular biology server.
By use of the PubMed website of the N.C.B.I., it was found at the protein level that 158P3D2 v.17 shows 60% homology and 40% identity with human otoferlin, a member of the ferlin family of plasma membrane proteins. Further, 158P3D2 v.17 shows 50% homology and 30% identity with dysferlin, another member of the ferlin family, and 80% homology and 75% identity with the murine gene Fer-1-like 4.
The ferlins are a family of transmembrane proteins that have function in membrane trafficking, including the repair of cell membranes. Mutation of human otoferlin leads to a specific form of nonsyndromic autosomal recessive deafness (DFNB9) and mutations in dysferlin lead to two subtypes of muscular dystrophies (reviewed in Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213). The major feature of the ferlin family includes multiple C2 domains (conserved PKC homologous region) that function in both Ca2+ dependent and Ca2+ independent phospholipid binding, as well as protein binding. The mechanism of action for dysferlin includes the repair of muscle cell membrane disruptions through dysferlin-containing cell vesicles. Such vesicles are tethered to the site of membrane tears (where Ca2+ concentrations are increased) via dysferlin molecules that interact with plasma membrane associated annexin A1 and annexin A2 molecules. The vesicles provide the lipid bilayer material to seal the wound.
158P3D2 associates with cell vesicles and the plasma membrane thereby providing a means for tumor cells to repair membranes during tumor growth and metastasis. Such a functional advantage can be exemplified by the increased stress that tumors experience, including increased hypoxia, decreased nutrition and increases in free radical formation. These stresses can alter membrane integrity, thereby increasing the need for robust plasma membrane repair mechanisms. In addition, the C2 domains of 158P3D2, and variants thereof, regulate migration of tumor cells expressing this protein. The regulation of the C2 domains occurs through the phosphorylation of threonine residues (Raftopolou, M., et al., 2004, Science, 303, 1179-81) and modulates the ability of the expressing cells to migrate. This signal transduction property of the 158P3D2 protein expressed in tumor cells enhances their ability to migrate during metastases, facilitates their homing to distal sites (lymph nodes), and promotes interactions with other cells during the formation of tumor masses. Further, the C2 domains of 158P3D2 play a role in membrane trafficking though interaction with Ca2+, phosphatidylserine and phosphatidylcholine (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998). These interactions are crucial for subsequent membrane metabolism and interaction. Taken together, the 158P3D2 protein significantly promotes unregulated growth of cancer cells, contributing to their viability and metastatic advantage in vivo.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
Tables







TABLE I





TISSUES THAT EXPRESS 158P3D2 WHEN MALIGNANT.

















Prostate



Bladder



Kidney



Colon



Ovary



Lung



Breast



Pancreas



Stomach



Cervix



Lymph node



Uterus
















TABLE II







AMINO ACID ABBREVIATIONS











SINGLE LETTER
THREE LETTER
FULL NAME






F
Phe
phenylalanine



L
Leu
leucine



S
Ser
serine



Y
Tyr
tyrosine



C
Cys
cysteine



W
Trp
tryptophan



P
Pro
proline



H
His
histidine



Q
Gln
glutamine



R
Arg
arginine



I
Ile
isoleucine



M
Met
methionine



T
Thr
threonine



N
Asn
asparagine



K
Lys
lysine



V
Val
valine



A
Ala
alanine



D
Asp
aspartic acid



E
Glu
glutamic acid



G
Gly
glycine
















TABLE III







Amino Acid Substitution Matrix


Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block


substitution matrix). The higher the value, the more likely a substitution is found in related,


natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html)



























A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
.






























4
0
−2
−1
−2
0
−2
−1
−1
−1
−1
−2
−1
−1
−1
1
0
0
−3
−2
A



9
−3
−4
−2
−3
−3
−1
−3
−1
−1
−3
−3
−3
−3
−1
−1
−1
−2
−2
C




6
2
−3
−1
−1
−3
−1
−4
−3
1
−1
0
−2
0
−1
−3
−4
−3
D





5
−3
−2
0
−3
1
−3
−2
0
−1
2
0
0
−1
−2
−3
−2
E






6
−3
−1
0
−3
0
0
−3
−4
−3
−3
−2
−2
−1
1
3
F







6
−2
−4
−2
−4
−3
0
−2
−2
−2
0
−2
−3
−2
−3
G








8
−3
−1
−3
−2
1
−2
0
0
−1
−2
−3
−2
2
H









4
−3
2
1
−3
−3
−3
−3
−2
−1
3
−3
−1
I










5
−2
−1
0
−1
1
2
0
−1
−2
−3
−2
K











4
2
−3
−3
−2
−2
−2
−1
1
−2
−1
L












5
−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















5
1
0
−1
−2
−2
−1
Q
















5
−1
−1
−3
−3
−2
R

















4
1
−2
−3
−2
S


















5
0
−2
−2
T



















4
−3
−1
V




















11
2
W





















7
Y









Table Iv
HLA Class I/II Motifs/Supermotifs







TABLE IV (A)







HLA Class I Supermotifs/Motifs













POSITION



POSITION
POSITION
C Terminus



2 (Primary Anchor)
3 (Primary Anchor)
(Primary Anchor)





SUPERMOTIF





A1
TILVMS

FWY


 


A2
LIVMATQ

IVMATL


 


A3
VSMATLI

RK


 


A24
YFWIVLMT

FIYWLM


 


B7
P

VILFMWYA


 


B27
RHK

FYLWMIVA


 


B44
ED

FWYLIMVA


 


B58
ATS

FWYLIVMA


 


B62
QLIVMP

FWYMIVLA


 


MOTIFS





A1
TSM

Y


 


A1

DEAS
Y


 


A2.1
LMVQIAT

VLIMAT


 


A3
LMVISATFCGD

KYRHFA


 


A11
VTMLISAGNCDF

KRYH


 


A24
YFWM

FLIW


 


A*3101
MVTALIS

RK


 


A*3301
MVALFIST

RK


 


A*6801
AVTMSLI

RK


 


B*0702
P

LMFWYAIV


 


B*3501
P

LMFWYIVA


 


B51
P

LIVFWYAM


 


B*5301
P

IMFWYALV


 


B*5401
P

ATIVLMFWY





Bolded residuess are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.













TABLE IV (B)







HLA Class II Supermotif









1
6
9





W, F, Y, V, .I, L
A, V, I, L, P,
A, V, I, L, C,



C, S, T
S, T, M, Y
















TABLE IV (C)





HLA Class II Motifs

























1°



1° anchor


















MOTIFS

anchor 1
2 3
4
5
6
7
8
9





DR4
preferred
FMYLI
M T

I
VSTCPA
M

MH




VW



LIM
H





deleterious


W


R

W











DE


 


DR1
preferred
MFLIV

PAMQ

VMATS
M

AV




WY



PLIC


M



deleterious

C C
FD
CW

GD
D






H

D

E




 


DR7
preferred
MFLIV
M W
A

IVMSAC
M

IV




WY



TPL






deleterious

C
G


GR
N
G









D



















1°


1°

1° anchor


DR3
MOTIFS
anchor 1
2
3
anchor 4
5
6





Motif a

LIVMF


D




preferred

Y







 


Motif b

LIVMF


DNQES

KRH


preferred

AY


T




 


DR

MFLIV




VMSTA


Supermotif

WY




CPLI





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (D)







HLA Class I Supermotifs








SUPER-
POSITION:

















MOTIFS

1
2
3
4
5
6
7
8
C-terminus





A1


1° Anchor






1° Anchor





TILVMS






FWY


 


A2


1° Anchor






1° Anchor





LIVMATQ






LIVMAT


 


A3
Preferred

1° Anchor
YFW


YFW
YFW
P
1° Anchor





VSMATLI
(4/5)


(3/5)
(4/5)
(4/5)
RK



deleterious
DE (3/5);

DE










P (5/5)

(4/5)








 


A24


1° Anchor






1° Anchor





YFWIVLMT






FIYWLM


 


B7
Preferred
FWY (5/5)
1° Anchor
FWY




FWY
1° Anchor




LIVM
P
(4/5)




(3/5)
VILFMWYA




(3/5)











deleterious
DE (3/5);



DE
G
QN
DE





P(5/5);



(3/5)
(4/5)
(4/5)
(4/5)





G(4/5);












A(3/5);












QN(3/5)










 


B27


1° Anchor






1° Anchor





RHK






FYLWMIVA


 


B44


1° Anchor






1° Anchor





ED






FWYLIMVA


 


B58


1° Anchor






1° Anchor





ATS






FWYLIVMA


 


B62


1° Anchor






1° Anchor





QLIVMP






FWYMIVLA





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (E)







HLA Class I Motifs









POSITION


























9 or C-
C-



1
2
3
4
5
6
7
8
terminus
terminus





















A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor



9-mer


STM






Y




deleterious
DE

RHKLIV
A
G
A










MP









 


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor



9-mer



DEAS





Y




deleterious
A
RHKDEP

DE
PQN
RHK
PG
GP







YFW










 


A1
preferred
YFW
1° Anchor
DEAQN
A
YFWQN

PASTC
GDE
P
1° Anchor


10-mer


STM







Y



deleterious
GP

RHKGLI
DE
RHK
QNA
RHKYFW
RHK
A







VM









 


A1
preferred
YFW
STCLIVM
1° Anchor
A
YFW

PG
G
YFW
1° Anchor


10-mer



DEAS






Y



deleterious
RHK
RHKDEP


P
G

PRHK
QN






YFW










 


A2.1
preferred
YFW
1° Anchor
YFW
STC
YFW

A
P
1° Anchor



9-mer


LMIVQAT






VLIMAT




deleterious
DEP

DERKH


RKH
DERKH





 


A2.1
preferred
AYFW
1° Anchor
LVIM
G

G

FYWL

1° Anchor


10-mer


LMIVQAT





VIM

VLIMAT



deleterious
DEP

DE
RKHA
P

RKH
DER
RKH












KH




 


A3
preferred
RHK
1° Anchor
YFW
PRHKY
A
YFW

P
1° Anchor






LMVISA

FW




KYRHFA






TFCGD











deleterious
DEP

DE









 


A11
preferred
A
1° Anchor
YFW
YFW
A
YFW
YFW
P
1° Anchor






VTLMIS






KRYH






AGNCDF











deleterious
DEP





A
G




 


A24
preferred
YFWRHK
1° Anchor

STC


YFW
YFW
1° Anchor



9-mer


YFWM






FLIW




deleterious
DEG

DE
G
QNP
DER
G
AQN











HK






 


A24
preferred

1° Anchor

P
YFWP

P


1° Anchor


10-mer


YFWM







FLIW



deleterious


GDE
QN
RHK
DE
A
QN
DEA



 


A3101
preferred
RHK
1° Anchor
YFW
P

YFW
YFW
AP
1° Anchor






MVTALIS






RK




deleterious
DEP

DE

ADE
DE
DE
DE




 


A3301
preferred

1° Anchor
YFW



AYFW

1° Anchor






MVALFI






RK






ST











deleterious
GP

DE









 


A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor



9-mer


STM






Y




deleterious
DE

RHKLIV
A
G
A










MP









 


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor



9-mer



DEAS





Y




deleterious
A
RHKDEP

DE
PQN
RHK
PG
GP







YFW










 


A6801
preferred
YFWSTC
1° Anchor


YFWLI

YFW
P
1° Anchor






AVTMSLI


VM



RK




deleterious
GP

DEG

RHK


A




 


B0702
preferred
RHKFWY
1° Anchor
RHK

RHK
RHK
RHK
PA
1° Anchor






P






LMFWY













AIV




deleterious
DEQNP

DEP
DE
DE
GDE
QN
DE




 


B3501
preferred
FWYLI
1° Anchor
FWY



FWY

1° Anchor





VM
P






LMFWY













IVA




deleterious
AGP



G
G






 


B51
preferred
LIVMF
1° Anchor
FWY
STC
FWY

G
FWY
1° Anchor





WY
P






LIVFWY













AM




deleterious
AGPDE



DE
G
DEQN
GDE






RHKSTC











 


B5301
preferred
LIVMF
1° Anchor
FWY
STC
FWY

LIVMF
FWY
1° Anchor





WY
P




WY

IMFWY













ALV




deleterious
AGPQN




G
RHKQN
DE




 


B5401
preferred
FWY
1° Anchor
FWYLIVM

LIVM

ALIVM
FWY
1° Anchor






P





AP
ATIVLM













FWY




deleterious
GPQNDE

GDESTC

RHKDE
DE
QNDGE
DE
















TABLE IV (F)







Summary of HLA-supertypes


Overall phenotypic frequencies of HLA-supertypes in different ethnic populations








Specificity
Phenotypic frequency















Supertype
Position 2
C-Terminus
Caucasian
N.A. Black
Japanese
Chinese
Hispanic
Average


















B7
P
AILMVFWY
43.2
55.1
57.1
43.0
49.3
49.5


A3
AILMVST
RK
37.5
42.1
45.8
52.7
43.1
44.2


A2
AILMVT
AILMVT
45.8
39.0
42.4
45.9
43.0
42.2


A24
YF (WIVLMT)
FI (YWLM)
23.9
38.9
58.6
40.1
38.3
40.0


B44
E (D)
FWYLIMVA
43.0
21.2
42.9
39.1
39.0
37.0


A1
TI (LVMS)
FWY
47.1
16.1
21.8
14.7
26.3
25.2


B27
RHK
FYL (WMI)
28.4
26.1
13.3
13.9
35.3
23.4


B62
QL (IVMP)
FWY (MIV)
12.6
4.8
36.5
25.4
11.1
18.1


B58
ATS
FWY (LIV)
10.0
25.1
1.6
9.0
5.9
10.3
















TABLE IV (G)







Calculated population coverage afforded by different


HLA-supertype combinations









Phenotypic frequency













HLA-
Cau-
N.A


His-



supertypes
casian
Blacks
Japanese
Chinese
panic
Average
















A2, A3 and B7
83.0
86.1
87.5
88.4
86.3
86.2


A2, A3, B7,
99.5
98.1
100.0
99.5
99.4
99.3


A24, B44 and


A1


A2, A3, B7,
99.9
99.6
100.0
99.8
99.9
99.8


A24, B44, A1,


B27, B62, and


B 58









Motifs indicate the residues defining supertype specificities. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.








TABLE V







Frequently Occurring Motifs











avrg. %




Name
identity
Description
Potential Function





zf-C2H2
34%
Zinc finger, C2H2 type
Nucleic acid-binding protein functions as





transcription factor, nuclear location probable


cytochrome_b_N
68%
Cytochrome b(N-
membrane bound oxidase, generate superoxide




terminal)/b6/petB



Ig
19%
Immunoglobulin domain
domains are one hundred amino acids long and





include a conserved intradomain disulfide bond.


WD40
18%
WD domain, G-beta repeat
tandem repeats of about 40 residues, each





containing a Trp-Asp motif. Function in signal





transduction and protein interaction


PDZ
23%
PDZ domain
may function in targeting signaling molecules to





sub-membranous sites


LRR
28%
Leucine Rich Repeat
short sequence motifs involved in protein-protein





interactions


Pkinase
23%
Protein kinase domain
conserved catalytic core common to both





serine/threonine and tyrosine protein kinases





containing an ATP binding site and a catalytic





site


PH
16%
PH domain
pleckstrin homology involved in intracellular





signaling or as constituents of the cytoskeleton


EGF
34%
EGF-like domain
30-40 amino-acid long found in the extracellular





domain of membrane-bound proteins or in





secreted proteins


Rvt
49%
Reverse transcriptase





(RNA-dependent DNA





polymerase)



Ank
25%
Ank repeat
Cytoplasmic protein, associates integral





membrane proteins to the cytoskeleton


Oxidored_q1
32%
NADH-
membrane associated. Involved in proton




Ubiquinone/plastoquinone
translocation across the membrane




(complex I), various chains



Efhand
24%
EF hand
calcium-binding domain, consists of a12 residue





loop flanked on both sides by a 12 residue alpha-





helical domain


Rvp
79%
Retroviral aspartyl
Aspartyl or acid proteases, centered on a




protease
catalytic aspartyl residue


Collagen
42%
Collagen triple helix repeat
extracellular structural proteins involved in




(20 copies)
formation of connective tissue. The sequence





consists of the G-X-Y and the polypeptide chains





forms a triple helix.


Fn3
20%
Fibronectin type III domain
Located in the extracellular ligand-binding





region of receptors and is about 200 amino acid





residues long with two pairs of cysteines





involved in disulfide bonds


7tm_1
19%
7 transmembrane receptor
seven hydrophobic transmembrane regions, with




(rhodopsin family)
the N-terminus located extracellularly while the





C-terminus is cytoplasmic. Signal through G





proteins
















TABLE VI





Motif-bearing subsequences and Post-Translational


Modifications of 158P3D2















Phosphorylation sites predicted:


Phosphoserine predictions








Pos.
Context





73
SRVFSLRPL (SEQ ID NO: 107)


107
NLQVSPIQV (SEQ ID NO: 108)


128
TGAWSEEDF (SEQ ID NO: 109)


173
RLARSLGQQ (SEQ ID NO: 110)


206
GVMFSPLKS (SEQ ID NO: 111)


210
SPLKSRARA (SEQ ID NO: 112)


268
QRGTSCPFY (SEQ ID NO: 113)


320
LTPPSPKAF (SEQ ID NO: 114)


380
KVTLSVRAR (SEQ ID NO: 115)


460
YVRVSFLGQ (SEQ ID NO: 116)


469
EGETSVSAE (SEQ ID NO: 117)


471
ETSVSAEAA (SEQ ID NO: 118)


484
NEQLSFVEL (SEQ ID NO: 119)


522
LRRISHPGR (SEQ ID NO: 120)


624
HLDASPGAE (SEQ ID NO: 121)


679
SQPISFEIS (SEQ ID NO: 122)


683
SFEISIGRA (SEQ ID NO: 123)


916
DVLFSVVEE (SEQ ID NO: 124)


982
GLPSSLHRD (SEQ ID NO: 125)


1012
DSGLSDPFA (SEQ ID NO: 126)


1127
ELDYSGRLE (SEQ ID NO: 127)


1136
PSVPSEVEP (SEQ ID NO: 128)


1151
VEPHSGRLS (SEQ ID NO: 129)


1155
SGRLSLPPN (SEQ ID NO: 130)


1208
EVLASYRES (SEQ ID NO: 131)


1212
SYRESPNFT (SEQ ID NO: 132)


1380
DPGDSDGVN (SEQ ID NO: 133)


1405
KGTVSPKKA (SEQ ID NO: 134)


1419
IYNRSLKEE (SEQ ID NO: 135)


1519
YVVVSAGRE (SEQ ID NO: 136)


1585
NRFYSHHRA (SEQ ID NO: 137)


1595
CGLASQYEV (SEQ ID NO: 138)


1652
LPPGSSSPT (SEQ ID NO: 139)


1654
PGSSSPTVA (SEQ ID NO: 140)


1699
YHPHSPGLL (SEQ ID NO: 141)


1733
RQPISYELR (SEQ ID NO: 142)


1809
EREVSVWRR (SEQ ID NO: 143)


1814
VWRRSGPFA (SEQ ID NO: 144)


1841
YDRISANDF (SEQ ID NO: 145)


1975
LEKPSRPKT (SEQ ID NO: 146)


 







Phosphothreonine predictions








29
FRGFTQKTR (SEQ ID NO: 147)


261
QRRVTATQR (SEQ ID NO: 148)


263
RVTATQRGT (SEQ ID NO: 149)


267
TQRGTSCPF (SEQ ID NO: 150)


338
TRIGTFRMD (SEQ ID NO: 151)


367
DPRDTRAGT (SEQ ID NO: 152)


601
LSRLTRKKK (SEQ ID NO: 153)


613
RRDQTPKAV (SEQ ID NO: 154)


704
AGEGTEGAA (SEQ ID NO: 155)


828
RRTMTRPNA (SEQ ID NO: 156)


1022
VLISTQCQT (SEQ ID NO: 157)


1026
TQCQTTRVL (SEQ ID NO: 158)


1033
VLEQTLSPL (SEQ ID NO: 159)


1223
VRHLTVVFK (SEQ ID NO: 160)


1412
KAVATLKIY (SEQ ID NO: 161)


1527
ERQDTKERY (SEQ ID NO: 162)


1557
ETELTVAVF (SEQ ID NO: 163)


1643
KVFLTPPET (SEQ ID NO: 164)


1743
VIWNTEDVV (SEQ ID NO: 165)


1804
DYLPTEREV (SEQ ID NO: 166)


1941
VYILTGKVE (SEQ ID NO: 167)


1952
FELLTVEEA (SEQ ID NO: 168)


1979
SRPKTSFNW (SEQ ID NO: 169)


 







Phosphotyrosine predictions








52
RWPHYGAPL (SEQ ID NO: 170)


456
LVEPYVRVS (SEQ ID NO: 171)


542
WVPLYGSPP (SEQ ID NO: 172)


990
DDFSYFQLR (SEQ ID NO: 173)


1095
MEDPYQRPE (SEQ ID NO: 174)


1243
PEQPYLQPP (SEQ ID NO: 175)


1515
KADPYVVVS (SEQ ID NO: 176)


1531
TKERYIPKQ (SEQ ID NO: 177)


1602
EVDGYNAWR (SEQ ID NO: 178)


1629
PAPEYRAGA (SEQ ID NO: 179)


1763
SSDIYVKSW (SEQ ID NO: 180)


 







N-glycosylation sites








67
NCSR (SEQ ID NO: 181)


1214
NFTE (SEQ ID NO: 182)


1417
NRSL (SEQ ID NO: 183)


 







O-glycosylation sites








1403
T


1647
T


1656
T


 







Dileucine motifs


LL at 290


LL at 291


LL at 406


LL at 407


LL at 438


LL at 571


LL at 572


LL at 643


LL at 658


LL at 714


LL at 840


LL at 847


LL at 855


LL at 874


LL at 971


LL at 1041


LL at 1184


LL at 1320


LL at 1333


LL at 1491


 


C2 domains


aa2-96


aa230-355


aa420-530


aa992-1100


aa1492-1591


aa1735-1865


 


Coiled-coil regions


aa168-186


aa1895-1922


 


Interaction motifs and specific kinase


phosphorylation sites


proteins interaction motif 2


RRTMTRP (SEQ ID NO: 184)


 


FHA domain interaction motif 1, threonine


phosphorylation is required


TPKA (SEQ ID NO: 185)


TEGA (SEQ ID NO: 186)


TWRL (SEQ ID NO: 187)


TSEL (SEQ ID NO: 188)


TAPL (SEQ ID NO: 189)


TLKI (SEQ ID NO: 190)


TRPL (SEQ ID NO: 191)


 


Motif found in p53 family members which confers


binding to the N-terminal domain of MDM2


FGPAWVPL (SEQ ID NO:192)


 


Class III PDZ domains binding motif


DENL (SEQ ID NO: 193)


FELI (SEQ ID NO: 194)


RDSL (SEQ ID NO: 195)


NEGV (SEQ ID NO: 196)


VEEL (SEQ ID NO: 197)


EELL (SEQ ID NO: 198)


PENV (SEQ ID NO: 199)


EDFL (SEQ ID NO: 200)


EEQL (SEQ ID NO: 201)


EEEL (SEQ ID NO: 202)


AERL (SEQ ID NO: 203)


VERL (SEQ ID NO: 204)


LEVL (SEQ ID NO: 205)


PDVL (SEQ ID NO: 206)


QDVL (SEQ ID NO: 207)


PDLL (SEQ ID NO: 208)


GELI (SEQ ID NO: 209)


VEVL (SEQ ID NO: 210)


SEVL (SEQ ID NO: 211)


TELV (SEQ ID NO: 212)


EEDI (SEQ ID NO: 213)


PEEL (SEQ ID NO: 214)


SDGV (SEQ ID NO: 215)


EDWL (SEQ ID NO: 216)


SEAV (SEQ ID NO: 217)


PETL (SEQ ID NO: 218)


PEHV (SEQ ID NO: 219)


PEDL (SEQ ID NO: 220)


FELL (SEQ ID NO: 221)


PEPL (SEQ ID NO: 222)


 


Src-family Src Homology 2 (SH2) domains binding


motif.


YQPP (SEQ ID NO: 223)


 


This is the motif recognized by class I SH3


domains


RAEPEPP (SEQ ID NO: 224)


RLEPSVP (SEQ ID NO: 225)


 


This is the motif recognized by those SH3 domains


with a non-canonical class I recognition


specificity


LPPPMLP (SEQ ID NO: 226)


PMLPPAP (SEQ ID NO: 227)


RAEPEPP (SEQ ID NO: 228)


AEGPEIP (SEQ ID NO: 229)


PENVLAP (SEQ ID NO: 230)


AEEPQPP (SEQ ID NO: 231)


EPQPPLP (SEQ ID NO: 232)


RLEPSVP (SEQ ID NO: 233)


LLEVEQP (SEQ ID NO: 234)


QDLPEQP (SEQ ID NO: 235)


DMMPKGP (SEQ ID NO: 236)


LYHPHSP (SEQ ID NO: 237)


PQDVPAP (SEQ ID NO: 238)


QDVPAPP (SEQ ID NO: 239)


DIKPRQP (SEQ ID NO: 240)


LEKPSRP (SEQ ID NO: 241)


 


This is the motif recognized by those SH3 domains


with a non-canonical class II recognition


specificity


KPPLKKLP (SEQ ID NO: 242)


 


Major TRAF2-binding consensus motif. Members of


the tumor necrosis factor receptor (TNFR)


superfamily initiate intracellular signaling by


recruiting the C-domain of the TNFR-associated


factors (TRAFs) through their cytoplasmic tails.


PEEE (SEQ ID NO: 243)


PPEE (SEQ ID NO: 244)


PEEE (SEQ ID NO: 245)


SLQE (SEQ ID NO: 246)


TVEE (SEQ ID NO: 247)


 


TRAF6 binding site. Members of the tumor necrosis


factor receptor (TNFR) superfamily initiate intra-


cellular signaling by recruiting the C-domain of


the TNFR-associated factors (TRAFs) through their


cytoplasmatic tails.


GVPAERPWA (SEQ ID NO: 248)


ARPEEEKEE (SEQ ID NO: 249)


DPPEEEGEM (SEQ ID NO: 250)


PDPEELDWG (SEQ ID NO: 251)


 


PPLP is the motif recognized by WW domains of


Group II


PPLP (SEQ ID NO: 252)


 


GSK3 phosphorylation recognition site


GATGAWS (SEQ ID NO: 253)


MFSPLKS (SEQ ID NO: 254)


RDTRAGT (SEQ ID NO: 255)


VASQPIS (SEQ ID NO: 256)


PISFEIS (SEQ ID NO: 257)


IQSLMLT (SEQ ID NO: 258)


PHSGRLS (SEQ ID NO: 259)


LASYRES (SEQ ID NO: 260)


RESPNFT (SEQ ID NO: 261) 


FLTPPET (SEQ ID NO: 262)


PGSSSPT (SEQ ID NO: 263)


KPSRPKT (SEQ ID NO: 264)


 


PKA phosphorylation site


RDT


RDS


RLT


RGS


RRT


RLS


RES


RFT


 


Site phosphorylated by the Polo-like-kinase


EATM (SEQ ID NO: 265)


DPTV (SEQ ID NO: 266)


EISI (SEQ ID NO: 267)


DHTW (SEQ ID NO: 268)


EPSA (SEQ ID NO: 269)


EPSV (SEQ ID NO: 270)


 


Proline-Directed Kinase (e.g. MAPK) phosphoryla- 


tion site in higher eukaryotes.


LQVSPIQ (SEQ ID NO: 271)


VMFSPLK (SEQ ID NO: 272)


LYGSPPG (SEQ ID NO: 273)


RDQTPKA (SEQ ID NO: 274)


LDASPGA (SEQ ID NO: 275)


YRESPNF (SEQ ID NO: 276)


GTVSPKK (SEQ ID NO: 277)


VFLTPPE (SEQ ID NO: 278)


GSSSPTV (SEQ ID NO: 279)


HPHSPGL (SEQ ID NO: 280)


 


SH-PTP2 and phospholipase C-gamma Src Homology 2 


(SH2) domains binding motif.


aa249-252


aa456-459


aa1515-1518


 


CK1 phosphorylation site


aa1009-1012


aa234-237


aa484-487


aa760-763


 


CK2 phosphorylation site


aa1085-1807


 


PKB Phosphorylation site


aa1522-1528
















TABLE VII





Search Peptides















158P3D2 v.1, 9-mers, 10-mers and 15-mers (SEQ ID NO: 281)








MWIDIFPQDV PAPPPVDIKP RQPISYELRV VIWNTEDVVL DDENPLTGEM SSDIYVKSWV
60


KGLEHDKQET DVHFNSLTGE GNFNWRFVFR FDYLPTEREV SVWRRSGPFA LEEAEFRQPA
120


VLVLQVWDYD RISANDFLGS LELQLPDMVR GARGPELCSV QLARNGAGPR CNLFRCRRLR
180


GWWPVVKLKE AEDVEREAQE AQAGKKKRKQ RRRKGRPEDL EFTDMGGNVY ILTGKVEAEF
240


ELLTVEEAEK RPVGKGRKQP EPLEKPSRPK TSFNWFVNPL KTFVFFIWRR YWRTLVLLLL
300


VLLTVFLLLV FYTIPGQISQ VIFRPLHK
328


 


158P3D2 v.2A, 9-mers, 10-mers and 15-mers (SEQ ID NO: 282)



MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL KIYNRSLEEE FNHFEDWLNV
60


FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVL FSEPQISRGI PQNRPIKLLV
120


RVYVVKATNL APADPNGKAD PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE
180


TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF
236


 


158P3D2 v.3



9-mers aa 95-111



PTEREVSVRRRSGPFAL (SEQ ID NO: 283)



10-mers aa 94-112



LPTEREVSVRRRSGPFALE (SEQ ID NO: 284)



15-mers aa 89-117



FRFDYLPTEREVSVRRRSGPFALEEAEFR (SEQ ID NO: 285)



 


158P3D2 v.4



9-mers aa 94-110



LPTEREVSIWRRSGPFA (SEQ ID NO: 286)



10-mers aa 93-111



YLPTEREVSIWRRSGPFAL(SEQ ID NO: 287)



15-mers aa 88-116



VFRFDYLPTEREVSIWRRSGPFALEEAEF (SEQ ID NO: 288)



 


158P3D2 v.5A (BCP2A) ORF: 849-1385



9-mers



LVLQVWDYTASLPMTSLDPWSCSYQTWCVGPGAPSSALCSWPAMGPGRGAICFAAAA (SEQ ID NO: 289)



10-mers



VLVLQVWDYTASLPMTSLDPWSCSYQTWCVGPGAPSSALCSWPAMGPGRGAICFAAAA (SEQ ID NO: 290)



15-mers



FRQPAVLVLQVWDYTASLPMTSLDPWSCSYQTWCVGPGAPSSALCSWPAMGPGRGAICFAAAA



(SEQ ID NO: 291)



 


158P3D2 v.10



9-mers aa 50-66



MSSDIYVKRWVKGLEHD (SEQ ID NO: 292)



10-mers aa 49-67



EMSSDIYVKRWVKGLEHDK (SEQ ID NO: 293)



15-mers aa 44-72



NPLTGEMSSDIYVKRWVKGLEHDKQETDV (SEQ ID NO: 294)



 


158P3D2 v.12



9-mers aa 273-287



FNWFVNPLNTFVFFIWR (SEQ ID NO: 295)



10-mers aa 272-288



SFNWFVNPLNTFVFFIWRR (SEQ ID NO: 296)



15-mers aa 267-293



SRPKTSFNWFVNPLNTFVFFIWRRYWRTL (SEQ ID NO: 297)



 


158P3D2 v.13



9-mers aa 274-288



NWFVNPLKAFVFFIWRR (SEQ ID NO: 298)



10-mers aa 273-287



FNWFVNPLKAFVFFIWRRY (SEQ ID NO: 299)



15-mers aa 268-294



RPKTSFNWFVNPLKAFVFFIWRRYWRTLV (SEQ ID NO: 300)



 


Combination of v12 and v13








9-mers aa 273-287








NWFVNPLNAFVFFIWR (SEQ ID NO: 301)



10-mers aa 272-288



FNWFVNPLNAFVFFIWRR (SEQ ID NO: 302)



15-mers aa 267-293



RPKTSFNWFVNPLNAFVFFIWRRYWRTL (SEQ ID NO: 303)



 


v.14 ORF:65-4246 Frame +2



Part A



9-mers



ELVRHLTVDLPEQPYL (SEQ ID NO: 304)



10-mers



ELVRHLTVDLPEQPYLQ (SEQ ID NO: 305)



15-mers



ESPNFTELVRHLTVDLPEQPYLQPPLSI (SEQ ID NO: 306)



 


Part B



9-mers



MVGEIQDQDLQQVPEGRI (SEQ ID NO: 307)



10-mers



SMVGEIQDQDLQQVPEGRI (SEQ ID NO: 308)



15-mers



GVNLISMVGEIQDQDLQQVPEGRI (SEQ ID NO: 309)



 


v.15 ORF: 65-3502 Frame +2



Part A



9-mers



KLRFLAEEHNFDEDEM (SEQ ID NO: 310)



10-mers



AKLRFLAEEHNFDEDEM (SEQ ID NO: 311)



15-mers



AKKLLAKLRFLAEEHNFDEDEMDDPGDS (SEQ ID NO: 312)



 


Part B



9-mers



LVRVYVVKLRNLCKIQGHEDFCLFSAATNLAPAD (SEQ ID NO: 313)



10-mers



LLVRVYVVKLRNLCKIQGHEDFCLFSAATNLAPADP (SEQ ID NO: 314)



15-mers



NRPIKLLVRVYVVKLRNLCKIQGHEDFCLFSAATNLAPADPNGKAD (SEQ ID NO: 315)



 


Part C



9-mers



GLASQYEVWVQQGPQEPF (SEQ ID NO: 316)



10-mers



CGLASQYEVWVQQGPQEPF (SEQ ID NO: 317)



15-mers



HHRANCGLASQYEVWVQQGPQEPF (SEQ ID NO: 318)



 


v.16 ORF:65-6037 Frame +2 1990 AA



Part A



9-mers



GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 319)



10-mers



GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 320)



15-mers



GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 321)



 


Part B



9-mers








VKLKEAEDGKVEAEFE (SEQ ID NO: 322)








10mers



VVKLKEAEDGKVEAEFEL (SEQ ID NO: 323)



15-mers



RGWWPVVKLKEAEDGKVEAEFELLTVEE (SEQ ID NO: 324)



 


v.17 ORF:65-6175 Frame +2 (SEQ ID NO: 325)



9-mers, 10-mers and 15-mers



MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC
60


LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP
120


EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE
180


ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK
240


LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG
300


VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP
360


LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR
420


LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN
480


EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP
540


LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL
600


TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF
660


GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE
720


EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE
780


RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL
840


LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ
900


RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA
960


KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI
1020


STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG
1080


RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP
1140


QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG
1200


VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH
1260


TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL
1320


LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS
1380


DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ
1440


GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA
1500


TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV
1560


FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ
1620


RCGLPAPEYR AGAVKVGSKV FLTPPETLPP GSSSPTVASG DPEEAQALLV LRRWQEMPGF
1680


GIQLVPEHVE TRPLYHPHSP GLLQGSLHMW IDIFPQDVPA PPPVDIKPRQ PISYELRVVI
1740


WNTEDVVLDD ENPLTGEMSS DIYVKSWVKG LEHDKQETDV HFNSLTGEGN FNWRFVFRFD
1800


YLPTEREVSV WRRSGPFALE EAEFRQPAVL VLQVWDYDRI SANDFLGSLE LQLPDMVRGA
1860


RGPELCSVQL ARNGAGPRCN LFRCRRLRGW WPVVKLKEAE DVEREAQEAQ AGKKKRKQRR
1920


RKGRPEDLEF TDMGGNVYIL TGKVEAEFEL LTVEEAEKRP VGKGRKQPEP LEKPSRPKTS
1980


FNWFVNPLKT FVFFIWRRYW RTLVLLLLVL LTVFLLLVFY TIPGQISQVI FRPLHK
2036


 


v.18 ORF:2932-4764 Frame +1 610 aa



Part A



9-mers



MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWSSSCGWAWASKPRPAPLSCPRICCP



SPQPGCPPAYTGTVLEQTLSP (SEQ ID NO: 326)



10-mers



MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWSSSCGWAWASKPRPAPLSCPRICCP



SPQPGCPPAYTGTVLEQTLSPL (SEQ ID NO: 327)



15-mers



MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWSSSCGWAWASKPRPAPLSCPRICCP



SPQPGCPPAYTGTVLEQTLSPLWDELL (SEQ ID NO: 328)



 


Part B



9-mers



GISRQLLKHNFDEDEM (SEQ ID NO: 329)



10-mers



AGISRQLLKHNFDEDEMD (SEQ ID NO: 330)



15-mers



PFLAEAGISRQLLKHNFDEDEMDDPGDS (SEQ ID NO: 331)



 


Part C



9-mers



GLASQYEVWVQQGPQEPF (SEQ ID NO: 332)



10-mers



CGLASQYEVWVQQGPQEPF (SEQ ID NO: 333)



15-mers



HHRANCGLASQYEVWVQQGPQEPF (SEQ ID NO: 334)









Tables VIII-XXI







TABLE VIII







158P3D2v.1-A1-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position for 


each peptide is the start position plus eight.









Pos
Subsequence
Score












222
FTDMGGNVY
62.500


 


47
TGEMSSDIY
11.250


 


219
DLEFTDMGG
4.500


 


110
ALEEAEFRQ
4.500


 


237
EAEFELLTV
4.500


 


247
EAEKRPVGK
3.600


 


198
AQEAQAGKK
2.700


 


78
TGEGNFNWR
2.250


 


259
QPEPLEKPS
2.250


 


113
EAEFRQPAV
1.800


 


140
SLELQLPDM
1.800


 


281
KTFVFFIWR
1.250


 


303
LTVFLLLVF
1.250


 


145
LPDMVRGAR
1.250


 


312
YTIPGQISQ
1.250


 


69
ETDVHFNSL
1.250


 


34
NTEDVVLDD
1.125


 


320
QVIFRPLHK
1.000


 


166
GAGPRCNLF
1.000


 


304
TVFLLLVFY
1.000


 


39
VLDDENPLT
1.000


 


188
LKEAEDVER
0.900


 


235
KVEAEFELL
0.900


 


190
EAEDVEREA
0.900


 


62
GLEHDKQET
0.900


 


51
SSDIYVKSW
0.750


 


2
WIDIFPQDV
0.500


 


257
RKQPEPLEK
0.500


 


142
ELQLPDMVR
0.500


 


283
FVFFIWRRY
0.500


 


121
VLVLQVWDY
0.500


 


156
ELCSVQLAR
0.500


 


154
GPELCSVQL
0.450


 


97
EREVSVWRR
0.450


 


242
LLTVEEAEK
0.400


 


197
EAQEAQAGK
0.400


 


243
LTVEEAEKR
0.250


 


90
RFDYLPTER
0.250


 


49
EMSSDIYVK
0.200


 


4
DIFPQDVPA
0.200


 


11
PAPPPVDIK
0.200


 


123
VLQVWDYDR
0.200


 


53
DIYVKSWVK
0.200


 


262
PLEKPSRPK
0.180


 


75
NSLTGEGNF
0.150


 


67
KQETDVHFN
0.135


 


126
VWDYDRISA
0.125


 


293
RTLVLLLLV
0.125


 


81
GNFNWRFVF
0.125


 


277
VNPLKTFVF
0.125


 


77
LTGEGNFNW
0.125


 


214
KGRPEDLEF
0.125


 


270
KTSFNWFVN
0.125


 


85
WRFVFRFDY
0.125


 


40
LDDENPLTG
0.125


 


216
RPEDLEFTD
0.113


 


298
LLLVLLTVF
0.100


 


200
EAQAGKKKR
0.100


 


170
RCNLFRCRR
0.100


 


109
FALEEAEFR
0.100


 


276
FVNPLKTFV
0.100


 


244
TVEEAEKRP
0.090


 


25
SYELRVVIW
0.090


 


193
DVEREAQEA
0.090


 


195
EREAQEAQA
0.090


 


132
ISANDFLGS
0.075


 


316
GQISQVIFR
0.075


 


105
RSGPFALEE
0.075


 


10
VPAPPPVDI
0.050


 


71
DVHFNSLTG
0.050


 


300
LVLLTVFLL
0.050


 


137
FLGSLELQL
0.050


 


232
LTGKVEAEF
0.050


 


294
TLVLLLLVL
0.050


 


301
VLLTVFLLL
0.050


 


302
LLTVFLLLV
0.050


 


227
GNVYILTGK
0.050


 


297
LLLLVLLTV
0.050


 


296
VLLLLVLLT
0.050


 


131
RISANDFLG
0.050


 


308
LLVFYTIPG
0.050


 


245
VEEAEKRPV
0.045


 


143
LQLPDMVRG
0.030


 


24
ISYELRVVI
0.030


 


201
AQAGKKKRK
0.030


 


50
MSSDIYVKS
0.030


 


116
FRQPAVLVL
0.025


 


46
LTGEMSSDI
0.025


 


191
AEDVEREAQ
0.025


 


95
PTEREVSVW
0.022


 


59
WVKGLEHDK
0.020


 


179
LRGWWPVVK
0.020


 


306
FLLLVFYTI
0.020


 


157
LCSVQLARN
0.020


 


230
YILTGKVEA
0.020


 


309
LVFYTIPGQ
0.020


 


299
LLVLLTVFL
0.020


 


17
DIKPRQPIS
0.020


 


295
LVLLLLVLL
0.020


 


158
CSVQLARNG
0.015
















TABLE IX







158P3D2v.1-A1-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












259
QPEPlEKPSR
45.000


 


276
FVNPlKTFVF
5.000


 


166
GAGPrCNLFR
5.000


 


235
KVEAeFELLT
4.500


 


198
AQEAqAGKKK
2.700


 


39
VLDDeNPLTG
2.500


 


303
LTVFlLLVFY
2.500


 


17
DIKPrQPISY
2.500


 


222
FTDMgGNVYI
2.500


 


78
TGEGnFNWRF
2.250


 


113
EAEFrQPAVL
1.800


 


46
LTGEmSSDIY
1.250


 


69
ETDVhFNSLT
1.250


 


47
TGEMsSDIYV
1.125


 


140
SLELqLPDMV
0.900


 


219
DLEFtDMGGN
0.900


 


190
EAEDvEREAQ
0.900


 


244
TVEEaEKRPV
0.900


 


51
SSDIyVKSWV
0.750


 


67
KQETdVHFNS
0.675


 


134
ANDFlGSLEL
0.625


 


120
AVLVlQVWDY
0.500


 


302
LLTVfLLLVF
0.500


 


10
VPAPpPVDIK
0.500


 


95
PTEReVSVWR
0.450


 


241
ELLTvEEAEK
0.400


 


312
YTIPgQISQV
0.250


 


281
KTFVfFIWRR
0.250


 


145
LPDMvRGARG
0.250


 


77
LTGEgNFNWR
0.250


 


12
APPPvDIKPR
0.250


 


154
GPELcSVQLA
0.225


 


216
RPEDlEFTDM
0.225


 


34
NTEDvVLDDE
0.225


 


25
SYELrVVIWN
0.225


 


122
LVLQvWDYDR
0.200


 


231
ILTGkVEAEF
0.200


 


197
EAQEaQAGKK
0.200


 


200
EAQAgKKKRK
0.200


 


100
VSVWrRSGPF
0.150


 


105
RSGPfALEEA
0.150


 


319
SQVIfRPLHK
0.150


 


80
EGNFnWRFVF
0.125


 


293
RTLVlLLLVL
0.125


 


297
LLLLvLLTVF
0.100


 


144
QLPDmVRGAR
0.100


 


242
LLTVeEAEKR
0.100


 


193
DVEReAQEAQ
0.090


 


247
EAEKrPVGKG
0.090


 


62
GLEHdKQETD
0.090


 


245
VEEAeKRPVG
0.090


 


110
ALEEaEFRQP
0.090


 


237
EAEFeLLTVE
0.090


 


107
GPFAlEEAEF
0.050


 


15
PVDIkPRQPI
0.050


 


304
TVFLlLVFYT
0.050


 


2
WIDIfPQDVP
0.050


 


76
SLTGeGNFNW
0.050


 


307
LLLVfYTIPG
0.050


 


300
LVLLtVFLLL
0.050


 


295
LVLLlLVLLT
0.050


 


301
VLLTvFLLLV
0.050


 


299
LLVLlTVFLL
0.050


 


261
EPLEkPSRPK
0.050


 


277
VNPLkTFVFF
0.050


 


109
FALEeAEFRQ
0.050


 


81
GNFNwRFVFR
0.050


 


296
VLLLlVLLTV
0.050


 


314
IPGQiSQVIF
0.050


 


226
GGNVyILTGK
0.050


 


131
RISAnDFLGS
0.050


 


97
EREVsVWRRS
0.045


 


239
EFELlTVEEA
0.045


 


111
LEEAeFRQPA
0.045


 


41
DDENpLTGEM
0.045


 


195
EREAqEAQAG
0.045


 


178
RLRGwWPVVK
0.040


 


24
ISYElRVVIW
0.030


 


139
GSLElQLPDM
0.030


 


318
ISQViFRPLH
0.030


 


224
DMGGnVYILT
0.025


 


165
NGAGpRCNLF
0.025


 


282
TFVFfIWRRY
0.025


 


280
LKTFvFFIWR
0.025


 


82
NFNWrFVFRF
0.025


 


171
CNLFrCRRLR
0.025


 


126
VWDYdRISAN
0.025


 


128
DYDRiSANDF
0.025


 


141
LELQlPDMVR
0.025


 


35
TEDVvLDDEN
0.025


 


74
FNSLtGEGNF
0.025


 


221
EFTDmGGNVY
0.025


 


294
TLVLlLLVLL
0.020


 


38
VVLDdENPLT
0.020


 


142
ELQLpDMVRG
0.020


 


53
DIYVkSWVKG
0.020


 


246
EEAEkRPVGK
0.020


 


187
KLKEaEDVER
0.020


 


272
SFNWfVNPLK
0.020


 


298
LLLVlLTVFL
0.020
















TABLE X







158P3D2v.1-A0201-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












302
LLTVFLLLV
1033.404


297
LLLLVLLTV
1006.209


286
FIWRRYWRT
440.113


306
FLLLVFYTI
337.376


301
VLLTVFLLL
255.302


299
LLVLLTVFL
199.738


300
LVLLTVFLL
156.843


276
FVNPLKTFV
153.971


296
VLLLLVLLT
107.808


137
FLGSLELQL
98.267


2
WIDIFPQDV
66.867


38
VVLDDENPL
48.205


48
GEMSSDIYV
27.521


31
VIWNTEDVV
27.109


295
LVLLLLVLL
27.042


313
TIPGQISQV
21.996


39
VLDDENPLT
20.776


294
TLVLLLLVL
20.145


230
YILTGKVEA
11.626


144
QLPDMVRGA
9.370


293
RTLVLLLLV
8.221


30
VVIWNTEDV
5.069


141
LELQLPDMV
4.168


236
VEAEFELLT
3.838


178
RLRGWWPVV
3.684


94
LPTEREVSV
3.165


180
RGWWPVVKL
2.662


228
NVYILTGKV
2.532


305
VFLLLVFYT
2.388


279
PLKTFVFFI
2.240


121
VLVLQVWDY
2.185


240
FELLTVEEA
1.853


133
SANDFLGSL
1.382


124
LQVWDYDRI
1.322


224
DMGGNVYIL
1.091


118
QPAVLVLQV
1.044


46
LTGEMSSDI
1.010


83
FNWRFVFRF
0.941


27
ELRVVIWNT
0.733


140
SLELQLPDM
0.731


234
GKVEAEFEL
0.706


55
YVKSWVKGL
0.692


114
AEFRQPAVL
0.630


24
ISYELRVVI
0.623


52
SDIYVKSWV
0.531


62
GLEHDKQET
0.477


177
RRLRGWWPV
0.456


22
QPISYELRV
0.454


298
LLLVLLTVF
0.442


159
SVQLARNGA
0.435


76
SLTGEGNFN
0.410


235
KVEAEFELL
0.390


183
WPVVKLKEA
0.343


269
PKTSFNWFV
0.333


26
YELRVVIWN
0.312


304
TVFLLLVFY
0.305


186
VKLKEAEDV
0.298


223
TDMGGNVYI
0.295


307
LLLVFYTIP
0.219


4
DIFPQDVPA
0.190


165
NGAGPRCNL
0.139


272
SFNWFVNPL
0.130


308
LLVFYTIPG
0.127


225
MGGNVYILT
0.124


10
VPAPPPVDI
0.116


112
EEAEFRQPA
0.113


135
NDFLGSLEL
0.110


143
LQLPDMVRG
0.109


281
KTFVFFIWR
0.106


171
CNLFRCRRL
0.103


8
QDVPAPPPV
0.097


318
ISQVIFRPL
0.090


87
FVFRFDYLP
0.084


86
RFVFRFDYL
0.076


93
YLPTEREVS
0.069


80
EGNFNWRFV
0.064


131
RISANDFLG
0.059


290
RYWRTLVLL
0.057


314
IPGQISQVI
0.047


77
LTGEGNFNW
0.042


79
GEGNFNWRF
0.041


23
PISYELRVV
0.040


70
TDVHFNSLT
0.039


109
FALEEAEFR
0.039


283
FVFFIWRRY
0.038


122
LVLQVWDYD
0.038


106
SGPFALEEA
0.037


68
QETDVHFNS
0.034


168
GPRCNLFRC
0.033


292
WRTLVLLLL
0.031


245
VEEAEKRPV
0.029


319
SQVIFRPLH
0.029


231
ILTGKVEAE
0.029


317
QISQVIFRP
0.027


120
AVLVLQVWD
0.027


215
GRPEDLEFT
0.026


242
LLTVEEAEK
0.025


123
VLQVWDYDR
0.025


16
VDIKPRQPI
0.025


258
KQPEPLEKP
0.024
















TABLE XI







158P3D2v.1-A0201-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












301
VLLTvFLLLV
3823.593


296
VLLLlVLLTV
1006.209


298
LLLVlLTVFL
739.032


299
LLVLlTVFLL
484.457


93
YLPTeREVSV
319.939


304
TVFLlLVFYT
177.011


278
NPLKtFVFFI
70.254


294
TLVLlLLVLL
49.134


26
YELRvVIWNT
42.542


286
FIWRrYWRTL
38.130


300
LVLLtVFLLL
22.339


236
VEAEfELLTV
21.680


101
SVWRrSGPFA
19.844


31
VIWNtEDVVL
16.993


38
VVLDdENPLT
16.816


87
FVFRfDYLPT
16.647


117
RQPAvLVLQV
16.219


125
QVWDyDRISA
14.793


123
VLQVwDYDRI
13.036


312
YTIPgQISQV
10.220


295
LVLLlLVLLT
9.433


63
LEHDkQETDV
9.426


21
RQPIsYELRV
7.052


114
AEFRqPAVLV
5.004


271
TSFNwFVNPL
4.510


68
QETDvHFNSL
3.236


29
RVVIwNTEDV
2.982


61
KGLEhDKQET
2.583


79
GEGNfNWRFV
2.529


268
RPKTsFNWFV
2.491


140
SLELqLPDMV
2.181


30
VVIWnTEDVV
2.078


273
FNWFvNPLKT
1.857


222
FTDMgGNVYI
1.466


143
LQLPdMVRGA
1.457


275
WFVNpLKTFV
1.222


139
GSLElQLPDM
1.132


317
QISQvIFRPL
1.116


220
LEFTdMGGNV
1.106


293
RTLVlLLLVL
1.035


51
SSDIyVKSWV
0.999


309
LVFYtIPGQI
0.746


224
DMGGnVYILT
0.605


306
FLLLvFYTIP
0.593


313
TIPGqISQVI
0.588


153
RGPElCSVQL
0.572


235
KVEAeFELLT
0.555


307
LLLVfYTIPG
0.469


297
LLLLvLLTVF
0.442


167
AGPRcNLFRC
0.433


76
SLTGeGNFNW
0.432


120
AVLVlQVWDY
0.416


112
EEAEfRQPAV
0.416


244
TVEEaEKRPV
0.319


91
FDYLpTEREV
0.284


189
KEAEdVEREA
0.277


172
NLFRcRRLRG
0.276


132
ISANdFLGSL
0.269


285
FFIWrRYWRT
0.268


85
WRFVfRFDYL
0.259


1
MWIDiFPQDV
0.256


148
MVRGaRGPEL
0.242


45
PLTGeMSSDI
0.230


39
VLDDeNPLTG
0.208


185
VVKLkEAEDV
0.177


281
KTFVfFIWRR
0.176


151
GARGpELCSV
0.169


47
TGEMsSDIYV
0.160


137
FLGSlELQLP
0.158


37
DVVLdDENPL
0.140


164
RNGAgPRCNL
0.139


231
ILTGkVEAEF
0.127


283
FVFFiWRRYW
0.122


302
LLTVfLLLVF
0.119


121
VLVLqVWDYD
0.116


234
GKVEaEFELL
0.113


258
KQPEpLEKPS
0.108


223
TDMGgNVYIL
0.104


292
WRTLvLLLLV
0.102


305
VFLLlVFYTI
0.087


22
QPISyELRVV
0.086


109
FALEeAEFRQ
0.084


214
KGRPeDLEFT
0.080


276
FVNPlKTFVF
0.071


9
DVPApPPVDI
0.068


7
PQDVpAPPPV
0.062


227
GNVYiLTGKV
0.059


308
LLVFyTIPGQ
0.058


290
RYWRtLVLLL
0.057


134
ANDFlGSLEL
0.056


194
VEREaQEAQA
0.051


111
LEEAeFRQPA
0.040


230
YILTgKVEAE
0.039


19
KPRQpISYEL
0.037


105
RSGPfALEEA
0.037


158
CSVQlARNGA
0.032


233
TGKVeAEFEL
0.028


129
YDRIsANDFL
0.028


170
RCNLfRCRRL
0.028


177
RRLRgWWPVV
0.025
















TABLE XII







158P3D2v.1-A3-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












281
KTFVFFIWR
54.000


121
VLVLQVWDY
54.000


123
VLQVWDYDR
36.000


49
EMSSDIYVK
27.000


242
LLTVEEAEK
20.000


306
FLLLVFYTI
12.150


53
DIYVKSWVK
9.000


301
VLLTVFLLL
8.100


320
QVIFRPLHK
6.000


298
LLLVLLTVF
4.500


142
ELQLPDMVR
3.600


156
ELCSVQLAR
3.600


316
GQISQVIFR
3.240


59
WVKGLEHDK
3.000


304
TVFLLLVFY
3.000


294
TLVLLLLVL
2.700


224
DMGGNVYIL
2.430


172
NLFRCRRLR
2.000


302
LLTVFLLLV
1.800


279
PLKTFVFFI
1.620


297
LLLLVLLTV
1.350


137
FLGSLELQL
1.200


181
GWWPVVKLK
1.013


299
LLVLLTVFL
0.900


296
VLLLLVLLT
0.900


178
RLRGWWPVV
0.900


300
LVLLTVFLL
0.810


81
GNFNWRFVF
0.540


235
KVEAEFELL
0.540


83
FNWRFVFRF
0.540


303
LTVFLLLVF
0.450


243
LTVEEAEKR
0.450


201
AQAGKKKRK
0.450


227
GNVYILTGK
0.405


62
GLEHDKQET
0.300


273
FNWFVNPLK
0.300


262
PLEKPSRPK
0.300


283
FVFFIWRRY
0.300


101
SVWRRSGPF
0.300


140
SLELQLPDM
0.300


55
YVKSWVKGL
0.270


27
ELRVVIWNT
0.203


222
FTDMGGNVY
0.200


85
WRFVFRFDY
0.180


308
LLVFYTIPG
0.180


198
AQEAQAGKK
0.180


79
GEGNFNWRF
0.162


286
FIWRRYWRT
0.150


232
LTGKVEAEF
0.150


295
LVLLLLVLL
0.135


11
PAPPPVDIK
0.135


21
RQPISYELR
0.120


170
RCNLFRCRR
0.120


31
VIWNTEDVV
0.100


39
VLDDENPLT
0.100


278
NPLKTFVFF
0.090


187
KLKEAEDVE
0.090


231
ILTGKVEAE
0.090


265
KPSRPKTSF
0.090


87
FVFRFDYLP
0.090


110
ALEEAEFRQ
0.090


307
LLLVFYTIP
0.090


38
VVLDDENPL
0.090


166
GAGPRCNLF
0.090


109
FALEEAEFR
0.090


197
EAQEAQAGK
0.090


282
TFVFFIWRR
0.081


179
LRGWWPVVK
0.060


257
RKQPEPLEK
0.060


144
QLPDMVRGA
0.060


268
RPKTSFNWF
0.060


247
EAEKRPVGK
0.060


2
WIDIFPQDV
0.060


46
LTGEMSSDI
0.045


293
RTLVLLLLV
0.045


4
DIFPQDVPA
0.045


77
LTGEGNFNW
0.045


313
TIPGQISQV
0.045


93
YLPTEREVS
0.040


230
YILTGKVEA
0.030


76
SLTGEGNFN
0.030


228
NVYILTGKV
0.030


57
KSWVKGLEH
0.030


276
FVNPLKTFV
0.030


30
VVIWNTEDV
0.030


199
QEAQAGKKK
0.030


69
ETDVHFNSL
0.027


319
SQVIFRPLH
0.027


168
GPRCNLFRC
0.027


124
LQVWDYDRI
0.027


96
TEREVSVWR
0.027


24
ISYELRVVI
0.022


159
SVQLARNGA
0.020


161
QLARNGAGP
0.020


285
FFIWRRYWR
0.018


250
KRPVGKGRK
0.018


214
KGRPEDLEF
0.018


78
TGEGNFNWR
0.018


154
GPELCSVQL
0.018


22
QPISYELRV
0.018
















TABLE XIII







158P3D2v.1-A3-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












178
RLRGwWPVVK
90.000


281
KTFVfFIWRR
40.500


187
KLKEaEDVER
18.000


241
ELLTvEEAEK
9.000


299
LLVLlTVFLL
8.100


302
LLTVfLLLVF
6.000


122
LVLQvWDYDR
5.400


120
AVLVlQVWDY
5.400


297
LLLLvLLTVF
4.500


231
ILTGkVEAEF
4.500


242
LLTVeEAEKR
4.000


301
VLLTvFLLLV
2.700


144
QLPDmVRGAR
1.800


319
SQVIfRPLHK
1.800


296
VLLLlVLLTV
1.350


294
TLVLlLLVLL
1.350


10
VPAPpPVDIK
1.350


48
GEMSsDIYVK
1.215


161
QLARnGAGPR
1.200


298
LLLVlLTVFL
0.900


77
LTGEgNFNWR
0.900


276
FVNPlKTFVF
0.900


76
SLTGeGNFNW
0.900


300
LVLLtVFLLL
0.810


123
VLQVwDYDRI
0.600


303
LTVFlLLVFY
0.450


304
TVFLlLVFYT
0.450


81
GNFNwRFVFR
0.360


17
DIKPrQPISY
0.360


166
GAGPrCNLFR
0.360


31
VIWNtEDVVL
0.300


107
GPFAlEEAEF
0.300


46
LTGEmSSDIY
0.300


198
AQEAqAGKKK
0.300


279
PLKTfVFFIW
0.270


278
NPLKtFVFFI
0.243


180
RGWWpVVKLK
0.225


93
YLPTeREVSV
0.200


140
SLELqLPDMV
0.200


172
NLFRcRRLRG
0.200


125
QVWDyDRISA
0.200


307
LLLVfYTIPG
0.180


235
KVEAeFELLT
0.180


96
TEREvSVWRR
0.162


226
GGNVyILTGK
0.135


293
RTLVlLLLVL
0.135


309
LVFYtIPGQI
0.135


224
DMGGnVYILT
0.135


271
TSFNwFVNPL
0.135


313
TIPGqISQVI
0.135


256
GRKQpEPLEK
0.120


87
FVFRfDYLPT
0.100


101
SVWRrSGPFA
0.100


52
SDIYvKSWVK
0.090


295
LVLLlLVLLT
0.090


148
MVRGaRGPEL
0.090


306
FLLLvFYTIP
0.090


45
PLTGeMSSDI
0.090


286
FIWRrYWRTL
0.090


19
KPRQpISYEL
0.081


280
LKTFvFFIWR
0.072


62
GLEHdKQETD
0.060


259
QPEPlEKPSR
0.060


284
VFFIwRRYWR
0.060


196
REAQeAQAGK
0.060


82
NFNWrFVFRF
0.054


141
LELQlPDMVR
0.054


121
VLVLqVWDYD
0.045


308
LLVFyTIPGQ
0.045


12
APPPvDIKPR
0.045


39
VLDDeNPLTG
0.040


84
NWRFvFRFDY
0.036


168
GPRCnLFRCR
0.036


117
RQPAvLVLQV
0.036


21
RQPIsYELRV
0.036


312
YTIPgQISQV
0.034


272
SFNWfVNPLK
0.030


58
SWVKgLEHDK
0.030


200
EAQAgKKKRK
0.030


30
VVIWnTEDVV
0.030


283
FVFFiWRRYW
0.030


137
FLGSlELQLP
0.030


222
FTDMgGNVYI
0.030


29
RVVIwNTEDV
0.030


95
PTEReVSVWR
0.030


37
DVVLdDENPL
0.027


78
TGEGnFNWRF
0.027


9
DVPApPPVDI
0.027


317
QISQvIFRPL
0.027


270
KTSFnWFVNP
0.027


246
EEAEkRPVGK
0.027


197
EAQEaQAGKK
0.027


131
RISAnDFLGS
0.024


24
ISYElRVVIW
0.022


261
EPLEkPSRPK
0.020


314
IPGQiSQVIF
0.020


202
QAGKkKRKQR
0.020


89
FRFDyLPTER
0.020


185
VVKLkEAEDV
0.020


316
GQISqVIFRP
0.018
















TABLE XIV







158P3D2v.1-A1101-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












320
QVIFRPLHK
6.000


281
KTFVFFIWR
2.400


59
WVKGLEHDK
2.000


316
GQISQVIFR
1.080


198
AQEAQAGKK
0.600


53
DIYVKSWVK
0.480


242
LLTVEEAEK
0.400


21
RQPISYELR
0.360


243
LTVEEAEKR
0.300


201
AQAGKKKRK
0.300


49
EMSSDIYVK
0.240


227
GNVYILTGK
0.180


123
VLQVWDYDR
0.160


257
RKQPEPLEK
0.120


90
RFDYLPTER
0.120


282
TFVFFIWRR
0.120


170
RCNLFRCRR
0.120


285
FFIWRRYWR
0.120


293
RTLVLLLLV
0.090


300
LVLLTVFLL
0.090


273
FNWFVNPLK
0.080


181
GWWPVVKLK
0.060


250
KRPVGKGRK
0.060


109
FALEEAEFR
0.060


247
EAEKRPVGK
0.060


197
EAQEAQAGK
0.060


235
KVEAEFELL
0.060


142
ELQLPDMVR
0.048


156
ELCSVQLAR
0.048


145
LPDMVRGAR
0.040


304
TVFLLLVFY
0.040


82
NFNWRFVFR
0.040


101
SVWRRSGPF
0.040


228
NVYILTGKV
0.040


162
LARNGAGPR
0.040


295
LVLLLLVLL
0.030


77
LTGEGNFNW
0.030


199
QEAQAGKKK
0.030


303
LTVFLLLVF
0.030


38
VVLDDENPL
0.030


30
VVIWNTEDV
0.030


290
RYWRTLVLL
0.024


276
FVNPLKTFV
0.020


179
LRGWWPVVK
0.020


11
PAPPPVDIK
0.020


159
SVQLARNGA
0.020


172
NLFRCRRLR
0.016


204
GKKKRKQRR
0.012


306
FLLLVFYTI
0.012


301
VLLTVFLLL
0.012


121
VLVLQVWDY
0.012


96
TEREVSVWR
0.012


178
RLRGWWPVV
0.012


297
LLLLVLLTV
0.012


294
TLVLLLLVL
0.012


232
LTGKVEAEF
0.010


222
FTDMGGNVY
0.010


55
YVKSWVKGL
0.010


46
LTGEMSSDI
0.010


29
RVVIWNTED
0.009


124
LQVWDYDRI
0.009


270
KTSFNWFVN
0.009


86
RFVFRFDYL
0.009


319
SQVIFRPLH
0.009


302
LLTVFLLLV
0.008


87
FVFRFDYLP
0.008


137
FLGSLELQL
0.008


167
AGPRCNLFR
0.008


31
VIWNTEDVV
0.008


81
GNFNWRFVF
0.007


48
GEMSSDIYV
0.007


208
RKQRRRKGR
0.006


206
KKRKQRRRK
0.006


154
GPELCSVQL
0.006


230
YILTGKVEA
0.006


22
QPISYELRV
0.006


299
LLVLLTVFL
0.006


193
DVEREAQEA
0.006


298
LLLVLLTVF
0.006


265
KPSRPKTSF
0.006


166
GAGPRCNLF
0.006


200
EAQAGKKKR
0.006


175
RCRRLRGWW
0.006


268
RPKTSFNWF
0.006


262
PLEKPSRPK
0.004


25
SYELRVVIW
0.004


2
WIDIFPQDV
0.004


78
TGEGNFNWR
0.004


188
LKEAEDVER
0.004


309
LVFYTIPGQ
0.004


118
QPAVLVLQV
0.004


313
TIPGQISQV
0.004


283
FVFFIWRRY
0.004


310
VFYTIPGQI
0.004


140
SLELQLPDM
0.004


131
RISANDFLG
0.004


79
GEGNFNWRF
0.004


312
YTIPGQISQ
0.003


278
NPLKTFVFF
0.003


120
AVLVLQVWD
0.003
















TABLE XV







158P3D2v.1-A1101-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












281
KTFVfFIWRR
2.400


319
SQVIfRPLHK
1.800


122
LVLQvWDYDR
1.200


178
RLRGwWPVVK
1.200


48
GEMSsDIYVK
0.720


198
AQEAqAGKKK
0.300


166
GAGPrCNLFR
0.240


187
KLKEaEDVER
0.240


272
SFNWfVNPLK
0.200


10
VPAPpPVDIK
0.200


77
LTGEgNFNWR
0.200


241
ELLTvEEAEK
0.180


196
REAQeAQAGK
0.180


284
VFFIwRRYWR
0.160


256
GRKQpEPLEK
0.120


29
RVVIwNTEDV
0.090


293
RTLVlLLLVL
0.090


125
QVWDyDRISA
0.080


144
QLPDmVRGAR
0.080


161
QLARnGAGPR
0.080


242
LLTVeEAEKR
0.080


226
GGNVyILTGK
0.060


180
RGWWpVVKLK
0.060


52
SDIYvKSWVK
0.060


120
AVLVlQVWDY
0.060


300
LVLLtVFLLL
0.060


197
EAQEaQAGKK
0.060


276
FVNPlKTFVF
0.060


81
GNFNwRFVFR
0.048


290
RYWRtLVLLL
0.048


101
SVWRrSGPFA
0.040


259
QPEPlEKPSR
0.040


309
LVFYtIPGQI
0.040


21
RQPIsYELRV
0.036


141
LELQlPDMVR
0.036


117
RQPAvLVLQV
0.036


58
SWVKgLEHDK
0.030


200
EAQAgKKKRK
0.030


30
VVIWnTEDVV
0.030


96
TEREvSVWRR
0.024


12
APPPvDIKPR
0.020


148
MVRGaRGPEL
0.020


202
QAGKkKRKQR
0.020


185
VVKLkEAEDV
0.020


95
PTEReVSVWR
0.020


299
LLVLlTVFLL
0.018


246
EEAEkRPVGK
0.018


303
LTVFlLLVFY
0.015


312
YTIPgQISQV
0.015


168
GPRCnLFRCR
0.012


235
KVEAeFELLT
0.012


19
KPRQpISYEL
0.012


304
TVFLlLVFYT
0.012


296
VLLLlVLLTV
0.012


107
GPFAlEEAEF
0.012


76
SLTGeGNFNW
0.012


301
VLLTvFLLLV
0.012


268
RPKTsFNWFV
0.012


222
FTDMgGNVYI
0.010


46
LTGEmSSDIY
0.010


37
DVVLdDENPL
0.009


261
EPLEkPSRPK
0.009


278
NPLKtFVFFI
0.009


316
GQISqVIFRP
0.008


280
LKTFvFFIWR
0.008


87
FVFRfDYLPT
0.008


302
LLTVfLLLVF
0.008


89
FRFDyLPTER
0.008


31
VIWNtEDVVL
0.008


207
KRKQrRRKGR
0.006


205
KKKRkQRRRK
0.006


216
RPEDlEFTDM
0.006


249
EKRPvGKGRK
0.006


294
TLVLlLLVLL
0.006


305
VFLLlVFYTI
0.006


82
NFNWrFVFRF
0.006


297
LLLLvLLTVF
0.006


199
QEAQaGKKKR
0.006


295
LVLLlLVLLT
0.006


154
GPELcSVQLA
0.006


151
GARGpELCSV
0.006


9
DVPApPPVDI
0.006


298
LLLVlLTVFL
0.006


248
AEKRpVGKGR
0.006


229
VYILtGKVEA
0.006


67
KQETdVHFNS
0.005


123
VLQVwDYDRI
0.004


93
YLPTeREVSV
0.004


283
FVFFiWRRYW
0.004


203
AGKKkRKQRR
0.004


231
ILTGkVEAEF
0.004


108
PFALeEAEFR
0.004


140
SLELqLPDMV
0.004


313
TIPGqISQVI
0.004


155
PELCsVQLAR
0.004


38
VVLDdENPLT
0.003


275
WFVNpLKTFV
0.003


270
KTSFnWFVNP
0.003


54
IYVKsWVKGL
0.003


131
RISAnDFLGS
0.002
















TABLE XVI







158P3D2v.1-A24-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












290
RYWRTLVLL
400.000


86
RFVFRFDYL
72.000


272
SFNWFVNPL
36.000


275
WFVNPLKTF
15.000


235
KVEAEFELL
14.400


318
ISQVIFRPL
10.080


301
VLLTVFLLL
10.080


92
DYLPTEREV
9.900


180
RGWWPVVKL
8.800


38
VVLDDENPL
8.640


255
KGRKQPEPL
8.000


25
SYELRVVIW
7.500


299
LLVLLTVFL
7.200


294
TLVLLLLVL
7.200


295
LVLLLLVLL
7.200


133
SANDFLGSL
7.200


310
VFYTIPGQI
7.000


171
CNLFRCRRL
6.000


32
IWNTEDVVL
6.000


154
GPELCSVQL
6.000


311
FYTIPGQIS
6.000


300
LVLLTVFLL
6.000


268
RPKTSFNWF
5.760


69
ETDVHFNSL
5.760


214
KGRPEDLEF
5.280


137
FLGSLELQL
4.800


291
YWRTLVLLL
4.800


55
YVKSWVKGL
4.000


224
DMGGNVYIL
4.000


165
NGAGPRCNL
4.000


287
IWRRYWRTL
4.000


265
KPSRPKTSF
4.000


303
LTVFLLLVF
3.600


298
LLLVLLTVF
3.600


278
NPLKTFVFF
3.600


232
LTGKVEAEF
3.080


277
VNPLKTFVF
3.000


75
NSLTGEGNF
3.000


166
GAGPRCNLF
2.880


306
FLLLVFYTI
2.520


83
FNWRFVFRF
2.000


101
SVWRRSGPF
2.000


81
GNFNWRFVF
2.000


314
IPGQISQVI
1.680


124
LQVWDYDRI
1.500


24
ISYELRVVI
1.440


10
VPAPPPVDI
1.200


46
LTGEMSSDI
1.200


108
PFALEEAEF
1.100


305
VFLLLVFYT
0.900


54
IYVKSWVKG
0.825


289
RRYWRTLVL
0.800


212
RRKGRPEDL
0.800


234
GKVEAEFEL
0.792


229
VYILTGKVE
0.750


140
SLELQLPDM
0.750


116
FRQPAVLVL
0.720


128
DYDRISAND
0.700


221
EFTDMGGNV
0.600


130
DRISANDFL
0.600


292
WRTLVLLLL
0.560


115
EFRQPAVLV
0.500


88
VFRFDYLPT
0.500


284
VFFIWRRYW
0.500


149
VRGARGPEL
0.440


135
NDFLGSLEL
0.440


114
AEFRQPAVL
0.400


103
WRRSGPFAL
0.400


66
DKQETDVHF
0.360


293
RTLVLLLLV
0.360


315
PGQISQVIF
0.300


67
KQETDVHFN
0.300


190
EAEDVEREA
0.277


129
YDRISANDF
0.240


175
RCRRLRGWW
0.240


259
QPEPLEKPS
0.216


276
FVNPLKTFV
0.216


297
LLLLVLLTV
0.210


150
RGARGPELC
0.200


270
KTSFNWFVN
0.200


164
RNGAGPRCN
0.200


79
GEGNFNWRF
0.200


178
RLRGWWPVV
0.200


193
DVEREAQEA
0.198


62
GLEHDKQET
0.198


50
MSSDIYVKS
0.185


16
VDIKPRQPI
0.180


296
VLLLLVLLT
0.180


159
SVQLARNGA
0.180


144
QLPDMVRGA
0.180


106
SGPFALEEA
0.165


183
WPVVKLKEA
0.165


230
YILTGKVEA
0.165


80
EGNFNWRFV
0.150


237
EAEFELLTV
0.150


43
ENPLTGEMS
0.150


44
NPLTGEMSS
0.150


30
VVIWNTEDV
0.150


223
TDMGGNVYI
0.150


22
QPISYELRV
0.150
















TABLE XVII







158P3D2v.1-A24-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












290
RYWRtLVLLL
480.000


54
IYVKsWVKGL
300.000


128
DYDRiSANDF
120.000


136
DFLGsLELQL
36.000


115
EFRQpAVLVL
20.000


82
NFNWrFVFRF
15.000


153
RGPElCSVQL
14.400


293
RTLVlLLLVL
14.400


305
VFLLlVFYTI
12.600


19
KPRQpISYEL
12.320


170
RCNLfRCRRL
12.000


25
SYELrVVIWN
10.500


300
LVLLtVFLLL
10.080


92
DYLPtEREVS
9.000


229
VYILtGKVEA
8.250


164
RNGAgPRCNL
8.000


37
DVVLdDENPL
7.200


294
TLVLlLLVLL
7.200


298
LLLVlLTVFL
7.200


317
QISQvIFRPL
6.720


299
LLVLlTVFLL
6.000


113
EAEFrQPAVL
6.000


291
YWRTlVLLLL
5.600


271
TSFNwFVNPL
4.800


134
ANDFlGSLEL
4.400


233
TGKVeAEFEL
4.400


148
MVRGaRGPEL
4.400


31
VIWNtEDVVL
4.000


286
FIWRrYWRTL
4.000


132
ISANdFLGSL
4.000


102
VWRRsGPFAL
4.000


277
VNPLkTFVFF
3.600


276
FVNPlKTFVF
3.600


297
LLLLvLLTVF
3.600


231
ILTGkVEAEF
3.080


80
EGNFnWRFVF
3.000


78
TGEGnFNWRF
3.000


100
VSVWrRSGPF
3.000


313
TIPGqISQVI
2.520


302
LLTVfLLLVF
2.400


165
NGAGpRCNLF
2.400


107
GPFAlEEAEF
2.200


216
RPEDlEFTDM
2.160


314
IPGQiSQVIF
2.000


74
FNSLtGEGNF
2.000


274
NWFVnPLKTF
2.000


9
DVPApPPVDI
1.500


278
NPLKtFVFFI
1.500


123
VLQVwDYDRI
1.500


309
LVFYtIPGQI
1.400


282
TFVFfIWRRY
1.050


222
FTDMgGNVYI
1.000


275
WFVNpLKTFV
0.900


139
GSLElQLPDM
0.900


234
GKVEaEFELL
0.864


239
EFELlTVEEA
0.825


211
RRRKgRPEDL
0.800


289
RRYWrTLVLL
0.800


285
FFIWrRYWRT
0.750


73
HFNSlTGEGN
0.750


221
EFTDmGGNVY
0.720


68
QETDvHFNSL
0.691


223
TDMGgNVYIL
0.600


310
VFYTiPGQIS
0.600


311
FYTIpGQISQ
0.500


173
LFRCrRLRGW
0.500


85
WRFVfRFDYL
0.480


61
KGLEhDKQET
0.475


213
RKGRpEDLEF
0.440


179
LRGWwPVVKL
0.440


267
SRPKtSFNWF
0.432


258
KQPEpLEKPS
0.432


67
KQETdVHFNS
0.420


129
YDRIsANDFL
0.400


254
GKGRkQPEPL
0.400


288
WRRYwRTLVL
0.400


117
RQPAvLVLQV
0.360


29
RVVIwNTEDV
0.300


235
KVEAeFELLT
0.300


264
EKPSrPKTSF
0.300


21
RQPIsYELRV
0.300


105
RSGPfALEEA
0.264


214
KGRPeDLEFT
0.240


131
RISAnDFLGS
0.240


1
MWIDiFPQDV
0.216


296
VLLLlVLLTV
0.210


268
RPKTsFNWFV
0.200


265
KPSRpKTSFN
0.200


65
HDKQeTDVHF
0.200


150
RGARgPELCS
0.200


227
GNVYiLTGKV
0.198


154
GPELcSVQLA
0.180


303
LTVFlLLVFY
0.180


38
VVLDdENPLT
0.180


312
YTIPgQISQV
0.180


158
CSVQlARNGA
0.180


143
LQLPdMVRGA
0.180


295
LVLLlLVLLT
0.180


244
TVEEaEKRPV
0.180


75
NSLTgEGNFN
0.180
















TABLE XVIII







158P3D2v.1-B7-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












255
KGRKQPEPL
40.000


154
GPELCSVQL
24.000


300
LVLLTVFLL
20.000


55
YVKSWVKGL
20.000


168
GPRCNLFRC
20.000


295
LVLLLLVLL
20.000


38
VVLDDENPL
20.000


133
SANDFLGSL
12.000


10
VPAPPPVDI
12.000


165
NGAGPRCNL
9.000


314
IPGQISQVI
8.000


180
RGWWPVVKL
6.000


235
KVEAEFELL
6.000


294
TLVLLLLVL
4.000


94
LPTEREVSV
4.000


22
QPISYELRV
4.000


301
VLLTVFLLL
4.000


291
YWRTLVLLL
4.000


318
ISQVIFRPL
4.000


103
WRRSGPFAL
4.000


299
LLVLLTVFL
4.000


118
QPAVLVLQV
4.000


137
FLGSLELQL
4.000


287
IWRRYWRTL
4.000


171
CNLFRCRRL
4.000


224
DMGGNVYIL
4.000


19
KPRQPISYE
3.000


178
RLRGWWPVV
2.000


183
WPVVKLKEA
2.000


114
AEFRQPAVL
1.200


69
ETDVHFNSL
1.200


276
FVNPLKTFV
1.000


27
ELRVVIWNT
1.000


30
VVIWNTEDV
1.000


228
NVYILTGKV
1.000


151
GARGPELCS
0.900


159
SVQLARNGA
0.750


148
MVRGARGPE
0.750


24
ISYELRVVI
0.600


265
KPSRPKTSF
0.600


12
APPPVDIKP
0.600


292
WRTLVLLLL
0.400


32
IWNTEDVVL
0.400


289
RRYWRTLVL
0.400


149
VRGARGPEL
0.400


46
LTGEMSSDI
0.400


306
FLLLVFYTI
0.400


272
SFNWFVNPL
0.400


234
GKVEAEFEL
0.400


278
NPLKTFVFF
0.400


130
DRISANDFL
0.400


86
RFVFRFDYL
0.400


135
NDFLGSLEL
0.400


44
NPLTGEMSS
0.400


212
RRKGRPEDL
0.400


268
RPKTSFNWF
0.400


290
RYWRTLVLL
0.400


116
FRQPAVLVL
0.400


124
LQVWDYDRI
0.400


140
SLELQLPDM
0.300


288
WRRYWRTLV
0.300


162
LARNGAGPR
0.300


115
EFRQPAVLV
0.300


175
RCRRLRGWW
0.300


214
KGRPEDLEF
0.200


80
EGNFNWRFV
0.200


302
LLTVFLLLV
0.200


297
LLLLVLLTV
0.200


261
EPLEKPSRP
0.200


107
GPFALEEAE
0.200


31
VIWNTEDVV
0.200


313
TIPGQISQV
0.200


251
RPVGKGRKQ
0.200


293
RTLVLLLLV
0.200


6
FPQDVPAPP
0.200


237
EAEFELLTV
0.180


113
EAEFRQPAV
0.180


193
DVEREAQEA
0.150


120
AVLVLQVWD
0.150


259
QPEPLEKPS
0.120


223
TDMGGNVYI
0.120


283
FVFFIWRRY
0.100


106
SGPFALEEA
0.100


101
SVWRRSGPF
0.100


150
RGARGPELC
0.100


304
TVFLLLVFY
0.100


42
DENPLTGEM
0.100


296
VLLLLVLLT
0.100


125
QVWDYDRIS
0.100


225
MGGNVYILT
0.100


144
QLPDMVRGA
0.100


102
VWRRSGPFA
0.100


4
DIFPQDVPA
0.100


88
VFRFDYLPT
0.100


209
KQRRRKGRP
0.100


286
FIWRRYWRT
0.100


230
YILTGKVEA
0.100


16
VDIKPRQPI
0.090


145
LPDMVRGAR
0.090


190
EAEDVEREA
0.090
















TABLE XIX







158P3D2v.1-B7-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












19
KPRQpISYEL
800.000


148
MVRGaRGPEL
200.000


37
DVVLdDENPL
20.000


300
LVLLtVFLLL
20.000


164
RNGAgPRCNL
9.000


278
NPLKtFVFFI
8.000


151
GARGpELCSV
6.000


216
RPEDlEFTDM
6.000


31
VIWNtEDVVL
4.000


298
LLLVlLTVFL
4.000


294
TLVLlLLVLL
4.000


129
YDRIsANDFL
4.000


132
ISANdFLGSL
4.000


288
WRRYwRTLVL
4.000


170
RCNLfRCRRL
4.000


22
QPISyELRVV
4.000


115
EFRQpAVLVL
4.000


153
RGPElCSVQL
4.000


293
RTLVlLLLVL
4.000


291
YWRTlVLLLL
4.000


286
FIWRrYWRTL
4.000


271
TSFNwFVNPL
4.000


233
TGKVeAEFEL
4.000


268
RPKTsFNWFV
4.000


299
LLVLlTVFLL
4.000


211
RRRKgRPEDL
4.000


102
VWRRsGPFAL
4.000


317
QISQvIFRPL
4.000


134
ANDFlGSLEL
3.600


113
EAEFrQPAVL
3.600


9
DVPApPPVDI
3.000


162
LARNgAGPRC
3.000


309
LVFYtIPGQI
2.000


168
GPRCnLFRCR
2.000


223
TDMGgNVYIL
1.200


30
VVIWnTEDVV
1.000


29
RVVIwNTEDV
1.000


214
KGRPeDLEFT
1.000


185
VVKLkEAEDV
1.000


139
GSLElQLPDM
1.000


125
QVWDyDRISA
0.750


12
APPPvDIKPR
0.600


154
GPELcSVQLA
0.600


179
LRGWwPVVKL
0.600


295
LVLLlLVLLT
0.500


38
VVLDdENPLT
0.500


87
FVFRfDYLPT
0.500


101
SVWRrSGPFA
0.500


304
TVFLlLVFYT
0.500


54
IYVKsWVKGL
0.400


313
TIPGqISQVI
0.400


289
RRYWrTLVLL
0.400


136
DFLGsLELQL
0.400


234
GKVEaEFELL
0.400


254
GKGRkQPEPL
0.400


118
QPAVlVLQVW
0.400


314
IPGQiSQVIF
0.400


68
QETDvHFNSL
0.400


107
GPFAlEEAEF
0.400


123
VLQVwDYDRI
0.400


290
RYWRtLVLLL
0.400


94
LPTErEVSVW
0.400


265
KPSRpKTSFN
0.400


85
WRFVfRFDYL
0.400


261
EPLEkPSRPK
0.300


10
VPAPpPVDIK
0.300


120
AVLVlQVWDY
0.300


167
AGPRcNLFRC
0.300


287
IWRRyWRTLV
0.300


244
TVEEaEKRPV
0.300


251
RPVGkGRKQP
0.300


6
FPQDvPAPPP
0.300


296
VLLLlVLLTV
0.200


117
RQPAvLVLQV
0.200


44
NPLTgEMSSD
0.200


176
CRRLrGWWPV
0.200


183
WPVVkLKEAE
0.200


301
VLLTvFLLLV
0.200


227
GNVYiLTGKV
0.200


21
RQPIsYELRV
0.200


312
YTIPgQISQV
0.200


93
YLPTeREVSV
0.200


235
KVEAeFELLT
0.150


158
CSVQlARNGA
0.150


283
FVFFiWRRYW
0.150


255
KGRKqPEPLE
0.150


15
PVDIkPRQPI
0.135


222
FTDMgGNVYI
0.120


209
KQRRrKGRPE
0.100


105
RSGPfALEEA
0.100


27
ELRVvIWNTE
0.100


273
FNWFvNPLKT
0.100


143
LQLPdMVRGA
0.100


175
RCRRlRGWWP
0.100


276
FVNPlKTFVF
0.100


61
KGLEhDKQET
0.100


224
DMGGnVYILT
0.100


178
RLRGwWPVVK
0.100


194
VEREaQEAQA
0.100


114
AEFRqPAVLV
0.090
















TABLE XX







158P3D2v.1-B3501-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












268
RPKTSFNWF
120.000


265
KPSRPKTSF
40.000


278
NPLKTFVFF
20.000


214
KGRPEDLEF
9.000


314
IPGQISQVI
8.000


94
LPTEREVSV
8.000


10
VPAPPPVDI
8.000


154
GPELCSVQL
6.000


168
GPRCNLFRC
6.000


255
KGRKQPEPL
6.000


133
SANDFLGSL
6.000


318
ISQVIFRPL
5.000


75
NSLTGEGNF
5.000


118
QPAVLVLQV
4.000


24
ISYELRVVI
4.000


22
QPISYELRV
4.000


38
VVLDDENPL
3.000


55
YVKSWVKGL
3.000


175
RCRRLRGWW
3.000


166
GAGPRCNLF
3.000


180
RGWWPVVKL
2.000


183
WPVVKLKEA
2.000


283
FVFFIWRRY
2.000


304
TVFLLLVFY
2.000


121
VLVLQVWDY
2.000


44
NPLTGEMSS
2.000


19
KPRQPISYE
1.200


178
RLRGWWPVV
1.200


299
LLVLLTVFL
1.000


165
NGAGPRCNL
1.000


224
DMGGNVYIL
1.000


277
VNPLKTFVF
1.000


298
LLLVLLTVF
1.000


294
TLVLLLLVL
1.000


137
FLGSLELQL
1.000


171
CNLFRCRRL
1.000


101
SVWRRSGPF
1.000


81
GNFNWRFVF
1.000


300
LVLLTVFLL
1.000


50
MSSDIYVKS
1.000


83
FNWRFVFRF
1.000


232
LTGKVEAEF
1.000


303
LTVFLLLVF
1.000


301
VLLTVFLLL
1.000


295
LVLLLLVLL
1.000


77
LTGEGNFNW
1.000


235
KVEAEFELL
0.900


151
GARGPELCS
0.900


46
LTGEMSSDI
0.800


51
SSDIYVKSW
0.750


132
ISANDFLGS
0.750


222
FTDMGGNVY
0.600


47
TGEMSSDIY
0.600


259
QPEPLEKPS
0.600


140
SLELQLPDM
0.600


212
RRKGRPEDL
0.600


124
LQVWDYDRI
0.600


293
RTLVLLLLV
0.400


306
FLLLVFYTI
0.400


251
RPVGKGRKQ
0.400


6
FPQDVPAPP
0.400


261
EPLEKPSRP
0.400


129
YDRISANDF
0.300


291
YWRTLVLLL
0.300


17
DIKPRQPIS
0.300


27
ELRVVIWNT
0.300


287
IWRRYWRTL
0.300


69
ETDVHFNSL
0.300


103
WRRSGPFAL
0.300


237
EAEFELLTV
0.270


216
RPEDLEFTD
0.240


164
RNGAGPRCN
0.200


234
GKVEAEFEL
0.200


30
VVIWNTEDV
0.200


313
TIPGQISQV
0.200


18
IKPRQPISY
0.200


150
RGARGPELC
0.200


297
LLLLVLLTV
0.200


42
DENPLTGEM
0.200


107
GPFALEEAE
0.200


290
RYWRTLVLL
0.200


302
LLTVFLLLV
0.200


12
APPPVDIKP
0.200


31
VIWNTEDVV
0.200


276
FVNPLKTFV
0.200


228
NVYILTGKV
0.200


125
QVWDYDRIS
0.200


86
RFVFRFDYL
0.200


144
QLPDMVRGA
0.200


66
DKQETDVHF
0.200


80
EGNFNWRFV
0.200


85
WRFVFRFDY
0.200


289
RRYWRTLVL
0.200


270
KTSFNWFVN
0.200


113
EAEFRQPAV
0.180


190
EAEDVEREA
0.180


76
SLTGEGNFN
0.150


266
PSRPKTSFN
0.150


32
IWNTEDVVL
0.150


119
PAVLVLQVW
0.150
















TABLE XXI







158P3D2v.1-B3501-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












19
KPRQpISYEL
120.000


216
RPEDlEFTDM
72.000


94
LPTErEVSVW
30.000


107
GPFAlEEAEF
30.000


268
RPKTsFNWFV
24.000


139
GSLElQLPDM
20.000


314
IPGQiSQVIF
20.000


118
QPAVlVLQVW
10.000


278
NPLKtFVFFI
8.000


17
DIKPrQPISY
6.000


22
QPISyELRVV
6.000


271
TSFNwFVNPL
5.000


24
ISYElRVVIW
5.000


50
MSSDiYVKSW
5.000


132
ISANdFLGSL
5.000


100
VSVWrRSGPF
5.000


46
LTGEmSSDIY
4.000


153
RGPElCSVQL
4.000


265
KPSRpKTSFN
4.000


148
MVRGaRGPEL
3.000


233
TGKVeAEFEL
3.000


151
GARGpELCSV
2.700


164
RNGAgPRCNL
2.000


120
AVLVlQVWDY
2.000


293
RTLVlLLLVL
2.000


303
LTVFlLLVFY
2.000


170
RCNLfRCRRL
2.000


37
DVVLdDENPL
1.500


31
VIWNtEDVVL
1.500


298
LLLVlLTVFL
1.000


317
QISQvIFRPL
1.000


294
TLVLlLLVLL
1.000


286
FIWRrYWRTL
1.000


299
LLVLlTVFLL
1.000


300
LVLLtVFLLL
1.000


277
VNPLkTFVFF
1.000


105
RSGPfALEEA
1.000


302
LLTVfLLLVF
1.000


74
FNSLtGEGNF
1.000


231
ILTGkVEAEF
1.000


80
EGNFnWRFVF
1.000


297
LLLLvLLTVF
1.000


165
NGAGpRCNLF
1.000


276
FVNPlKTFVF
1.000


113
EAEFrQPAVL
0.900


185
VVKLkEAEDV
0.900


214
KGRPeDLEFT
0.900


162
LARNgAGPRC
0.900


75
NSLTgEGNFN
0.750


266
PSRPkTSFNW
0.750


123
VLQVwDYDRI
0.600


154
GPELcSVQLA
0.600


84
NWRFvFRFDY
0.600


211
RRRKgRPEDL
0.600


61
KGLEhDKQET
0.600


168
GPRCnLFRCR
0.600


158
CSVQlARNGA
0.500


283
FVFFiWRRYW
0.500


76
SLTGeGNFNW
0.500


9
DVPApPPVDI
0.400


261
EPLEkPSRPK
0.400


29
RVVIwNTEDV
0.400


21
RQPIsYELRV
0.400


251
RPVGkGRKQP
0.400


309
LVFYtIPGQI
0.400


6
FPQDvPAPPP
0.400


258
KQPEpLEKPS
0.400


117
RQPAvLVLQV
0.400


313
TIPGqISQVI
0.400


221
EFTDmGGNVY
0.400


213
RKGRpEDLEF
0.300


125
QVWDyDRISA
0.300


129
YDRIsANDFL
0.300


102
VWRRsGPFAL
0.300


115
EFRQpAVLVL
0.300


288
WRRYwRTLVL
0.300


134
ANDFlGSLEL
0.300


78
TGEGnFNWRF
0.300


38
VVLDdENPLT
0.300


234
GKVEaEFELL
0.300


65
HDKQeTDVHF
0.300


291
YWRTlVLLLL
0.300


12
APPPvDIKPR
0.300


131
RISAnDFLGS
0.300


44
NPLTgEMSSD
0.300


51
SSDIyVKSWV
0.300


290
RYWRtLVLLL
0.200


282
TFVFfIWRRY
0.200


183
WPVVkLKEAE
0.200


10
VPAPpPVDIK
0.200


68
QETDvHFNSL
0.200


227
GNVYiLTGKV
0.200


93
YLPTeREVSV
0.200


30
VVIWnTEDVV
0.200


296
VLLLlVLLTV
0.200


150
RGARgPELCS
0.200


301
VLLTvFLLLV
0.200


289
RRYWrTLVLL
0.200


312
YTIPgQISQV
0.200


187
KLKEaEDVER
0.180
















TABLE VIII







158P3D2v.17, ORF: 65-6175, Frame +2, A1-9-mers


Each peptide is a portion of SEQ ID NO: 29;


each start position is Specified, the length


of the peptide is 9 amino acids and the end


position for each peptide is the start position


plus eight.









Pos
Subsequence
Score












1930
FTDMGGNVY
62.500


626
GAEGPEIPR
45.000


1553
ETELTVAVF
45.000


1576
HIDLENRFY
25.000


40
EADIGELFR
25.000


217
HGDPFQVSR
25.000


1349
ELDWGSKYY
25.000


151
ELEPGEAQL
18.000


427
RAEGLPALR
18.000


197
DVELSGVMF
18.000


583
RAEPEPPQA
18.000


1474
FSEPQISRG
13.500


1202
ESEVLASYR
13.500


272
YNEYFLFEF
11.250


1755
TGEMSSDIY
11.250


1306
SLDPFLAEA
10.000


668
MIDPTVASQ
10.000


1598
EVDGYNAWR
10.000


1511
KADPYVVVS
10.000


452
LVEPYVRVS
9.000


1146
LVEPHSGRL
9.000


1121
LIELDYSGR
9.000


267
TSCPFYNEY
7.500


1011
LSDPFARVL
7.500


786
LQEVERLQR
6.750


1344
IPDPEELDW
6.250


346
ILDQPDGQF
5.000


1267
HIVPHMLRF
5.000


327
QTLPFMATR
5.000


1927
DLEFTDMGG
4.500


703
GTEGAAVEA
4.500


821
GAERRTMTR
4.500


1466
YPESEAVLF
4.500


1945
EAEFELLTV
4.500


1200
GVESEVLAS
4.500


1688
HVETRPLYH
4.500


708
AVEAQPLLG
4.500


1137
EVEPQDLAP
4.500


585
EPEPPQAQQ
4.500


235
VLEAQKLVG
4.500


1818
ALEEAEFRQ
4.500


1379
DSDGVNLIS
3.750


1955
EAEKRPVGK
3.600


1906
AQEAQAGKK
2.700


316
LTPPSPKAF
2.500


1659
SGDPEEAQA
2.500


349
QPDGQFYQR
2.500


1215
FTELVRHLT
2.250


1967
QPEPLEKPS
2.250


1786
TGEGNFNWR
2.250


734
RPEPMDGSG
2.250


745
FCLPLCHCK
2.000


485
FVELFPPLT
1.800


807
ALEVLVAGS
1.800


1186
EVEQPQVVL
1.800


1578
DLENRFYSH
1.800


1821
EAEFRQPAV
1.800


471
SAEAAAPEW
1.800


1129
RLEPSVPSE
1.800


881
LAEEPQPPL
1.800


1184
LLEVEQPQV
1.800


1848
SLELQLPDM
1.800


543
GSPPGAGLR
1.500


964
TSELPPDLL
1.350


827
MTRPNALDR
1.250


1989
KTFVFFIWR
1.250


977
AGLPSSLHR
1.250


1777
ETDVHFNSL
1.250


1853
LPDMVRGAR
1.250


512
ATHVPDLRR
1.250


2020
YTIPGQISQ
1.250


1073
FGPPVFLGR
1.250


2011
LTVFLLLVF
1.250


1097
RPELQFFPL
1.125


1540
FGEILELSI
1.125


1742
NTEDVVLDD
1.125


1874
GAGPRCNLF
1.000


368
RAGTKGFIK
1.000


1159
NVCPVLREF
1.000


2012
TVFLLLVFY
1.000


2028
QVIFRPLHK
1.000


1099
ELQFFPLRK
1.000


506
LVDAALATH
1.000


1621
RCGLPAPEY
1.000


710
EAQPLLGAR
1.000


1747
VLDDENPLT
1.000


1029
VLEQTLSPL
0.900


1057
QEEPPLVII
0.900


1896
LKEAEDVER
0.900


110
QVELDLKYQ
0.900


291
LLEITVSGV
0.900


947
KLELFLRLG
0.900


689
RLEEQLGRG
0.900


1397
EAEVKGTVS
0.900


1193
VLEVAGQGV
0.900


1943
KVEAEFELL
0.900


1311
LAEAGISRQ
0.900


1898
EAEDVEREA
0.900


1770
GLEHDKQET
0.900


1168
RVEVLFWGL
0.900
















TABLE IX







158P3D2v.17, ORF: 65-6175, Frame +2, A1-10-mers


Each peptide is a portion of SEQ ID NO: 29;


each start position is Specified, the length


of the peptide is 10 amino acids and the end


position for each peptide is the start position


plus nine.









Pos
Subsequence
Score












452
LVEPyVRVSF
180.000


1200
GVESeVLASY
90.000


485
FVELfPPLTR
45.000


1967
QPEPlEKPSR
45.000


346
ILDQpDGQFY
25.000


43
IGELfRWPHY
22.500


1137
EVEPqDLAPL
18.000


1011
LSDPfARVLI
15.000


1474
FSEPqISRGI
13.500


972
LPEPsAGLPS
11.250


1215
FTELvRHLTV
11.250


476
APEWnEQLSF
11.250


1511
KADPyVVVSA
10.000


1626
APEYrAGAVK
9.000


807
ALEVlVAGSR
9.000


1168
RVEVlFWGLR
9.000


583
RAEPePPQAQ
9.000


1129
RLEPsVPSEV
9.000


1146
LVEPhSGRLS
9.000


1306
SLDPfLAEAG
5.000


1874
GAGPrCNLFR
5.000


1984
FVNPlKTFVF
5.000


1346
DPEElDWGSK
4.500


401
DIEKnLLLPR
4.500


708
AVEAqPLLGA
4.500


427
RAEGlPALRL
4.500


1097
RPELqFFPLR
4.500


1290
MEETgDMMPK
4.500


637
EVEVeELLPL
4.500


1943
KVEAeFELLT
4.500


1543
ILELsISLPA
4.500


197
DVELsGVMFS
4.500


947
KLELfLRLGL
4.500


1379
DSDGvNLISM
3.750


981
SSLHrDDFSY
3.750


399
CSDIeKNLLL
3.750


521
ISHPgRAAGF
3.000


1906
AQEAqAGKKK
2.700


127
WSEEdFGAPI
2.700


786
LQEVeRLQRK
2.700


1725
DIKPrQPISY
2.500


1576
HIDLeNRFYS
2.500


1747
VLDDeNPLTG
2.500


2011
LTVFlLLVFY
2.500


1117
AAFQlIELDY
2.500


1930
FTDMgGNVYI
2.500


40
EADIgELFRW
2.500


193
DDEPdVELSG
2.250


1786
TGEGnFNWRF
2.250


703
GTEGaAVEAQ
2.250


38
GPEAdIGELF
2.250


585
EPEPpQAQQG
2.250


1057
QEEPpLVIIN
2.250


1502
NLAPaDPNGK
2.000


938
GAAPgEVCAK
2.000


235
VLEAqKLVGV
1.800


1029
VLEQtLSPLW
1.800


1821
EAEFrQPAVL
1.800


626
GAEGpEIPRA
1.800


1392
IQDQgEAEVK
1.500


267
TSCPfYNEYF
1.500


1360
LQELqGQHNF
1.350


1754
LTGEmSSDIY
1.250


340
RMDLgIILDQ
1.250


1344
IPDPeELDWG
1.250


266
GTSCpFYNEY
1.250


217
HGDPfQVSRA
1.250


1659
SGDPeEAQAL
1.250


1777
ETDVhFNSLT
1.250


1572
IGEThIDLEN
1.125


629
GPEIpRAMEV
1.125


181
ENELeLELEQ
1.125


1755
TGEMsSDIYV
1.125


1466
YPESeAVLFS
1.125


1661
DPEEaQALLV
1.125


1112
AGELiAAFQL
1.125


620
HLDAsPGAEG
1.000


841
LVHSlNLLAK
1.000


1313
EAGIsRQLLK
1.000


1481
RGIPqNRPIK
1.000


949
ELFLrLGLGK
1.000


1683
QLVPeHVETR
1.000


868
VALAkKLLAK
1.000


191
DLDDePDVEL
1.000


315
GLTPpSPKAF
1.000


1159
NVCPvLREFR
1.000


1349
ELDWgSKYYA
1.000


1121
LIELdYSGRL
0.900


917
VVEEeRGRDC
0.900


1927
DLEFtDMGGN
0.900


1578
DLENrFYSHH
0.900


689
RLEEqLGRGS
0.900


725
EEELgTHAQR
0.900


1952
TVEEaEKRPV
0.900


183
ELELeLEQDL
0.900


291
LLEItVSGVG
0.900


1184
LLEVeQPQVV
0.900


471
SAEAaAPEWN
0.900


1848
SLELqLPDMV
0.900


1164
LREFrVEVLF
0.900
















TABLE X







158P3D2v.16, ORF: 65-6175, Frame +2, A0201-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












2010
LLTVFLLLV
1033.404


2005
LLLLVLLTV
1006.209


1490
KLLVRVYVV
849.359


451
VLVEPYVRV
727.166


873
KLLAKLRFL
699.907


1010
GLSDPFARV
541.810


1994
FIWRRYWRT
440.113


571
LLLAVSMQV
437.482


658
LLFGVLFEA
416.858


2014
FLLLVFYTI
337.376


98
ALVDENLQV
285.163


445
ALHDQRVLV
285.163


839
KLLVHSLNL
276.643


2009
VLLTVFLLL
255.302


2007
LLVLLTVFL
199.738


895
WMLSGQRRV
170.990


1155
SLPPNVCPV
159.970


2008
LVLLTVFLL
156.843


1984
FVNPLKTFV
153.971


971
LLPEPSAGL
148.896


1163
VLREFRVEV
140.004


2004
VLLLLVLLT
107.808


286
RLQDLLLEI
98.381


1845
FLGSLELQL
98.267


650
VLAPCEDFL
97.872


1243
YLQPPLSIL
92.666


234
TVLEAQKLV
92.322


840
LLVHSLNLL
83.527


1332
GLLNQGPGL
79.041


435
RLGLLGSLV
69.552


86
QLQNAGHLV
69.552


103
NLQVSPIQV
69.552


1710
WIDIFPQDV
66.867


781
RLDQGLQEV
63.988


1171
VLFWGLRGL
61.810


2
ALTVSVQRL
49.134


510
ALATHVPDL
49.134


79
TLVISLQQL
49.134


874
LLAKLRFLA
48.984


1041
LLVFEQLIV
48.478


1746
VVLDDENPL
48.205


437
GLLGSLVRA
42.278


1003
VLAADDSGL
36.316


847
LLAKQGLRL
36.316


572
LLAVSMQVL
34.246


553
SLQGLNEGV
34.080


406
LLLPRGVPA
31.249


1635
KVGSKVFLT
30.444


1756
GEMSSDIYV
27.521


1739
VIWNTEDVV
27.109


2003
LVLLLLVLL
27.042


802
AQLKQALEV
26.092


1464
LIYPESEAV
25.492


1463
FLIYPESEA
22.853


2021
TIPGQISQV
21.996


499
QLRDDAPLV
21.672


1082
ALAAPRVKL
21.362


430
GLPALRLGL
21.362


1747
VLDDENPLT
20.776


1571
LIGETHIDL
20.473


2002
TLVLLLLVL
20.145


931
SLMLTAPGA
18.382


1701
GLLQGSLHM
18.382


1033
TLSPLWDEL
17.795


291
LLEITVSGV
17.405


1306
SLDPFLAEA
17.368


438
LLGSLVRAL
16.705


549
GLRDSLQGL
15.310


1531
YIPKQLNPI
15.177


225
RAQDFQVGV
15.050


643
LLPLPENVL
14.890


498
LQLRDDAPL
13.624


1018
VLISTQCQT
12.668


1244
LQPPLSILV
11.988


63
VQVVNCSRV
11.988


1224
VVFKDTAPL
11.757


1938
YILTGKVEA
11.626


60
CLSVQVVNC
11.426


335
RIGTFRMDL
11.162


1040
ELLVFEQLI
11.001


184
LELELEQDL
10.712


328
TLPFMATRI
10.433


1703
LQGSLHMWI
9.890


1055
HLQEEPPLV
9.696


215
LAHGDPFQV
9.525


642
ELLPLPENV
9.457


1852
QLPDMVRGA
9.370


1261
TVLVGSHIV
9.232


1113
GELIAAFQL
8.914


1614
ILAGLCQRC
8.446


1639
KVFLTPPET
8.444


2001
RTLVLLLLV
8.221


1193
VLEVAGQGV
7.567


1184
LLEVEQPQV
7.567


1683
QLVPEHVET
7.452


654
CEDFLLFGV
7.216


1557
TVAVFDHDL
7.103


638
VEVEELLPL
6.659


867
KVALAKKLL
6.542


888
PLPDVLVWM
5.669
















TABLE XI







158P3D2v.17, ORF: 65-6175, Frame +2, A0201-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












290
LLLEiTVSGV
4125.456


2009
VLLTvFLLLV
3823.593


276
FLFEfHDTRL
1490.711


657
FLLFgVLFEA
1127.969


2004
VLLLlVLLTV
1006.209


2006
LLLVlLTVFL
739.032


839
KLLVhSLNLL
636.279


2007
LLVLlTVFLL
484.457


214
ALAHgDPFQV
403.402


880
FLAEePQPPL
402.895


873
KLLAkLRFLA
373.146


1243
YLQPpLSILV
319.939


1801
YLPTeREVSV
319.939


461
FLGQeGETSV
319.939


570
RLLLaVSMQV
257.342


650
VLAPcEDFLL
210.633


1319
QLLKpPLKKL
181.794


2012
TVFLlLVFYT
177.011


1178
GLGRvHLLEV
159.970


1192
VVLEvAGQGV
156.947


1463
FLIYpESEAV
156.770


571
LLLAvSMQVL
126.710


1702
LLQGsLHMWI
97.547


846
NLLAkQGLRL
79.041


1986
NPLKtFVFFI
70.254


1549
SLPAeTELTV
69.552


1028
RVLEqTLSPL
65.219


241
LVGVnINPYV
56.902


869
ALAKkLLAKL
49.134


2002
TLVLlLLVLL
49.134


810
VLVAgSRQFC
46.451


498
LQLRdDAPLV
44.356


658
LLFGvLFEAT
43.639


556
GLNEgVGQGI
42.774


1734
YELRvVIWNT
42.542


65
VVNCsRVFSL
42.390


1994
FIWRrYWRTL
38.130


437
GLLGsLVRAL
36.359


1183
HLLEvEQPQV
35.874


760
SCWEdHTWRL
35.591


222
QVSRaQDFQV
35.298


450
RVLVePYVRV
33.776


888
PLPDvLVWML
33.239


157
AQLErRAVAL
32.857


1010
GLSDpFARVL
27.292


777
KVAErLDQGL
26.823


85
QQLQnAGHLV
26.092


564
GIWFrGRLLL
24.380


203
VMFSpLKSRA
22.569


2008
LVLLtVFLLL
22.339


1944
VEAEfELLTV
21.680


497
RLQLrDDAPL
21.362


844
SLNLlAKQGL
21.362


1809
SVWRrSGPFA
19.844


991
FQLRaHLYQA
19.718


1491
LLVRvYVVKA
19.425


649
NVLApCEDFL
18.639


405
NLLLpRGVPA
18.382


1455
LVGKfKGSFL
17.477


235
VLEAqKLVGV
17.405


1739
VIWNtEDVVL
16.993


1746
VVLDdENPLT
16.816


847
LLAKqGLRLL
16.705


1795
FVFRfDYLPT
16.647


1535
QLNPiFGEIL
16.308


1825
RQPAvLVLQV
16.219


424
RLYRaEGLPA
15.898


1305
KSLDpFLAEA
15.049


1833
QVWDyDRISA
14.793


1668
LLVLrRWQEM
14.358


286
RLQDlLLEIT
14.118


295
TVSGvGVTSV
13.997


12
GLTGtHDRQV
13.910


352
GQFYqRWVPL
13.624


1831
VLQVwDYDRI
13.036


1018
VLIStQCQTT
12.668


1639
KVFLtPPETL
11.861


530
FNPTfGPAWV
11.487


1115
LIAAfQLIEL
11.485


1082
ALAApRVKLM
11.426


1033
TLSPlWDELL
10.468


908
IPAQdVLFSV
10.296


2020
YTIPgQISQV
10.220


1427
FEDWlNVFPL
10.196


1047
LIVDgRREHL
10.032


245
NINPyVAVQV
9.563


143
IIPNvGFQEL
9.488


2003
LVLLlLVLLT
9.433


1771
LEHDkQETDV
9.426


243
GVNInPYVAV
9.129


453
VEPYvRVSFL
8.933


175
GQQDdEENEL
8.880


1155
SLPPnVCPVL
8.759


86
QLQNaGHLVL
8.759


1676
EMPGfGIQLV
8.665


832
ALDRcRGKLL
8.545


643
LLPLpENVLA
8.446


840
LLVHsLNLLA
8.446


1040
ELLVfEQLIV
7.913


72
FSLRpLGTLV
7.727
















TABLE XII







158P3D2v.17, ORF: 65-6175, Frame +2, A3-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












1319
QLLKPPLKK
90.000


276
FLFEFHDTR
90.000


1089
KLMEDPYQR
81.000


869
ALAKKLLAK
60.000


858
GLRRRNVQK
60.000


1829
VLVLQVWDY
54.000


1989
KTFVFFIWR
54.000


1491
LLVRVYVVK
45.000


1099
ELQFFPLRK
36.000


1831
VLQVWDYDR
36.000


1757
EMSSDIYVK
27.000


1430
WLNVFPLYR
24.000


203
VMFSPLKSR
22.500


304
VLQRRGDEK
20.000


953
RLGLGKQAK
20.000


1950
LLTVEEAEK
20.000


23
LTFRGFTQK
15.000


2014
FLLLVFYTI
12.150


982
SLHRDDFSY
12.000


1310
FLAEAGISR
12.000


658
LLFGVLFEA
10.125


599
RLTRKKKKK
10.000


1761
DIYVKSWVK
9.000


1706
SLHMWIDIF
9.000


1419
SLKEEFNHF
9.000


1490
KLLVRVYVV
8.100


2009
VLLTVFLLL
8.100


2028
QVIFRPLHK
6.000


1064
IINVFDHNK
6.000


232
GVTVLEAQK
6.000


1482
GIPQNRPIK
6.000


560
GVGQGIWFR
5.400


839
KLLVHSLNL
5.400


2006
LLLVLLTVF
4.500


768
RLQSSNCVR
4.000


852
GLRLLRGLR
3.600


1864
ELCSVQLAR
3.600


1850
ELQLPDMVR
3.600


430
GLPALRLGL
3.600


2024
GQISQVIFR
3.240


1767
WVKGLEHDK
3.000


1036
PLWDELLVF
3.000


2012
TVFLLLVFY
3.000


1399
EVKGTVSPK
2.700


1623
GLPAPEYRA
2.700


1332
GLLNQGPGL
2.700


2002
TLVLLLLVL
2.700


549
GLRDSLQGL
2.700


300
GVTSVLQRR
2.700


1010
GLSDPFARV
2.700


1932
DMGGNVYIL
2.430


1247
PLSILVIER
2.400


595
STLSRLTRK
2.250


1243
YLQPPLSIL
2.025


437
GLLGSLVRA
2.025


327
QTLPFMATR
2.025


1880
NLFRCRRLR
2.000


596
TLSRLTRKK
2.000


2
ALTVSVQRL
1.800


635
AMEVEVEEL
1.800


1701
GLLQGSLHM
1.800


2010
LLTVFLLLV
1.800


450
RVLVEPYVR
1.800


846
NLLAKQGLR
1.800


1684
LVPEHVETR
1.800


510
ALATHVPDL
1.800


286
RLQDLLLEI
1.800


864
VQKKVALAK
1.800


899
GQRRVAWAR
1.620


1676
EMPGFGIQL
1.620


1987
PLKTFVFFI
1.620


141
ELIIPNVGF
1.350


315
GLTPPSPKA
1.350


1267
HIVPHMLRF
1.350


1145
PLVEPHSGR
1.350


971
LLPEPSAGL
1.350


840
LLVHSLNLL
1.350


79
TLVISLQQL
1.350


2005
LLLLVLLTV
1.350


1306
SLDPFLAEA
1.350


1535
QLNPIFGEI
1.215


284
RLRLQDLLL
1.200


1845
FLGSLELQL
1.200


847
LLAKQGLRL
1.200


1889
GWWPVVKLK
1.013


1320
LLKPPLKKL
1.012


854
RLLRGLRRR
0.900


1526
DTKERYIPK
0.900


1029
VLEQTLSPL
0.900


643
LLPLPENVL
0.900


420
RLRVRLYRA
0.900


571
LLLAVSMQV
0.900


650
VLAPCEDFL
0.900


1033
TLSPLWDEL
0.900


939
AAPGEVCAK
0.900


337
GTFRMDLGI
0.900


1318
RQLLKPPLK
0.900


992
QLRAHLYQA
0.900


996
HLYQARGVL
0.900


331
FMATRIGTF
0.900
















TABLE XIII







158P3D2v.17, ORF: 65-6175, Frame +2, A3-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












858
GLRRrNVQKK
180.000


22
KLTFrGFTQK
180.000


1490
KLLVrVYVVK
135.000


1886
RLRGwWPVVK
90.000


949
ELFLrLGLGK
60.000


1472
VLFSePQISR
60.000


1989
KTFVfFIWRR
40.500


240
KLVGvNINPY
40.500


785
GLQEvERLQR
36.000


1502
NLAPaDPNGK
30.000


913
VLFSvVEEER
30.000


714
LLGArPEEEK
30.000


1683
QLVPeHVETR
27.000


852
GLRLlRGLRR
24.000


1412
TLKIyNRSLK
20.000


768
RLQSsNCVRK
20.000


1895
KLKEaEDVER
18.000


596
TLSRlTRKKK
10.000


1949
ELLTvEEAEK
9.000


1063
VIINvFDHNK
9.000


1120
QLIElDYSGR
9.000


1296
MMPKgPQGQK
9.000


2007
LLVLlTVFLL
8.100


826
TMTRpNALDR
8.000


435
RLGLlGSLVR
8.000


298
GVGVtSVLQR
7.200


2010
LLTVfLLLVF
6.000


510
ALAThVPDLR
6.000


863
NVQKkVALAK
6.000


407
LLPRgVPAER
6.000


1218
LVRHlTVVFK
6.000


441
SLVRaLHDQR
6.000


331
FMATrIGTFR
6.000


841
LVHSlNLLAK
6.000


982
SLHRdDFSYF
6.000


1828
AVLVlQVWDY
5.400


1830
LVLQvWDYDR
5.400


315
GLTPpSPKAF
4.500


276
FLFEfHDTRL
4.500


1939
ILTGkVEAEF
4.500


2005
LLLLvLLTVF
4.500


1454
HLVGkFKGSF
4.050


839
KLLVhSLNLL
4.050


556
GLNEgVGQGI
4.050


1950
LLTVeEAEKR
4.000


1078
FLGRaLAAPR
4.000


992
QLRAhLYQAR
4.000


346
ILDQpDGQFY
4.000


947
KLELfLRLGL
3.600


411
GVPAeRPWAR
3.600


303
SVLQrRGDEK
3.000


2009
VLLTvFLLLV
2.700


938
GAAPgEVCAK
2.700


1535
QLNPiFGEIL
2.700


650
VLAPcEDFLL
2.700


1491
LLVRvYVVKA
2.700


1318
RQLLkPPLKK
2.700


1200
GVESeVLASY
2.700


873
KLLAkLRFLA
2.700


1055
HLQEePPLVI
2.700


1163
VLREfRVEVL
2.700


1175
GLRGlGRVHL
2.700


1033
TLSPlWDELL
2.700


658
LLFGvLFEAT
2.250


202
GVMFsPLKSR
2.025


657
FLLFgVLFEA
2.025


424
RLYRaEGLPA
2.000


893
LVWMlSGQRR
2.000


846
NLLAkQGLRL
1.800


892
VLVWmLSGQR
1.800


73
SLRPlGTLVI
1.800


1852
QLPDmVRGAR
1.800


2027
SQVIfRPLHK
1.800


266
GTSCpFYNEY
1.800


1178
GLGRvHLLEV
1.800


1409
AVATlKIYNR
1.800


1702
LLQGsLHMWI
1.800


564
GIWFrGRLLL
1.800


807
ALEVlVAGSR
1.800


880
FLAEePQPPL
1.350


1639
KVFLtPPETL
1.350


571
LLLAvSMQVL
1.350


290
LLLEiTVSGV
1.350


1399
EVKGtVSPKK
1.350


45
ELFRwPHYGA
1.350


1701
GLLQgSLHMW
1.350


1718
VPAPpPVDIK
1.350


1155
SLPPnVCPVL
1.350


2004
VLLLlVLLTV
1.350


2002
TLVLlLLVLL
1.350


437
GLLGsLVRAL
1.215


1756
GEMSsDIYVK
1.215


1617
GLCQrCGLPA
1.200


1869
QLARnGAGPR
1.200


485
FVELfPPLTR
1.200


86
QLQNaGHLVL
1.200


517
DLRRiSHPGR
1.200


1319
QLLKpPLKKL
1.012


1224
VVFKdTAPLF
1.000


13
LTGThDRQVK
1.000
















TABLE XIV







158P3D2v.17, ORF: 65-6175, Frame +2, A1101-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












2028
QVIFRPLHK
6.000


232
GVTVLEAQK
6.000


450
RVLVEPYVR
3.600


1318
RQLLKPPLK
2.700


560
GVGQGIWFR
2.400


1989
KTFVFFIWR
2.400


23
LTFRGFTQK
2.000


1767
WVKGLEHDK
2.000


368
RAGTKGFIK
1.800


595
STLSRLTRK
1.500


953
RLGLGKQAK
1.200


858
GLRRRNVQK
1.200


864
VQKKVALAK
1.200


1319
QLLKPPLKK
1.200


300
GVTSVLQRR
1.200


1482
GIPQNRPIK
1.200


2024
GQISQVIFR
1.080


1089
KLMEDPYQR
0.960


869
ALAKKLLAK
0.800


893
LVWMLSGQR
0.800


816
RQFCHGAER
0.720


899
GQRRVAWAR
0.720


769
LQSSNCVRK
0.600


1399
EVKGTVSPK
0.600


599
RLTRKKKKK
0.600


950
LFLRLGLGK
0.600


797
GPGACAQLK
0.600


607
KARRDQTPK
0.600


1906
AQEAQAGKK
0.600


1526
DTKERYIPK
0.600


1491
LLVRVYVVK
0.600


1761
DIYVKSWVK
0.480


512
ATHVPDLRR
0.400


1684
LVPEHVETR
0.400


827
MTRPNALDR
0.400


304
VLQRRGDEK
0.400


1950
LLTVEEAEK
0.400


62
SVQVVNCSR
0.400


1064
IINVFDHNK
0.400


251
AVQVGGQRR
0.400


442
LVRALHDQR
0.400


1729
RQPISYELR
0.360


327
QTLPFMATR
0.300


745
FCLPLCHCK
0.300


1951
LTVEEAEKR
0.300


1909
AQAGKKKRK
0.300


1757
EMSSDIYVK
0.240


786
LQEVERLQR
0.240


591
AQQGSTLSR
0.240


1099
ELQFFPLRK
0.240


852
GLRLLRGLR
0.240


1665
AQALLVLRR
0.240


768
RLQSSNCVR
0.240


821
GAERRTMTR
0.240


626
GAEGPEIPR
0.240


939
AAPGEVCAK
0.200


1297
MPKGPQGQK
0.200


1503
LAPADPNGK
0.200


1935
GNVYILTGK
0.180


1430
WLNVFPLYR
0.160


1831
VLQVWDYDR
0.160


1310
FLAEAGISR
0.160


276
FLFEFHDTR
0.160


26
RGFTQKTRK
0.120


1965
RKQPEPLEK
0.120


337
GTFRMDLGI
0.120


376
KVTLSVRAR
0.120


1798
RFDYLPTER
0.120


1168
RVEVLFWGL
0.120


427
RAEGLPALR
0.120


1878
RCNLFRCRR
0.120


243
GVNINPYVA
0.120


902
RVAWARIPA
0.120


1598
EVDGYNAWR
0.120


945
CAKLELFLR
0.120


164
VALGRRLAR
0.120


1990
TFVFFIWRR
0.120


1993
FFIWRRYWR
0.120


846
NLLAKQGLR
0.120


1613
QILAGLCQR
0.120


661
GVLFEATMI
0.090


1081
RALAAPRVK
0.090


2008
LVLLTVFLL
0.090


1062
LVIINVFDH
0.090


2001
RTLVLLLLV
0.090


787
QEVERLQRK
0.090


1121
LIELDYSGR
0.080


203
VMFSPLKSR
0.080


760
SCWEDHTWR
0.080


412
VPAERPWAR
0.080


1410
VATLKIYNR
0.080


1473
LFSEPQISR
0.080


1212
SPNFTELVR
0.080


349
QPDGQFYQR
0.080


374
FIKVTLSVR
0.080


1981
FNWFVNPLK
0.080


1958
KRPVGKGRK
0.060


1314
AGISRQLLK
0.060


1889
GWWPVVKLK
0.060


1087
RVKLMEDPY
0.060
















TABLE XV







158P3D2v.17, ORF: 65-6175, Frame +2, A1101-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












1318
RQLLkPPLKK
5.400


841
LVHSlNLLAK
4.000


863
NVQKkVALAK
4.000


303
SVLQrRGDEK
3.000


298
GVGVtSVLQR
2.400


411
GVPAeRPWAR
2.400


1989
KTFVfFIWRR
2.400


1218
LVRHlTVVFK
2.000


2027
SQVIfRPLHK
1.800


1490
KLLVrVYVVK
1.800


1886
RLRGwWPVVK
1.200


202
GVMFsPLKSR
1.200


768
RLQSsNCVRK
1.200


1168
RVEVlFWGLR
1.200


858
GLRRrNVQKK
1.200


22
KLTFrGFTQK
1.200


1830
LVLQvWDYDR
1.200


13
LTGThDRQVK
1.000


893
LVWMlSGQRR
0.800


1409
AVATlKIYNR
0.800


163
AVALgRRLAR
0.800


485
FVELfPPLTR
0.800


816
RQFChGAERR
0.720


1756
GEMSsDIYVK
0.720


1063
VIINvFDHNK
0.600


864
VQKKvALAKK
0.600


938
GAAPgEVCAK
0.600


868
VALAkKLLAK
0.600


786
LQEVeRLQRK
0.600


1392
IQDQgEAEVK
0.600


1630
RAGAvKVGSK
0.600


1399
EVKGtVSPKK
0.600


785
GLQEvERLQR
0.480


435
RLGLlGSLVR
0.480


949
ELFLrLGLGK
0.480


852
GLRLlRGLRR
0.480


249
YVAVqVGGQR
0.400


714
LLGArPEEEK
0.400


1296
MMPKgPQGQK
0.400


574
AVSMqVLEGR
0.400


1412
TLKIyNRSLK
0.400


1159
NVCPvLREFR
0.400


1502
NLAPaDPNGK
0.400


395
APGHcSDIEK
0.400


256
GQRRvTATQR
0.360


1472
VLFSePQISR
0.320


1906
AQEAqAGKKK
0.300


107
SPIQvELDLK
0.300


1895
KLKEaEDVER
0.240


1072
KFGPpVFLGR
0.240


1874
GAGPrCNLFR
0.240


1980
SFNWfVNPLK
0.200


10
LTGLtGTHDR
0.200


492
LTRSlRLQLR
0.200


596
TLSRlTRKKK
0.200


744
YFCLpLCHCK
0.200


1519
SAGReRQDTK
0.200


1626
APEYrAGAVK
0.200


313
AAGLtPPSPK
0.200


1785
LTGEgNFNWR
0.200


1718
VPAPpPVDIK
0.200


1405
SPKKaVATLK
0.200


373
GFIKvTLSVR
0.180


1612
SQILaGLCQR
0.180


1949
ELLTvEEAEK
0.180


1904
REAQeAQAGK
0.180


1172
LFWGlRGLGR
0.160


1992
VFFIwRRYWR
0.160


913
VLFSvVEEER
0.160


826
TMTRpNALDR
0.160


595
STLSrLTRKK
0.150


1964
GRKQpEPLEK
0.120


944
VCAKlELFLR
0.120


1290
MEETgDMMPK
0.120


892
VLVWmLSGQR
0.120


87
LQNAgHLVLR
0.120


1621
RCGLpAPEYR
0.120


867
KVALaKKLLA
0.120


1494
RVYVvKATNL
0.120


1313
EAGIsRQLLK
0.120


1120
QLIElDYSGR
0.120


326
SQTLpFMATR
0.120


1639
KVFLtPPETL
0.120


1097
RPELqFFPLR
0.120


1683
QLVPeHVETR
0.120


441
SLVRaLHDQR
0.120


600
LTRKkKKKAR
0.100


1481
RGIPqNRPIK
0.090


857
RGLRrRNVQK
0.090


2001
RTLVlLLLVL
0.090


1002
GVLAaDDSGL
0.090


1737
RVVIwNTEDV
0.090


1028
RVLEqTLSPL
0.090


450
RVLVePYVRV
0.090


1251
LVIErRAFGH
0.090


1398
AEVKgTVSPK
0.090


407
LLPRgVPAER
0.080


1950
LLTVeEAEKR
0.080


510
ALAThVPDLR
0.080


331
FMATrIGTFR
0.080
















TABLE XVI







158P3D2v.17, ORF: 65-6175, Frame +2, A24-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












1355
KYYASLQEL
528.000


1998
RYWRTLVLL
400.000


1465
IYPESEAVL
360.000


1495
VYVVKATNL
300.000


1694
LYHPHSPGL
240.000


989
SYFQLRAHL
240.000


1601
GYNAWRDAF
150.000


1794
RFVFRFDYL
72.000


484
SFVELFPPL
51.840


488
LFPPLTRSL
43.200


1980
SFNWFVNPL
36.000


277
LFEFHDTRL
30.000


1423
EFNHFEDWL
30.000


1640
VFLTPPETL
30.000


228
DFQVGVTVL
30.000


1214
NFTELVRHL
28.800


533
TFGPAWVPL
24.000


279
EFHDTRLRL
24.000


1426
HFEDWLNVF
21.600


353
QFYQRWVPL
20.000


116
KYQPPEGAT
18.000


1168
RVEVLFWGL
17.280


656
DFLLFGVLF
15.000


1983
WFVNPLKTF
15.000


1094
PYQRPELQF
15.000


162
RAVALGRRL
14.400


1097
RPELQFFPL
14.400


825
RTMTRPNAL
14.400


1943
KVEAEFELL
14.400


1107
KGPWAAGEL
13.200


1322
KPPLKKLPL
12.000


48
RWPHYGAPL
12.000


873
KLLAKLRFL
12.000


839
KLLVHSLNL
12.000


862
RNVQKKVAL
12.000


1225
VFKDTAPLF
12.000


1177
RGLGRVHLL
12.000


444
RALHDQRVL
12.000


1299
KGPQGQKSL
12.000


2026
ISQVIFRPL
10.080


2009
VLLTVFLLL
10.080


323
AFHSQTLPF
10.000


1800
DYLPTEREV
9.900


308
RGDEKAAGL
9.600


686
RAGRLEEQL
9.600


837
RGKLLVHSL
9.600


144
IPNVGFQEL
9.504


1888
RGWWPVVKL
8.800


778
VAERLDQGL
8.640


1746
VVLDDENPL
8.640


1542
EILELSISL
8.640


297
SGVGVTSVL
8.400


107
SPIQVELDL
8.400


503
DAPLVDAAL
8.400


1156
LPPNVCPVL
8.400


384
RGDLPPPML
8.000


284
RLRLQDLLL
8.000


867
KVALAKKLL
8.000


335
RIGTFRMDL
8.000


1963
KGRKQPEPL
8.000


1548
ISLPAETEL
7.920


233
VTVLEAQKL
7.920


1242
PYLQPPLSI
7.500


1596
QYEVDGYNA
7.500


997
LYQARGVLA
7.500


1733
SYELRVVIW
7.500


105
QVSPIQVEL
7.392


940
APGEVCAKL
7.392


643
LLPLPENVL
7.200


845
LNLLAKQGL
7.200


1240
EQPYLQPPL
7.200


1230
APLFHPQDL
7.200


1243
YLQPPLSIL
7.200


2007
LLVLLTVFL
7.200


1841
SANDFLGSL
7.200


840
LLVHSLNLL
7.200


1146
LVEPHSGRL
7.200


971
LLPEPSAGL
7.200


2002
TLVLLLLVL
7.200


851
QGLRLLRGL
7.200


151
ELEPGEAQL
7.200


881
LAEEPQPPL
7.200


1661
DPEEAQALL
7.200


430
GLPALRLGL
7.200


1208
SYRESPNFT
7.200


707
AAVEAQPLL
7.200


79
TLVISLQQL
7.200


2003
LVLLLLVLL
7.200


761
CWEDHTWRL
7.200


475
AAPEWNEQL
7.200


2018
VFYTIPGQI
7.000


398
HCSDIEKNL
6.720


613
TPKAVPQHL
6.720


134
APIQDSFEL
6.600


38
GPEADIGEL
6.600


1483
IPQNRPIKL
6.600


635
AMEVEVEEL
6.600


1033
TLSPLWDEL
6.336


176
QQDDEENEL
6.336


2019
FYTIPGQIS
6.000
















TABLE XVII







158P3D2v.17, ORF: 65-6175, Frame +2, A24-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












1998
RYWRtLVLLL
480.000


1762
IYVKsWVKGL
300.000


1694
LYHPhSPGLL
240.000


1530
RYIPkQLNPI
216.000


425
LYRAeGLPAL
200.000


541
LYGSpPGAGL
200.000


271
FYNEyFLFEF
198.000


1415
IYNRsLKEEF
198.000


1465
IYPEsEAVLF
180.000


1836
DYDRiSANDF
120.000


1844
DFLGsLELQL
36.000


1242
PYLQpPLSIL
30.000


338
TFRMdLGIIL
24.000


71
VFSLrPLGTL
20.000


1823
EFRQpAVLVL
20.000


1094
PYQRpELQFF
18.000


634
RAMEvEVEEL
15.840


1790
NFNWrFVFRF
15.000


2001
RTLVlLLLVL
14.400


1861
RGPElCSVQL
14.400


75
RPLGtLVISL
14.400


947
KLELfLRLGL
14.400


839
KLLVhSLNLL
14.400


1028
RVLEqTLSPL
14.400


131
DFGApIQDSF
14.000


777
KVAErLDQGL
13.824


1081
RALAaPRVKL
13.200


2013
VFLLlVFYTI
12.600


1727
KPRQpISYEL
12.320


1878
RCNLfRCRRL
12.000


427
RAEGlPALRL
12.000


497
RLQLrDDAPL
12.000


1733
SYELrVVIWN
10.500


612
QTPKaVPQHL
10.080


2008
LVLLtVFLLL
10.080


1155
SLPPnVCPVL
10.080


795
KPGPgACAQL
9.600


850
KQGLrLLRGL
9.600


416
RPWArLRVRL
9.600


175
GQQDdEENEL
9.504


143
IIPNvGFQEL
9.504


104
LQVSpIQVEL
9.240


939
AAPGeVCAKL
9.240


1800
DYLPtEREVS
9.000


1495
VYVVkATNLA
9.000


1596
QYEVdGYNAW
9.000


958
KQAKaCTSEL
8.800


429
EGLPaLRLGL
8.640


1418
RSLKeEFNHF
8.640


642
ELLPlPENVL
8.640


183
ELELeLEQDL
8.640


884
EPQPpLPDVL
8.640


1609
FWPSqILAGL
8.400


106
VSPIqVELDL
8.400


1311
LAEAgISRQL
8.400


773
NCVRkVAERL
8.400


635
AMEVeVEELL
8.400


1937
VYILtGKVEA
8.250


1494
RVYVvKATNL
8.000


1872
RNGAgPRCNL
8.000


1639
KVFLtPPETL
8.000


422
RVRLyRAEGL
8.000


479
WNEQlSFVEL
7.920


1032
QTLSpLWDEL
7.920


37
CGPEaDIGEL
7.920


831
NALDrCRGKL
7.920


1601
GYNAwRDAFW
7.500


997
LYQArGVLAA
7.500


1074
GPPVfLGRAL
7.200


1229
TAPLfHPQDL
7.200


1539
IFGEiLELSI
7.200


1047
LIVDgRREHL
7.200


548
AGLRdSLQGL
7.200


1137
EVEPqDLAPL
7.200


1535
QLNPiFGEIL
7.200


1698
HSPGlLQGSL
7.200


1302
QGQKsLDPFL
7.200


1745
DVVLdDENPL
7.200


571
LLLAvSMQVL
7.200


1
MALTvSVQRL
7.200


437
GLLGsLVRAL
7.200


157
AQLErRAVAL
7.200


2002
TLVLlLLVLL
7.200


970
DLLPePSAGL
7.200


78
GTLViSLQQL
7.200


2006
LLLVlLTVFL
7.200


19
RQVKlTFRGF
7.200


844
SLNLlAKQGL
7.200


1446
GGEEeGSGHL
7.200


2025
QISQvIFRPL
6.720


51
HYGApLAGEC
6.600


1537
NPIFgEILEL
6.600


1319
QLLKpPLKKL
6.600


133
GAPIqDSFEL
6.600


1482
GIPQnRPIKL
6.600


1483
IPQNrPIKLL
6.000


1821
EAEFrQPAVL
6.000


2007
LLVLlTVFLL
6.000


1002
GVLAaDDSGL
6.000


1556
LTVAvFDHDL
6.000
















TABLE XVIII







158P3D2v.17, ORF: 65-6175, Frame +2, B7-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












940
APGEVCAKL
240.000


134
APIQDSFEL
240.000


1230
APLFHPQDL
240.000


774
CVRKVAERL
200.000


1148
EPHSGRLSL
120.000


269
CPFYNEYFL
80.000


107
SPIQVELDL
80.000


1699
SPGLLQGSL
80.000


1610
WPSQILAGL
80.000


431
LPALRLGLL
80.000


613
TPKAVPQHL
80.000


454
EPYVRVSFL
80.000


1483
IPQNRPIKL
80.000


144
IPNVGFQEL
80.000


1156
LPPNVCPVL
80.000


1322
KPPLKKLPL
80.000


1405
SPKKAVATL
80.000


492
LTRSLRLQL
60.000


284
RLRLQDLLL
40.000


1026
TTRVLEQTL
40.000


549
GLRDSLQGL
40.000


282
DTRLRLQDL
40.000


1963
KGRKQPEPL
40.000


166
LGRRLARSL
40.000


632
IPRAMEVEV
40.000


707
AAVEAQPLL
36.000


475
AAPEWNEQL
36.000


333
ATRIGTFRM
30.000


363
DPRDTRAGT
30.000


1082
ALAAPRVKL
27.000


1661
DPEEAQALL
24.000


1862
GPELCSVQL
24.000


38
GPEADIGEL
24.000


889
LPDVLVWML
24.000


1097
RPELQFFPL
24.000


1763
YVKSWVKGL
20.000


1876
GPRCNLFRC
20.000


867
KVALAKKLL
20.000


2008
LVLLTVFLL
20.000


2003
LVLLLLVLL
20.000


105
QVSPIQVEL
20.000


1282
DPPEEEGEM
20.000


1746
VVLDDENPL
20.000


1557
TVAVFDHDL
20.000


1224
VVFKDTAPL
20.000


1313
EAGISRQLL
18.000


1616
AGLCQRCGL
18.000


1607
DAFWPSQIL
18.000


651
LAPCEDFLL
12.000


800
ACAQLKQAL
12.000


1841
SANDFLGSL
12.000


959
QAKACTSEL
12.000


162
RAVALGRRL
12.000


870
LAKKLLAKL
12.000


53
GAPLAGECL
12.000


936
APGAAPGEV
12.000


503
DAPLVDAAL
12.000


706
GAAVEAQPL
12.000


1116
IAAFQLIEL
12.000


848
LAKQGLRLL
12.000


2
ALTVSVQRL
12.000


686
RAGRLEEQL
12.000


444
RALHDQRVL
12.000


825
RTMTRPNAL
12.000


1718
VPAPPPVDI
12.000


510
ALATHVPDL
12.000


1048
IVDGRREHL
9.000


1873
NGAGPRCNL
9.000


1504
APADPNGKA
9.000


1186
EVEQPQVVL
9.000


905
WARIPAQDV
9.000


1308
DPFLAEAGI
8.000


490
PPLTRSLRL
8.000


676
QPISFEISI
8.000


670
DPTVASQPI
8.000


1108
GPWAAGELI
8.000


1245
QPPLSILVI
8.000


2022
IPGQISQVI
8.000


1075
PPVFLGRAL
8.000


624
SPGAEGPEI
8.000


1320
LLKPPLKKL
6.000


1471
AVLFSEPQI
6.000


643
LLPLPENVL
6.000


884
EPQPPLPDV
6.000


996
HLYQARGVL
6.000


1146
LVEPHSGRL
6.000


563
QGIWFRGRL
6.000


430
GLPALRLGL
6.000


72
FSLRPLGTL
6.000


542
YGSPPGAGL
6.000


971
LLPEPSAGL
6.000


1085
APRVKLMED
6.000


504
APLVDAALA
6.000


335
RIGTFRMDL
6.000


1168
RVEVLFWGL
6.000


739
DGSGPYFCL
6.000


379
LSVRARGDL
6.000


1943
KVEAEFELL
6.000


1888
RGWWPVVKL
6.000


886
QPPLPDVLV
6.000
















TABLE XIX







158P3D2v.17, ORF: 65-6175, Frame +2, B7-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












1727
KPRQpISYEL
800.000


1856
MVRGaRGPEL
200.000


422
RVRLyRAEGL
200.000


1483
IPQNrPIKLL
120.000


433
ALRLgLLGSL
120.000


416
RPWArLRVRL
120.000


412
VPAErPWARL
120.000


905
WARIpAQDVL
120.000


974
EPSAgLPSSL
80.000


884
EPQPpLPDVL
80.000


75
RPLGtLVISL
80.000


1376
DPGDsDGVNL
80.000


1074
GPPVfLGRAL
80.000


1134
VPSEvEPQDL
80.000


1537
NPIFgEILEL
80.000


320
SPKAfHSQTL
80.000


489
FPPLtRSLRL
80.000


206
SPLKsRARAL
80.000


795
KPGPgACAQL
80.000


1175
GLRGlGRVHL
60.000


1633
AVKVgSKVFL
60.000


1163
VLREfRVEVL
40.000


282
DTRLrLQDLL
40.000


1316
ISRQlLKPPL
40.000


68
CSRVfSLRPL
40.000


634
RAMEvEVEEL
36.000


939
AAPGeVCAKL
36.000


474
AAAPeWNEQL
36.000


509
AALAtHVPDL
36.000


382
RARGdLPPPM
30.000


1639
KVFLtPPETL
30.000


1081
RALAaPRVKL
27.000


1685
VPEHvETRPL
24.000


134
APIQdSFELI
24.000


1028
RVLEqTLSPL
20.000


777
KVAErLDQGL
20.000


649
NVLApCEDFL
20.000


65
VVNCsRVFSL
20.000


1223
TVVFkDTAPL
20.000


2008
LVLLtVFLLL
20.000


4
TVSVqRLTGL
20.000


1002
GVLAaDDSGL
20.000


1745
DVVLdDENPL
20.000


1494
RVYVvKATNL
20.000


1170
EVLFwGLRGL
20.000


747
LPLChCKPCM
20.000


1455
LVGKfKGSFL
20.000


232
GVTVlEAQKL
20.000


943
EVCAkLELFL
20.000


831
NALDrCRGKL
18.000


90
AGHLvLREAL
18.000


1615
LAGLcQRCGL
18.000


869
ALAKkLLAKL
12.000


1411
ATLKiYNRSL
12.000


133
GAPIqDSFEL
12.000


799
GACAqLKQAL
12.000


157
AQLErRAVAL
12.000


591
AQQGsTLSRL
12.000


165
ALGRrLARSL
12.000


1241
QPYLqPPLSI
12.000


706
GAAVeAQPLL
12.000


1229
TAPLfHPQDL
12.000


54
APLAgECLSV
12.000


545
PPGAgLRDSL
12.000


548
AGLRdSLQGL
12.000


945
CAKLeLFLRL
12.000


962
ACTSeLPPDL
12.000


802
AQLKqALEVL
12.000


1
MALTvSVQRL
12.000


1197
AGQGvESEVL
12.000


442
LVRAlHDQRV
10.000


568
RGRLlLAVSM
10.000


936
APGAaPGEVC
9.000


1872
RNGAgPRCNL
9.000


588
PPQAqQGSTL
8.000


1986
NPLKtFVFFI
8.000


1070
HNKFgPPVFL
6.000


429
EGLPaLRLGL
6.000


562
GQGIwFRGRL
6.000


1859
GARGpELCSV
6.000


1047
LIVDgRREHL
6.000


637
EVEVeELLPL
6.000


963
CTSElPPDLL
6.000


642
ELLPlPENVL
6.000


1137
EVEPqDLAPL
6.000


1319
QLLKpPLKKL
6.000


970
DLLPePSAGL
6.000


504
APLVdAALAT
6.000


988
FSYFqLRAHL
6.000


378
TLSVrARGDL
6.000


1085
APRVkLMEDP
6.000


880
FLAEePQPPL
6.000


195
EPDVeLSGVM
6.000


1924
RPEDlEFTDM
6.000


564
GIWFrGRLLL
6.000


427
RAEGlPALRL
5.400


1263
LVGShIVPHM
5.000


1492
LVRVyVVKAT
5.000


2001
RTLVlLLLVL
4.000


1535
QLNPiFGEIL
4.000
















TABLE XX







158P3D2v.17, ORF: 65-6175, Frame +2, B3501-9-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 9 amino acids and the end position


for each peptide is the start position plus eight.









Pos
Subsequence
Score












1282
DPPEEEGEM
120.000


1976
RPKTSFNWF
120.000


736
EPMDGSGPY
80.000


1532
IPKQLNPIF
60.000


1405
SPKKAVATL
60.000


613
TPKAVPQHL
60.000


652
APCEDFLLF
60.000


1507
DPNGKADPY
40.000


1322
KPPLKKLPL
40.000


1973
KPSRPKTSF
40.000


940
APGEVCAKL
40.000


134
APIQDSFEL
30.000


1156
LPPNVCPVL
20.000


1610
WPSQILAGL
20.000


454
EPYVRVSFL
20.000


1230
APLFHPQDL
20.000


1148
EPHSGRLSL
20.000


1986
NPLKTFVFF
20.000


1699
SPGLLQGSL
20.000


107
SPIQVELDL
20.000


1269
VPHMLRFTF
20.000


431
LPALRLGLL
20.000


269
CPFYNEYFL
20.000


144
IPNVGFQEL
20.000


1483
IPQNRPIKL
20.000


418
WARLRVRLY
18.000


1594
ASQYEVDGY
15.000


363
DPRDTRAGT
12.000


632
IPRAMEVEV
12.000


1087
RVKLMEDPY
12.000


624
SPGAEGPEI
12.000


1408
KAVATLKIY
12.000


1661
DPEEAQALL
12.000


1097
RPELQFFPL
12.000


267
TSCPFYNEY
10.000


1922
KGRPEDLEF
9.000


1419
SLKEEFNHF
9.000


444
RALHDQRVL
9.000


959
QAKACTSEL
9.000


38
GPEADIGEL
9.000


848
LAKQGLRLL
9.000


870
LAKKLLAKL
9.000


1466
YPESEAVLF
9.000


1308
DPFLAEAGI
8.000


1802
LPTEREVSV
8.000


1108
GPWAAGELI
8.000


1245
QPPLSILVI
8.000


1139
EPQDLAPLV
8.000


670
DPTVASQPI
8.000


1718
VPAPPPVDI
8.000


1487
RPIKLLVRV
8.000


676
QPISFEISI
8.000


1550
LPAETELTV
8.000


2022
IPGQISQVI
8.000


1207
ASYRESPNF
7.500


1841
SANDFLGSL
6.000


889
LPDVLVWML
6.000


1876
GPRCNLFRC
6.000


686
RAGRLEEQL
6.000


333
ATRIGTFRM
6.000


213
RALAHGDPF
6.000


1035
SPLWDELLV
6.000


475
AAPEWNEQL
6.000


320
SPKAFHSQT
6.000


1111
AAGELIAAF
6.000


284
RLRLQDLLL
6.000


1862
GPELCSVQL
6.000


1963
KGRKQPEPL
6.000


526
RAAGFNPTF
6.000


837
RGKLLVHSL
6.000


448
DQRVLVEPY
6.000


162
RAVALGRRL
6.000


1083
LAAPRVKLM
6.000


549
GLRDSLQGL
6.000


707
AAVEAQPLL
6.000


1265
GSHIVPHML
5.000


1451
GSGHLVGKF
5.000


1783
NSLTGEGNF
5.000


72
FSLRPLGTL
5.000


200
LSGVMFSPL
5.000


379
LSVRARGDL
5.000


1034
LSPLWDELL
5.000


5
VSVQRLTGL
5.000


682
ISIGRAGRL
5.000


2026
ISQVIFRPL
5.000


1548
ISLPAETEL
5.000


1095
YQRPELQFF
4.500


706
GAAVEAQPL
4.500


651
LAPCEDFLL
4.500


1344
IPDPEELDW
4.500


886
QPPLPDVLV
4.000


1161
CPVLREFRV
4.000


531
NPTFGPAWV
4.000


884
EPQPPLPDV
4.000


936
APGAAPGEV
4.000


1059
EPPLVIINV
4.000


1621
RCGLPAPEY
4.000


109
IQVELDLKY
4.000


1677
MPGFGIQLV
4.000


118
QPPEGATGA
4.000
















TABLE XXI







158P3D2v.17, ORF: 65-6175, Frame +2, B3501-10-mers


Each peptide is a portion of SEQ ID NO: 29; each


start position is Specified, the length of the


peptide is 10 amino acids and the end position


for each peptide is the start position plus nine.









Pos
Subsequence
Score












1727
KPRQpISYEL
120.000


1234
HPQDlPEQPY
80.000


1487
RPIKlLVRVY
80.000


1924
RPEDlEFTDM
72.000


1134
VPSEvEPQDL
60.000


320
SPKAfHSQTL
60.000


1376
DPGDsDGVNL
60.000


75
RPLGtLVISL
40.000


412
VPAErPWARL
40.000


747
LPLChCKPCM
40.000


795
KPGPgACAQL
40.000


736
EPMDgSGPYF
40.000


416
RPWArLRVRL
40.000


382
RARGdLPPPM
36.000


1035
SPLWdELLVF
30.000


1802
LPTErEVSVW
30.000


1815
GPFAlEEAEF
30.000


1537
NPIFgEILEL
30.000


269
CPFYnEYFLF
30.000


1976
RPKTsFNWFV
24.000


974
EPSAgLPSSL
20.000


1093
DPYQrPELQF
20.000


2022
IPGQiSQVIF
20.000


1059
EPPLvIINVF
20.000


753
KPCMhVWSCW
20.000


884
EPQPpLPDVL
20.000


1483
IPQNrPIKLL
20.000


1074
GPPVfLGRAL
20.000


206
SPLKsRARAL
20.000


118
QPPEgATGAW
20.000


1847
GSLElQLPDM
20.000


979
LPSSlHRDDF
20.000


489
FPPLtRSLRL
20.000


634
RAMEvEVEEL
18.000


68
CSRVfSLRPL
15.000


1316
ISRQlLKPPL
15.000


886
QPPLpDVLVW
15.000


981
SSLHrDDFSY
15.000


1418
RSLKeEFNHF
15.000


568
RGRLlLAVSM
12.000


1588
RANCgLASQY
12.000


195
EPDVeLSGVM
12.000


1826
QPAVlVLQVW
10.000


1685
VPEHvETRPL
9.000


945
CAKLeLFLRL
9.000


476
APEWnEQLSF
9.000


905
WARIpAQDVL
9.000


1593
LASQyEVDGY
9.000


1986
NPLKtFVFFI
8.000


1241
QPYLqPPLSI
8.000


134
APIQdSFELI
8.000


1725
DIKPrQPISY
6.000


263
TQRGtSCPFY
6.000


54
APLAgECLSV
6.000


1081
RALAaPRVKL
6.000


1730
QPISyELRVV
6.000


831
NALDrCRGKL
6.000


38
GPEAdIGELF
6.000


1117
AAFQlIELDY
6.000


1550
LPAEtELTVA
6.000


322
KAFHsQTLPF
6.000


332
MATRiGTFRM
6.000


422
RVRLyRAEGL
6.000


1297
MPKGpQGQKS
6.000


1163
VLREfRVEVL
6.000


1698
HSPGlLQGSL
5.000


521
ISHPgRAAGF
5.000


470
VSAEaAAPEW
5.000


740
GSGPyFCLPL
5.000


296
VSGVgVTSVL
5.000


1808
VSVWrRSGPF
5.000


267
TSCPfYNEYF
5.000


1758
MSSDiYVKSW
5.000


1840
ISANdFLGSL
5.000


1404
VSPKkAVATL
5.000


1732
ISYElRVVIW
5.000


483
LSFVeLFPPL
5.000


106
VSPIqVELDL
5.000


988
FSYFqLRAHL
5.000


1979
TSFNwFVNPL
5.000


1705
GSLHmWIDIF
5.000


1584
YSHHrANCGL
5.000


133
GAPIqDSFEL
4.500


651
LAPCeDFLLF
4.500


1206
LASYrESPNF
4.500


1648
LPPGsSSPTV
4.000


777
KVAErLDQGL
4.000


1507
DPNGkADPYV
4.000


1861
RGPElCSVQL
4.000


1754
LTGEmSSDIY
4.000


967
LPPDlLPEPS
4.000


908
IPAQdVLFSV
4.000


1028
RVLEqTLSPL
4.000


1973
KPSRpKTSFN
4.000


887
PPLPdVLVWM
4.000


240
KLVGvNINPY
4.000


1643
TPPEtLPPGS
4.000


398
HCSDiEKNLL
3.000


1110
WAAGeLIAAF
3.000


1365
GQHNfDEDEM
3.000









Tables XXII-XLIX







TABLE XXII





158P3D2v.1-A1-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316491


188
0GWWPVVKL
1441796


314
0VLLTVFLL
1441795


62
0KGLEHDKQ
1441794


251
0ELLTVEEA
1441793


125
0VLVLQVWD
1441792


313
00VLLTVFL
1441792


250
40ELLTVEE
262154


312
300VLLTVF
65803


230
FTDMGGNVY
36


34
NTEDVVLDD
25


47
TGEMSSDIY
25


18
IKPRQPISY
21


126
VLVLQVWDY
20


71
ETDVHFNSL
19


51
SSDIYVKSW
18


97
PTEREVSVW
18


326
YTIPGQISQ
18
















TABLE XXIII





158P3D2v.1-A0201-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316492


188
0GWWPVVKL
1441812


314
0VLLTVFLL
1441812


313
00VLLTVFL
1441807


251
0ELLTVEEA
1441804


125
0VLVLQVWD
1441801


62
0KGLEHDKQ
1441794


250
40ELLTVEE
262150


312
300VLLTVF
65807


310
LL300VLLT
29


309
LLL300VLL
27


316
LLTVFLLLV
27


138
SANDFLGSL
25


232
DMGGNVYIL
24


307
VLLLL300V
24


308
LLLL300VL
24


315
VLLTVFLLL
24


320
FLLLVFYTI
24


327
TIPGQISQV
24


142
FLGSLELQL
23


238
YILTGKVEA
22


31
VIWNTEDVV
20


145
SLELQLPDM
20


149
QLPDMVRGA
20


157
ARGPELCSV
20


236
NVYILTGKV
20


248
F240ELLTV
20


2
WIDIFPQDV
19


30
VVIWNTEDV
19


38
VVLDDENPL
19


122
A120VLVLQ
19


239
ILTGKVEAE
19


283
SFNWFVNPL
19


287
FVNPLKTFV
19


290
PLKTFVFFI
19


311
L300VLLTV
19


23
PISYELRVV
18


118
FRQPA120V
18


121
PA120VLVL
18


302
YWRTLVLLL
18


335
VIFRPLHK3
18


39
VLDDENPLT
17


96
LPTEREVSV
17


301
RYWRTLVLL
17


4
DIFPQDVPA
16


10
VPAPPPVDI
16


24
ISYELRVVI
16


46
LTGEMSSDI
16


64
GLEHDKQET
16


140
NDFLGSLEL
16


305
TLVLLLL30
16


27
ELRVVIWNT
15


32
IWNTEDVVL
15


94
DYLPTEREV
15


126
VLVLQVWDY
15


146
LELQLPDMV
15


166
QLARNGAGP
15


170
NGAGPRCNL
15


231
TDMGGNVYI
15


246
AEF240ELL
15


253
LLTVEEAEK
15


298
IWRRYWRTL
15


321
LLLVFYTIP
15
















TABLE XXIV





158P3D2v.1-A0203-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















124
20VLVLQVW
1761625411


249
240ELLTVE
1761625411


123
120VLVLQV
1381322579


186
180GWWPVV
1381322579


62
0KGLEHDKQ
1346719859


125
0VLVLQVWD
1346719859


188
0GWWPVVKL
1346719859


251
0ELLTVEEA
1346719859


313
00VLLTVFL
1346719859


314
0VLLTVFLL
1346719859


312
300VLLTVF
1315204197


187
80GWWPVVK
609485069


250
40ELLTVEE
2911585
















TABLE XXV





158P3D2v.1-A3-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















124
20VLVLQVW
1761625417


249
240ELLTVE
1761625417


186
180GWWPVV
1381322589


123
120VLVLQV
1381322585


125
0VLVLQVWD
1346719876


314
0VLLTVFLL
1346719872


313
00VLLTVFL
1346719864


188
0GWWPVVKL
1346719863


251
0ELLTVEEA
1346719862


62
0KGLEHDKQ
1346719860


312
300VLLTVF
1315204206


187
80GWWPVVK
609485088


250
40ELLTVEE
2911590


334
QVIFRPLHK
31


183
RLR180GWW
23


253
LLTVEEAEK
23


55
YVKSWV60K
22


166
QLARNGAGP
22


59
WV60KGLEH
21


103
SVWRRSGPF
21


122
A120VLVLQ
21


268
RKQPEPLEK
21


318
TVFLLLVFY
21


147
ELQLPDMVR
20


273
PLEKPSRPK
20


161
ELCSVQLAR
19


195
KLKEAEDVE
19


206
AQEAQAGKK
19


177
NLFRCRRLR
18


320
FLLLVFYTI
18


9
DVPAPPPVD
17


45
PLTGEMSSD
17


53
DIYVKSWV6
17


73
DVHFNSLTG
17


126
VLVLQVWDY
17


153
MVRGARGPE
17


209
AQAGKKKRK
17


214
KKRKQRRRK
17


258
EAEKRPVGK
17


300
RRYWRTLVL
17


308
LLLL300VL
17


15
PVDIKPRQP
16


24
ISYELRVVI
16


29
RVVIWNTED
16


78
SLTGEGNFN
16


142
FLGSLELQL
16


193
VVKLKEAED
16


201
DVEREAQEA
16


207
QEAQAGKKK
16


222
KGRPEDLEF
16


236
NVYILTGKV
16


238
YILTGKVEA
16


239
ILTGKVEAE
16


261
KRPVGKGRK
16


263
PVGKGRKQP
16


294
FVFFIWRRY
16


309
LLL300VLL
16


315
VLLTVFLLL
16


327
TIPGQISQV
16
















TABLE XXVI





158P3D2v.1-A26-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316490


314
0VLLTVFLL
1441814


188
0GWWPVVKL
1441805


313
00VLLTVFL
1441805


125
0VLVLQVWD
1441803


251
0ELLTVEEA
1441796


62
0KGLEHDKQ
1441793


250
40ELLTVEE
262145


312
300VLLTVF
65815


318
TVFLLLVFY
30


71
ETDVHFNSL
27


294
FVFFIWRRY
26


317
LTVFLLLVF
26


37
DVVLDDENP
22


73
DVHFNSLTG
21


101
EVSVWRRSG
21


135
DRISANDFL
21


232
DMGGNVYIL
21


103
SVWRRSGPF
20


38
VVLDDENPL
19


230
FTDMGGNVY
19


245
EAEF240EL
19


9
DVPAPPPVD
18


68
DKQETDVHF
18


126
VLVLQVWDY
18


201
DVEREAQEA
18


323
LVFYTIPGQ
18


4
DIFPQDVPA
17


200
EDVEREAQE
17


286
WFVNPLKTF
17


138
SANDFLGSL
16


192
PVVKLKEAE
16


240
LTGKVEAEF
16


30
VVIWNTEDV
15


36
EDVVLDDEN
15


49
EMSSDIYVK
15


55
YVKSWV60K
15


254
LTVEEAEKR
15


304
RTLVLLLL3
15


306
LVLLLL300
15


326
YTIPGQISQ
15


334
QVIFRPLHK
15
















TABLE XXVII





158P3D2v.1-B0702-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















249
240ELLTVE
1761625413


124
20VLVLQVW
1761625412


186
180GWWPVV
1381322590


123
120VLVLQV
1381322588


188
0GWWPVVKL
1346719875


313
00VLLTVFL
1346719873


314
0VLLTVFLL
1346719870


251
0ELLTVEEA
1346719866


125
0VLVLQVWD
1346719861


62
0KGLEHDKQ
1346719860


312
300VLLTVF
1315204205


187
80GWWPVVK
609485073


250
40ELLTVEE
2911588


10
VPAPPPVDI
23


276
KPSRPKTSF
23


159
GPELCSVQL
22


289
NPLKTFVFF
21


120
QPA120VLV
19


328
IPGQISQVI
19


22
QPISYELRV
18


96
LPTEREVSV
18


279
RPKTSFNWF
18


170
NGAGPRCNL
17


19
KPRQPISYE
16


191
WPVVKLKEA
16


266
KGRKQPEPL
15


300
RRYWRTLVL
15


302
YWRTLVLLL
15


32
IWNTEDVVL
14


119
RQPA120VL
14


121
PA120VLVL
14


154
VRGARGPEL
14


232
DMGGNVYIL
14


262
RPVGKGRKQ
14


12
APPPVDIKP
13


59
WV60KGLEH
13


71
ETDVHFNSL
13


105
WRRSGPFAL
13


142
FLGSLELQL
13


150
LPDMVRGAR
13


220
RRKGRPEDL
13


298
IWRRYWRTL
13


301
RYWRTLVLL
13


309
LLL300VLL
13


315
VLLTVFLLL
13


332
ISQVIFRPL
13


6
FPQDVPAPP
12


16
VDIKPRQPI
12


57
KSWV60KGL
12


88
RFVFRFDYL
12


109
GPFALEEAE
12


140
NDFLGSLEL
12


173
GPRCNLFRC
12


222
KGRPEDLEF
12


246
AEF240ELL
12


270
QPEPLEKPS
12


283
SFNWFVNPL
12


303
WRTLVLLLL
12
















TABLE XXVIII





158P3D2v.1-B08-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316491


188
0GWWPVVKL
1441804


313
00VLLTVFL
1441804


314
0VLLTVFLL
1441804


251
0ELLTVEEA
1441795


125
0VLVLQVWD
1441794


62
0KGLEHDKQ
1441793


250
40ELLTVEE
262147


312
300VLLTVF
65809


220
RRKGRPEDL
28


181
CRRLR180G
25


193
VVKLKEAED
23


290
PLKTFVFFI
23


17
DIKPRQPIS
22


183
RLR180GWW
22


279
RPKTSFNWF
22


211
AGKKKRKQR
21


154
VRGARGPEL
20


213
KKKRKQRRR
20


272
EPLEKPSRP
20


159
GPELCSVQL
19


171
GAGPRCNLF
19


191
WPVVKLKEA
19


212
GKKKRKQRR
19


239
ILTGKVEAE
19


264
VGKGRKQPE
19


88
RFVFRFDYL
18


120
QPA120VLV
18


176
CNLFRCRRL
18


195
KLKEAEDVE
18


215
KRKQRRRKG
18


245
EAEF240EL
18


288
VNPLKTFVF
18


300
RRYWRTLVL
18


59
WV60KGLEH
17


96
LPTEREVSV
17


105
WRRSGPFAL
17


142
FLGSLELQL
17


298
IWRRYWRTL
17


302
YWRTLVLLL
17


308
LLLL300VL
17


309
LLL300VLL
17


315
VLLTVFLLL
17


27
ELRVVIWNT
16


103
SVWRRSGPF
16


138
SANDFLGSL
16


218
QRRRKGRPE
16


262
RPVGKGRKQ
16


266
KGRKQPEPL
16


277
PSRPKTSFN
16


53
DIYVKSWV6
15


115
EAEFRQPA1
15


121
PA120VLVL
15


258
EAEKRPVGK
15


10
VPAPPPVDI
14


178
LFRCRRLR1
14


210
QAGKKKRKQ
14


217
KQRRRKGRP
14


257
EEAEKRPVG
14


320
FLLLVFYTI
14
















TABLE XXIX





158P3D2v.1-B1510-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















249
240ELLTVE
1761625414


124
20VLVLQVW
1761625413


186
180GWWPVV
1381322583


123
120VLVLQV
1381322581


188
0GWWPVVKL
1346719873


313
00VLLTVFL
1346719871


314
0VLLTVFLL
1346719870


125
0VLVLQVWD
1346719862


251
0ELLTVEEA
1346719861


62
0KGLEHDKQ
1346719859


312
300VLLTVF
1315204206


187
80GWWPVVK
609485074


250
40ELLTVEE
2911589


32
IWNTEDVVL
16


298
IWRRYWRTL
15


332
ISQVIFRPL
15


159
GPELCSVQL
14


170
NGAGPRCNL
14


176
CNLFRCRRL
14


245
EAEF240EL
14


20
PRQPISYEL
13


57
KSWV60KGL
13


105
WRRSGPFAL
13


119
RQPA120VL
13


121
PA120VLVL
13


232
DMGGNVYIL
13


66
EHDKQETDV
12


71
ETDVHFNSL
12


140
NDFLGSLEL
12


154
VRGARGPEL
12


220
RRKGRPEDL
12


266
KGRKQPEPL
12


300
RRYWRTLVL
12


301
RYWRTLVLL
12


302
YWRTLVLLL
12


308
LLLL300VL
12


309
LLL300VLL
12


38
VVLDDENPL
11


138
SANDFLGSL
11


283
SFNWFVNPL
11


74
VHFNSLTGE
10


83
GNFNWRFVF
10


88
RFVFRFDYL
10


135
DRISANDFL
10


142
FLGSLELQL
10


246
AEF240ELL
10


303
WRTLVLLLL
10


315
VLLTVFLLL
10


42
DENPLTGEM
9


68
DKQETDVHF
9


81
GEGNFNWRF
9


85
FNWRFVFRF
9


171
GAGPRCNLF
9


222
KGRPEDLEF
9


276
KPSRPKTSF
9


289
NPLKTFVFF
9


24
ISYELRVVI
8


110
PFALEEAEF
8


145
SLELQLPDM
8


240
LTGKVEAEF
8


286
WFVNPLKTF
8


288
VNPLKTFVF
8


317
LTVFLLLVF
8


329
PGQISQVIF
8
















TABLE XXX





158P3D2v.1-B2705-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















249
240ELLTVE
1761625417


124
20VLVLQVW
1761625416


186
180GWWPVV
1381322587


123
120VLVLQV
1381322583


188
0GWWPVVKL
1346719874


313
00VLLTVFL
1346719873


314
0VLLTVFLL
1346719873


125
0VLVLQVWD
1346719865


251
0ELLTVEEA
1346719864


62
0KGLEHDKQ
1346719863


312
300VLLTVF
1315204212


187
80GWWPVVK
609485081


250
40ELLTVEE
2911589


300
RRYWRTLVL
28


261
KRPVGKGRK
27


220
RRKGRPEDL
26


20
PRQPISYEL
25


99
EREVSVWRR
25


303
WRTLVLLLL
24


135
DRISANDFL
23


105
WRRSGPFAL
22


154
VRGARGPEL
22


87
WRFVFRFDY
21


174
PRCNLFRCR
21


219
RRRKGRPED
20


140
NDFLGSLEL
19


185
R180GWWPV
19


92
RFDYLPTER
18


106
RRSGPFALE
18


119
RQPA120VL
18


182
RRLR180GW
18


212
GKKKRKQRR
18


235
GNVYILTGK
18


268
RKQPEPLEK
18


81
GEGNFNWRF
17


83
GNFNWRFVF
17


159
GPELCSVQL
17


175
RCNLFRCRR
17


209
AQAGKKKRK
17


213
KKKRKQRRR
17


222
KGRPEDLEF
17


267
GRKQPEPLE
17


276
KPSRPKTSF
17


293
TFVFFIWRR
17


330
GQISQVIFR
17


77
NSLTGEGNF
16


91
FRFDYLPTE
16


168
ARNGAGPRC
16


189
GWWPVVKLK
16


214
KKRKQRRRK
16


215
KRKQRRRKG
16


246
AEF240ELL
16


254
LTVEEAEKR
16


279
RPKTSFNWF
16


292
KTFVFFIWR
16


301
RYWRTLVLL
16


21
RQPISYELR
15


32
IWNTEDVVL
15


49
EMSSDIYVK
15


88
RFVFRFDYL
15


111
FALEEAEFR
15


121
PA120VLVL
15


147
ELQLPDMVR
15


157
ARGPELCSV
15


170
NGAGPRCNL
15


171
GAGPRCNLF
15


208
EAQAGKKKR
15


211
AGKKKRKQR
15


223
GRPEDLEFT
15


240
LTGKVEAEF
15


271
PEPLEKPSR
15


278
SRPKTSFNW
15


289
NPLKTFVFF
15


317
LTVFLLLVF
15


318
TVFLLLVFY
15


337
FRPLHK328
15


28
LRVVIWNTE
14


98
TEREVSVWR
14


110
PFALEEAEF
14


161
ELCSVQLAR
14


176
CNLFRCRRL
14


196
LKEAEDVER
14


205
EAQEAQAGK
14


206
AQEAQAGKK
14


207
QEAQAGKKK
14


216
RKQRRRKGR
14


232
DMGGNVYIL
14


266
KGRKQPEPL
14


273
PLEKPSRPK
14


286
WFVNPLKTF
14


294
FVFFIWRRY
14


308
LLLL300VL
14


315
VLLTVFLLL
14


329
PGQISQVIF
14
















TABLE XXXI





158P3D2v.1-B2709-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316491


314
0VLLTVFLL
1441805


188
0GWWPVVKL
1441804


313
00VLLTVFL
1441804


125
0VLVLQVWD
1441795


251
0ELLTVEEA
1441795


62
0KGLEHDKQ
1441792


250
40ELLTVEE
262145


312
300VLLTVF
65810


300
RRYWRTLVL
27


185
R180GWWPV
24


220
RRKGRPEDL
24


20
PRQPISYEL
23


135
DRISANDFL
22


303
WRTLVLLLL
22


105
WRRSGPFAL
21


154
VRGARGPEL
21


157
ARGPELCSV
20


118
FRQPA120V
19


299
WRRYWRTLV
18


182
RRLR180GW
16


88
RFVFRFDYL
15


159
GPELCSVQL
15


219
RRRKGRPED
15


246
AEF240ELL
15


301
RYWRTLVLL
15


106
RRSGPFALE
14


119
RQPA120VL
14


121
PA120VLVL
14


223
GRPEDLEFT
14


267
GRKQPEPLE
14
















TABLE XXXII





158P3D2v.1-B4402-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















124
20VLVLQVW
1761625424


249
240ELLTVE
1761625414


123
120VLVLQV
1381322582


186
180GWWPVV
1381322581


314
0VLLTVFLL
1346719873


188
0GWWPVVKL
1346719872


251
0ELLTVEEA
1346719872


313
00VLLTVFL
1346719872


62
0KGLEHDKQ
1346719861


125
0VLVLQVWD
1346719861


312
300VLLTVF
1315204209


187
80GWWPVVK
609485072


250
40ELLTVEE
2911588


246
AEF240ELL
28


81
GEGNFNWRF
21


116
AEFRQPA12
20


199
AEDVEREAQ
17


57
KSWV60KGL
16


171
GAGPRCNLF
16


259
AEKRPVGKG
16


25
SYELRVVIW
15


51
SSDIYVKSW
15


71
ETDVHFNSL
15


83
GNFNWRFVF
15


140
NDFLGSLEL
15


222
KGRPEDLEF
15


274
LEKPSRPKT
15


286
WFVNPLKTF
15


315
VLLTVFLLL
15


318
TVFLLLVFY
15


18
IKPRQPISY
14


20
PRQPISYEL
14


26
YELRVVIWN
14


42
DENPLTGEM
14


121
PA120VLVL
14


245
EAEF240EL
14


257
EEAEKRPVG
14


288
VNPLKTFVF
14


289
NPLKTFVFF
14


294
FVFFIWRRY
14


295
VFFIWRRYW
14


300
RRYWRTLVL
14


301
RYWRTLVLL
14


302
YWRTLVLLL
14


303
WRTLVLLLL
14


309
LLL300VLL
14
















TABLE XXXIII





158P3D2v.1-B5101-9-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















187
80GWWPVVK
1755316495


188
0GWWPVVKL
1441810


314
0VLLTVFLL
1441803


313
00VLLTVFL
1441799


251
0ELLTVEEA
1441797


125
0VLVLQVWD
1441795


62
0KGLEHDKQ
1441794


250
40ELLTVEE
262145


312
300VLLTVF
65807


328
IPGQISQVI
25


121
PA120VLVL
24


10
VPAPPPVDI
23


96
LPTEREVSV
23


24
ISYELRVVI
22


22
QPISYELRV
21


120
QPA120VLV
19


138
SANDFLGSL
18


320
FLLLVFYTI
18


159
GPELCSVQL
17


272
EPLEKPSRP
17


289
NPLKTFVFF
17


311
L300VLLTV
17


324
VFYTIPGQI
17


12
APPPVDIKP
16


94
DYLPTEREV
16


111
FALEEAEFR
16


245
EAEF240EL
16


6
FPQDVPAPP
15


46
LTGEMSSDI
15


82
EGNFNWRFV
15


210
QAGKKKRKQ
15


236
NVYILTGKV
15


248
F240ELLTV
15


262
RPVGKGRKQ
15


31
VIWNTEDVV
14


44
NPLTGEMSS
14


150
LPDMVRGAR
14


170
NGAGPRCNL
14


198
EAEDVEREA
14


208
EAQAGKKKR
14


231
TDMGGNVYI
14


266
KGRKQPEPL
14


279
RPKTSFNWF
14


300
RRYWRTLVL
14


315
VLLTVFLLL
14


11
PAPPPVDIK
13


13
PPPVDIKPR
13


23
PISYELRVV
13


32
IWNTEDVVL
13


129
LQVWDYDRI
13


146
LELQLPDMV
13


167
LARNGAGPR
13


191
WPVVKLKEA
13


194
VKLKEAEDV
13


224
RPEDLEFTD
13


232
DMGGNVYIL
13


258
EAEKRPVGK
13


290
PLKTFVFFI
13


308
LLLL300VL
13
















TABLE XXXIV





158P3D2v.1-A1-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















124
20VLVLQVWD
1761625411


249
240ELLTVEE
1761625411


186
180GWWPVVK
1381322581


123
120VLVLQVW
1381322580


125
0VLVLQVWDY
1346719875


314
0VLLTVFLLL
1346719870


188
0GWWPVVKLK
1346719862


313
00VLLTVFLL
1346719862


62
0KGLEHDKQE
1346719859


251
0ELLTVEEAE
1346719859


312
300VLLTVFL
1315204197


187
80GWWPVVKL
609485072


250
40ELLTVEEA
2911596


17
DIKPRQPISY
23


46
LTGEMSSDIY
21


317
LTVFLLLVFY
21


71
ETDVHFNSLT
19


39
VLDDENPLTG
18


230
FTDMGGNVYI
18


25
SYELRVVIWN
17


139
ANDFLGSLEL
17


229
EFTDMGGNVY
17


34
NTEDVVLDDE
16


51
SSDIYVKSWV
16


86
NWRFVFRFDY
16


97
PTEREVSVWR
16


112
ALEEAEFRQP
15


293
TFVFFIWRRY
15


47
TGEMSSDIYV
14


145
SLELQLPDMV
14


206
AQEAQAGKKK
14


270
QPEPLEKPSR
14


273
PLEKPSRPKT
14


159
GPELCSVQLA
13


224
RPEDLEFTDM
13


256
VEEAEKRPVG
13


258
EAEKRPVGKG
13


80
TGEGNFNWRF
12


115
EAEFRQPA12
12


189
GWWPVVKLKE
12


245
EAEF240ELL
12


304
RTLVLLLL30
12


326
YTIPGQISQV
12
















TABLE XXXV





158P3D2v.1-A0201-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















187
80GWWPVVKL
1755316508


314
0VLLTVFLLL
1441809


313
00VLLTVFLL
1441806


125
0VLVLQVWDY
1441802


188
0GWWPVVKLK
1441796


251
0ELLTVEEAE
1441795


62
0KGLEHDKQE
1441791


250
40ELLTVEEA
262153


312
300VLLTVFL
65814


310
LL300VLLTV
37


122
A120VLVLQV
28


315
VLLTVFLLLV
28


95
YLPTEREVSV
26


308
LLLL300VLL
26


247
EF240ELLTV
25


309
LLL300VLLT
24


326
YTIPGQISQV
24


156
GARGPELCSV
23


307
VLLLL300VL
23


31
VIWNTEDVVL
22


297
FIWRRYWRTL
22


145
SLELQLPDMV
21


137
ISANDFLGSL
20


128
VLQVWDYDRI
19


153
MVRGARGPEL
19


231
TDMGGNVYIL
18


306
LVLLLL300V
18


327
TIPGQISQVI
18


331
QISQVIFRPL
18
















TABLE XXXVI





158P3D2v.1-A0203-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















124
20VLVLQVWD
1761625411


249
240ELLTVEE
1761625411


123
120VLVLQVW
1381322579


186
180GWWPVVK
1381322579


251
0ELLTVEEAE
1346719868


62
0KGLEHDKQE
1346719859


125
0VLVLQVWDY
1346719859


188
0GWWPVVKLK
1346719859


313
00VLLTVFLL
1346719859


314
0VLLTVFLLL
1346719859


312
300VLLTVFL
1315204197


187
80GWWPVVKL
609485069


250
40ELLTVEEA
2911595


202
VEREAQEAQA
18


3
IDIFPQDVPA
10


103
SVWRRSGPFA
10


107
RSGPFALEEA
10


113
LEEAEFRQPA
10


130
QVWDYDRISA
10


148
LQLPDMVRGA
10


159
GPELCSVQLA
10


163
CSVQLARNGA
10


190
WWPVVKLKEA
10


197
KEAEDVEREA
10


200
EDVEREAQEA
10


237
VYILTGKVEA
10


4
DIFPQDVPAP
9


104
VWRRSGPFAL
9


108
SGPFALEEAE
9


114
EEAEFRQPA1
9


131
VWDYDRISAN
9


149
QLPDMVRGAR
9


160
PELCSVQLAR
9


164
SVQLARNGAG
9


191
WPVVKLKEAE
9


198
EAEDVEREAQ
9


201
DVEREAQEAQ
9


203
EREAQEAQAG
9


238
YILTGKVEAE
9
















TABLE XXXVII





158P3D2v.1-A3-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















124
20VLVLQVWD
1761625417


249
240ELLTVEE
1761625416


186
180GWWPVVK
1381322604


123
120VLVLQVW
1381322583


125
0VLVLQVWDY
1346719877


314
0VLLTVFLLL
1346719875


188
0GWWPVVKLK
1346719869


62
0KGLEHDKQE
1346719863


251
0ELLTVEEAE
1346719863


313
00VLLTVFLL
1346719860


312
300VLLTVFL
1315204201


187
80GWWPVVKL
609485073


250
40ELLTVEEA
2911586


183
RLR180GWWP
25


252
ELLTVEEAEK
25


166
QLARNGAGPR
24


195
KLKEAEDVER
23


122
A120VLVLQV
22


236
NVYILTGKVE
22


17
DIKPRQPISY
21


239
ILTGKVEAEF
21


287
FVNPLKTFVF
21


316
LLTVFLLLVF
21


149
QLPDMVRGAR
20


39
VLDDENPLTG
19


153
MVRGARGPEL
18


204
REAQEAQAGK
18


206
AQEAQAGKKK
18


213
KKKRKQRRRK
18


9
DVPAPPPVDI
17


23
PISYELRVVI
17


27
ELRVVIWNTE
17


30
VVIWNTEDVV
17


31
VIWNTEDVVL
17


103
SVWRRSGPFA
17


112
ALEEAEFRQP
17


136
RISANDFLGS
17


177
NLFRCRRLR1
17


247
EF240ELLTV
17


253
LLTVEEAEKR
17


257
EEAEKRPVGK
17


260
EKRPVGKGRK
17


307
VLLLL300VL
17


308
LLLL300VLL
17


310
LL300VLLTV
17


53
DIYVKSWV60
16


89
FVFRFDYLPT
16


95
YLPTEREVSV
16


127
LVLQVWDYDR
16


130
QVWDYDRISA
16


333
SQVIFRPLHK
16


334
QVIFRPLHK3
16


29
RVVIWNTEDV
15


45
PLTGEMSSDI
15


147
ELQLPDMVRG
15


193
VVKLKEAEDV
15


205
EAQEAQAGKK
15


243
KVEAEF240E
15


267
GRKQPEPLEK
15


297
FIWRRYWRTL
15


309
LLL300VLLT
15


315
VLLTVFLLLV
15


323
LVFYTIPGQI
15


10
VPAPPPVDIK
14


48
GEMSSDIYVK
14


64
GLEHDKQETD
14


101
EVSVWRRSGP
14


145
SLELQLPDMV
14


164
SVQLARNGAG
14


201
DVEREAQEAQ
14


221
RKGRPEDLEF
14


229
EFTDMGGNVY
14


259
AEKRPVGKGR
14


320
FLLLVFYTIP
14


327
TIPGQISQVI
14


2
WIDIFPQDVP
13


38
VVLDDENPLT
13


54
IYVKSWV60K
13


78
SLTGEGNFNW
13


157
ARGPELCSVQ
13


161
ELCSVQLARN
13


192
PVVKLKEAED
13


272
EPLEKPSRPK
13


306
LVLLLL300V
13
















TABLE XXXVIII





158P3D2v.1-A26-10-mers


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















187
80GWWPVVKL
1755316503


125
0VLVLQVWDY
1441816


314
0VLLTVFLLL
1441811


313
00VLLTVFLL
1441808


188
0GWWPVVKLK
1441796


62
0KGLEHDKQE
1441793


251
0ELLTVEEAE
1441793


250
40ELLTVEEA
262148


312
300VLLTVFL
65810


37
DVVLDDENPL
30


317
LTVFLLLVFY
26


17
DIKPRQPISY
25


229
EFTDMGGNVY
23


4
DIFPQDVPAP
22


73
DVHFNSLTGE
22


101
EVSVWRRSGP
21


46
LTGEMSSDIY
20


275
EKPSRPKTSF
20


334
QVIFRPLHK3
20


71
ETDVHFNSLT
19


153
MVRGARGPEL
19


201
DVEREAQEAQ
19


287
FVNPLKTFVF
19


318
TVFLLLVFYT
19


326
YTIPGQISQV
19


9
DVPAPPPVDI
18


82
EGNFNWRFVF
18


141
DFLGSLELQL
18


245
EAEF240ELL
18


282
TSFNWFVNPL
17


285
NWFVNPLKTF
17


133
DYDRISANDF
16


200
EDVEREAQEA
16


247
EF240ELLTV
16


258
EAEKRPVGKG
16


292
KTFVFFIWRR
16


323
LVFYTIPGQI
16


89
FVFRFDYLPT
15


161
ELCSVQLARN
15


293
TFVFFIWRRY
15
















TABLE XXXIX





158P3D2v.1-B0702-10-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 10 amino acids, and the


end position for each peptide is the start


position plus nine.

















249
240ELLTVEE
1761625414


124
20VLVLQVWD
1761625413


186
180GWWPVVK
1381322585


123
120VLVLQVW
1381322580


314
0VLLTVFLLL
1346719872


313
00VLLTVFLL
1346719870


62
0KGLEHDKQE
1346719860


125
0VLVLQVWDY
1346719860


188
0GWWPVVKLK
1346719860


251
0ELLTVEEAE
1346719860


312
300VLLTVFL
1315204211


187
80GWWPVVKL
609485083


250
40ELLTVEEA
2911592


120
QPA120VLVL
25


19
KPRQPISYEL
23


328
IPGQISQVIF
20


224
RPEDLEFTDM
19


289
NPLKTFVFFI
19


109
GPFALEEAEF
18


279
RPKTSFNWFV
18


22
QPISYELRVV
17


159
GPELCSVQLA
17


299
WRRYWRTLVL
16


139
ANDFLGSLEL
15


153
MVRGARGPEL
15


276
KPSRPKTSFN
15


12
APPPVDIKPR
14


141
DFLGSLELQL
14


169
RNGAGPRCNL
14


219
RRRKGRPEDL
14


231
TDMGGNVYIL
14


262
RPVGKGRKQP
14


301
RYWRTLVLLL
14


302
YWRTLVLLLL
14


331
QISQVIFRPL
14


10
VPAPPPVDIK
13


31
VIWNTEDVVL
13


104
VWRRSGPFAL
13


134
YDRISANDFL
13


158
RGPELCSVQL
13


173
GPRCNLFRCR
13


300
RRYWRTLVLL
13
















TABLE XL





158P3D2v.1-B08-10-mers







No Results Found.
















TABLE XLI





158P3D2v.1-B1510-10-mers







No Results Found.
















TABLE XLII





158P3D2v.1-B2705-10-mers







No Results Found.
















TABLE XLIII





158P3D2v.1-B2709-10-mers







No Results Found.
















TABLE XLIV





158P3D2v.1-B4402-10-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 10 amino acids, and the


end position for each peptide is the start


position plus nine.

















187
80GWWPVVKL
1755316503


314
0VLLTVFLLL
1441807


251
0ELLTVEEAE
1441806


313
00VLLTVFLL
1441805


125
0VLVLQVWDY
1441804


62
0KGLEHDKQE
1441796


188
0GWWPVVKLK
1441796


250
40ELLTVEEA
262146


312
300VLLTVFL
65814


70
QETDVHFNSL
23


244
VEAEF240EL
20


246
AEF240ELLT
19


116
AEFRQPA120
18


274
LEKPSRPKTS
17


285
NWFVNPLKTF
17


259
AEKRPVGKGR
16


17
DIKPRQPISY
15


26
YELRVVIWNT
15


48
GEMSSDIYVK
15


50
MSSDIYVKSW
15


114
EEAEFRQPA1
15


139
ANDFLGSLEL
15


199
AEDVEREAQE
15


229
EFTDMGGNVY
15


282
TSFNWFVNPL
15


287
FVNPLKTFVF
15


82
EGNFNWRFVF
14


104
VWRRSGPFAL
14


118
FRQPA120VL
14


120
QPA120VLVL
14


133
DYDRISANDF
14


141
DFLGSLELQL
14


160
PELCSVQLAR
14


170
NGAGPRCNLF
14


245
EAEF240ELL
14


257
EEAEKRPVGK
14


278
SRPKTSFNWF
14


288
VNPLKTFVFF
14


294
FVFFIWRRYW
14


301
RYWRTLVLLL
14


302
YWRTLVLLLL
14


308
LLLL300VLL
14


31
VIWNTEDVVL
13


42
DENPLTGEMS
13


56
VKSWV60KGL
13


87
WRFVFRFDYL
13


158
RGPELCSVQL
13


207
QEAQAGKKKR
13


225
PEDLEFTDMG
13


228
LEFTDMGGNV
13


231
TDMGGNVYIL
13


271
PEPLEKPSRP
13


275
EKPSRPKTSF
13


277
PSRPKTSFNW
13


297
FIWRRYWRTL
13


299
WRRYWRTLVL
13


300
RRYWRTLVLL
13


307
VLLLL300VL
13


316
LLTVFLLLVF
13


331
QISQVIFRPL
13


12
APPPVDIKPR
12


23
PISYELRVVI
12


24
ISYELRVVIW
12


37
DVVLDDENPL
12


76
FNSLTGEGNF
12


78
SLTGEGNFNW
12


81
GEGNFNWRFV
12


84
NFNWRFVFRF
12


86
NWRFVFRFDY
12


96
LPTEREVSVW
12


100
REVSVWRRSG
12


109
GPFALEEAEF
12


137
ISANDFLGSL
12


146
LELQLPDMVR
12


175
RCNLFRCRRL
12


182
RRLR180GWW
12


197
KEAEDVEREA
12


221
RKGRPEDLEF
12


256
VEEAEKRPVG
12


265
GKGRKQPEPL
12


290
PLKTFVFFIW
12


311
L300VLLTVF
12


317
LTVFLLLVFY
12


319
VFLLLVFYTI
12


323
LVFYTIPGQI
12
















TABLE XLV





158P3D2v.1-B5101-10-mers







No Results Found.
















TABLE XLVI





158P3D2v.1-DRB1 0101-15-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the start


position plus fourteen.

















124
20VLVLQVWDYDRIS
1761625427


249
240ELLTVEEAEKRP
1761625412


123
120VLVLQVWDYDRI
1381322595


186
180GWWPVVKLKEAE
1381322580


188
0GWWPVVKLKEAEDV
1346719884


313
00VLLTVFLLLVFYT
1346719881


251
0ELLTVEEAEKRPVG
1346719878


314
0VLLTVFLLLVFYTI
1346719876


125
0VLVLQVWDYDRISA
1346719873


62
0KGLEHDKQETDVHF
1346719867


312
300VLLTVFLLLVFY
1315204214


187
80GWWPVVKLKEAED
609485086


250
40ELLTVEEAEKRPV
2911600


300
RRYWRTLVLLLL300
36


131
VWDYDRISANDFLGS
32


322
LLVFYTIPGQISQVI
32


285
NWFVNPLKTFVFFIW
31


305
TLVLLLL300VLLTV
31


73
DVHFNSLTGEGNFNW
29


143
LGSLELQLPDMVRGA
29


234
GGNVYILTGKVEAEF
28


245
EAEF240ELLTVEEA
28


325
FYTIPGQISQVIFRP
28


102
VSVWRRSGPFALEEA
27


181
CRRLR180GWWPVVK
27


191
WPVVKLKEAEDVERE
27


317
LTVFLLLVFYTIPGQ
27


27
ELRVVIWNTEDVVLD
26


151
PDMVRGARGPELCSV
26


227
DLEFTDMGGNVYILT
26


40
LDDENPLTGEMSSDI
25


140
NDFLGSLELQLPDMV
25


237
VYILTGKVEAEF240
25


304
RTLVLLLL300VLLT
25


3
IDIFPQDVPAPPPVD
24


90
VFRFDYLPTEREVSV
24


101
EVSVWRRSGPFALEE
24


137
ISANDFLGSLELQLP
24


308
LLLL300VLLTVFLL
24


318
TVFLLLVFYTIPGQI
24


43
ENPLTGEMSSDIYVK
23


329
PGQISQVIFRPLHK3
23


2
WIDIFPQDVPAPPPV
22


4
DIFPQDVPAPPPVDI
22


7
PQDVPAPPPVDIKPR
22


12
APPPVDIKPRQPISY
22


120
QPA120VLVLQVWDY
22


156
GARGPELCSVQLARN
22


233
MGGNVYILTGKVEAE
22


310
LL300VLLTVFLLLV
22


321
LLLVFYTIPGQISQV
22


296
FFIWRRYWRTLVLLL
21


86
NWRFVFRFDYLPTER
20


108
SGPFALEEAEFRQPA
20


115
EAEFRQPA120VLVL
20


149
QLPDMVRGARGPELC
20


235
GNVYILTGKVEAEF2
20


284
FNWFVNPLKTFVFFI
20


13
PPPVDIKPRQPISYE
19


49
EMSSDIYVKSWV60K
19


52
SDIYVKSWV60KGLE
19


56
VKSWV60KGLEHDKQ
19


82
EGNFNWRFVFRFDYL
19


84
NFNWRFVFRFDYLPT
19


92
RFDYLPTEREVSVWR
19


161
ELCSVQLARNGAGPR
19


190
WWPVVKLKEAEDVER
19


283
SFNWFVNPLKTFVFF
19


35
TEDVVLDDENPLTGE
18


99
EREVSVWRRSGPFAL
18


110
PFALEEAEFRQPA12
18


117
EFRQPA120VLVLQV
18


134
YDRISANDFLGSLEL
18


139
ANDFLGSLELQLPDM
18


163
CSVQLARNGAGPRCN
18


198
EAEDVEREAQEAQAG
18


217
KQRRRKGRPEDLEFT
18


222
KGRPEDLEFTDMGGN
18


253
LLTVEEAEKRPVGKG
18


299
WRRYWRTLVLLLL30
18


303
WRTLVLLLL300VLL
18


306
LVLLLL300VLLTVF
18


311
L300VLLTVFLLLVF
18


331
QISQVIFRPLHK328
18
















TABLE XLVII





158P3D2v.1-DRB1 0301-15-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the start


position plus fourteen.

















187
80GWWPVVKLKEAED
1755316499


62
0KGLEHDKQETDVHF
1441819


314
0VLLTVFLLLVFYTI
1441813


313
00VLLTVFLLLVFYT
1441806


251
0ELLTVEEAEKRPVG
1441805


125
0VLVLQVWDYDRISA
1441804


188
0GWWPVVKLKEAEDV
1441793


250
40ELLTVEEAEKRPV
262164


312
300VLLTVFLLLVFY
65825


35
TEDVVLDDENPLTGE
37


237
VYILTGKVEAEF240
34


36
EDVVLDDENPLTGEM
30


139
ANDFLGSLELQLPDM
26


306
LVLLLL300VLLTVF
24


47
TGEMSSDIYVKSWV6
23


305
TLVLLLL300VLLTV
21


308
LLLL300VLLTVFLL
21


15
PVDIKPRQPISYELR
20


29
RVVIWNTEDVVLDDE
20


245
EAEF240ELLTVEEA
20


285
NWFVNPLKTFVFFIW
20


3
IDIFPQDVPAPPPVD
19


76
FNSLTGEGNFNWRFV
19


88
RFVFRFDYLPTEREV
19


151
PDMVRGARGPELCSV
19


190
WWPVVKLKEAEDVER
19


227
DLEFTDMGGNVYILT
19


13
PPPVDIKPRQPISYE
18


21
RQPISYELRVVIWNT
18


135
DRISANDFLGSLELQ
18


162
LCSVQLARNGAGPRC
18


175
RCNLFRCRRLR180G
18


221
RKGRPEDLEFTDMGG
18


253
LLTVEEAEKRPVGKG
18


271
PEPLEKPSRPKTSFN
18


295
VFFIWRRYWRTLVLL
18


329
PGQISQVIFRPLHK3
18
















TABLE XLVIII





158P3D2v.1-DRB1 0401-15-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the start


position plus fourteen.

















124
20VLVLQVWDYDRIS
1761625419


249
240ELLTVEEAEKRP
1761625411


123
120VLVLQVWDYDRI
1381322593


186
180GWWPVVKLKEAE
1381322585


188
0GWWPVVKLKEAEDV
1346719881


62
0KGLEHDKQETDVHF
1346719879


313
00VLLTVFLLLVFYT
1346719879


125
0VLVLQVWDYDRISA
1346719873


251
0ELLTVEEAEKRPVG
1346719873


314
0VLLTVFLLLVFYTI
1346719873


312
300VLLTVFLLLVFY
1315204211


187
80GWWPVVKLKEAED
609485085


250
40ELLTVEEAEKRPV
2911605


281
KTSFNWFVNPLKTFV
28


300
RRYWRTLVLLLL300
28


21
RQPISYELRVVIWNT
26


36
EDVVLDDENPLTGEM
26


43
ENPLTGEMSSDIYVK
26


199
AEDVEREAQEAQAGK
26


111
FALEEAEFRQPA120
24


245
EAEF240ELLTVEEA
24


73
DVHFNSLTGEGNFNW
22


84
NFNWRFVFRFDYLPT
22


90
VFRFDYLPTEREVSV
22


92
RFDYLPTEREVSVWR
22


129
LQVWDYDRISANDFL
22


296
FFIWRRYWRTLVLLL
22


308
LLLL300VLLTVFLL
22


317
LTVFLLLVFYTIPGQ
22


322
LLVFYTIPGQISQVI
22


323
LVFYTIPGQISQVIF
22


27
ELRVVIWNTEDVVLD
20


35
TEDVVLDDENPLTGE
20


47
TGEMSSDIYVKSWV6
20


76
FNSLTGEGNFNWRFV
20


87
WRFVFRFDYLPTERE
20


93
FDYLPTEREVSVWRR
20


120
QPA120VLVLQVWDY
20


128
VLQVWDYDRISANDF
20


151
PDMVRGARGPELCSV
20


159
GPELCSVQLARNGAG
20


162
LCSVQLARNGAGPRC
20


241
TGKVEAEF240ELLT
20


253
LLTVEEAEKRPVGKG
20


271
PEPLEKPSRPKTSFN
20


285
NWFVNPLKTFVFFIW
20


292
KTFVFFIWRRYWRTL
20


304
RTLVLLLL300VLLT
20


305
TLVLLLL300VLLTV
20


316
LLTVFLLLVFYTIPG
20


319
VFLLLVFYTIPGQIS
20


325
FYTIPGQISQVIFRP
20


329
PGQISQVIFRPLHK3
20


59
WV60KGLEHDKQETD
18


67
HDKQETDVHFNSLTG
18


112
ALEEAEFRQPA120V
18


137
ISANDFLGSLELQLP
18


156
GARGPELCSVQLARN
18


161
ELCSVQLARNGAGPR
18


196
LKEAEDVEREAQEAQ
18


202
VEREAQEAQAGKKKR
18


233
MGGNVYILTGKVEAE
18


242
GKVEAEF240ELLTV
18


310
LL300VLLTVFLLLV
18


331
QISQVIFRPLHK328
18


3
IDIFPQDVPAPPPVD
16


30
VVIWNTEDVVLDDEN
16


52
SDIYVKSWV60KGLE
16


88
RFVFRFDYLPTEREV
16


102
VSVWRRSGPFALEEA
16


108
SGPFALEEAEFRQPA
16


115
EAEFRQPA120VLVL
16


131
VWDYDRISANDFLGS
16


139
ANDFLGSLELQLPDM
16


235
GNVYILTGKVEAEF2
16


284
FNWFVNPLKTFVFFI
16


291
LKTFVFFIWRRYWRT
16


293
TFVFFIWRRYWRTLV
16


299
WRRYWRTLVLLLL30
16


13
PPPVDIKPRQPISYE
15


7
PQDVPAPPPVDIKPR
14


25
SYELRVVIWNTEDVV
14


28
LRVVIWNTEDVVLDD
14


29
RVVIWNTEDVVLDDE
14


37
DVVLDDENPLTGEMS
14


99
EREVSVWRRSGPFAL
14


110
PFALEEAEFRQPA12
14


126
VLVLQVWDYDRISAN
14


134
YDRISANDFLGSLEL
14


140
NDFLGSLELQLPDMV
14


143
LGSLELQLPDMVRGA
14


147
ELQLPDMVRGARGPE
14


150
LPDMVRGARGPELCS
14


175
RCNLFRCRRLR180G
14


190
WWPVVKLKEAEDVER
14


193
VVKLKEAEDVEREAQ
14


225
PEDLEFTDMGGNVYI
14


230
FTDMGGNVYILTGKV
14


234
GGNVYILTGKVEAEF
14


288
VNPLKTFVFFIWRRY
14


303
WRTLVLLLL300VLL
14


306
LVLLLL300VLLTVF
14


307
VLLLL300VLLTVFL
14


318
TVFLLLVFYTIPGQI
14


320
FLLLVFYTIPGQISQ
14


321
LLLVFYTIPGQISQV
14
















TABLE XLIX





158P3D2v.1-DRB1 1101-15-mers


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the start


position plus fourteen.

















187
80GWWPVVKLKEAED
1755316517


188
0GWWPVVKLKEAEDV
1441815


313
00VLLTVFLLLVFYT
1441805


125
0VLVLQVWDYDRISA
1441804


251
0ELLTVEEAEKRPVG
1441802


314
0VLLTVFLLLVFYTI
1441802


62
0KGLEHDKQETDVHF
1441798


250
40ELLTVEEAEKRPV
262156


312
300VLLTVFLLLVFY
65811


92
RFDYLPTEREVSVWR
25


84
NFNWRFVFRFDYLPT
24


235
GNVYILTGKVEAEF2
24


322
LLVFYTIPGQISQVI
23


147
ELQLPDMVRGARGPE
22


292
KTFVFFIWRRYWRTL
22


98
TEREVSVWRRSGPFA
21


99
EREVSVWRRSGPFAL
21


128
VLQVWDYDRISANDF
20


161
ELCSVQLARNGAGPR
20


178
LFRCRRLR180GWWP
20


140
NDFLGSLELQLPDMV
19


227
DLEFTDMGGNVYILT
19


293
TFVFFIWRRYWRTLV
19


296
FFIWRRYWRTLVLLL
19


300
RRYWRTLVLLLL300
19


305
TLVLLLL300VLLTV
19


318
TVFLLLVFYTIPGQI
19


3
IDIFPQDVPAPPPVD
18


90
VFRFDYLPTEREVSV
18


175
RCNLFRCRRLR180G
18


284
FNWFVNPLKTFVFFI
18


317
LTVFLLLVFYTIPGQ
18


86
NWRFVFRFDYLPTER
17


177
NLFRCRRLR180GWW
17


49
EMSSDIYVKSWV60K
16


67
HDKQETDVHFNSLTG
16


73
DVHFNSLTGEGNFNW
16


131
VWDYDRISANDFLGS
16


172
AGPRCNLFRCRRLR1
16


212
GKKKRKQRRRKGRPE
16


214
KKRKQRRRKGRPEDL
16


245
EAEF240ELLTVEEA
16


258
EAEKRPVGKGRKQPE
16


330
GQISQVIFRPLHK32
16


21
RQPISYELRVVIWNT
15


53
DIYVKSWV60KGLEH
15


59
WV60KGLEHDKQETD
15


253
LLTVEEAEKRPVGKG
15


254
LTVEEAEKRPVGKGR
15


271
PEPLEKPSRPKTSFN
15


13
PPPVDIKPRQPISYE
14


111
FALEEAEFRQPA120
14


145
SLELQLPDMVRGARG
14


148
LQLPDMVRGARGPEL
14


150
LPDMVRGARGPELCS
14


159
GPELCSVQLARNGAG
14


196
LKEAEDVEREAQEAQ
14


260
EKRPVGKGRKQPEPL
14


261
KRPVGKGRKQPEPLE
14


268
RKQPEPLEKPSRPKT
14


308
LLLL300VLLTVFLL
14


2
WIDIFPQDVPAPPPV
13


12
APPPVDIKPRQPISY
13


25
SYELRVVIWNTEDVV
13


34
NTEDVVLDDENPLTG
13


47
TGEMSSDIYVKSWV6
13


110
PFALEEAEFRQPA12
13


176
CNLFRCRRLR180GW
13


234
GGNVYILTGKVEAEF
13


281
KTSFNWFVNPLKTFV
13


285
NWFVNPLKTFVFFIW
13


291
LKTFVFFIWRRYWRT
13


316
LLTVFLLLVFYTIPG
13


319
VFLLLVFYTIPGQIS
13


329
PGQISQVIFRPLHK3
13
















TABLE XXII





158P3D2v.18, ORF: 2932-4764, Frame +1,


610 aa, Part A, A1-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length of


the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















62
PQPGCPPAY
16


22
RHPGQPLVR
10


46
KPRPAPLSC
10


70
YTGTVLEQT
10


44
ASKPRPAPL
9


69
AYTGTVLEQ
9


21
RRHPGQPLV
8


30
RSVPSWSSS
8


36
SSSCGWAWA
8


52
LSCPRICCP
8


61
SPQPGCPPA
8


72
GTVLEQTLS
8


73
TVLEQTLSP
8
















TABLE XXIII





158P3D2-v.18, ORF: 2932-4764, Frame +1,


610 aa, Part A, A0201-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















24
PGQPLVRSV
16


21
RRHPGQPLV
15


44
ASKPRPAPL
15


66
CPPAYTGTV
15


27
PLVRSVPSW
14


70
YTGTVLEQT
14


20
LRRHPGQPL
13


73
TVLEQTLSP
13


12
SWQNCAFWL
12


71
TGTVLEQTL
12


61
SPQPGCPPA
11


19
WLRRHPGQP
10


36
SSSCGWAWA
10


49
PAPLSCPRI
10


56
RICCPSPQP
10


69
AYTGTVLEQ
10


42
AWASKPRPA
9


31
SVPSWSSSC
8


51
PLSCPRICC
8


52
LSCPRICCP
8


65
GCPPAYTGT
8


67
PPAYTGTVL
8


68
PAYTGTVLE
8
















TABLE XXIV





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, A0203-9-mers







No Results Found.
















TABLE XXV





158P3D2 v.18, ORF: 2932-4764, Frame +1,


610 aa, Part A, A3-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















19
WLRRHPGQP
21


28
LVRSVPSWS
19


22
RHPGQPLVR
18


73
TVLEQTLSP
18


31
SVPSWSSSC
16


38
SCGWAWASK
16


27
PLVRSVPSW
15


46
KPRPAPLSC
15


56
RICCPSPQP
15


44
ASKPRPAPL
13


30
RSVPSWSSS
12


48
RPAPLSCPR
12


14
QNCAFWLRR
11


51
PLSCPRICC
11
















TABLE XXVI





158P3D2-v.18, ORF: 2932-4764, Frame +1,


Part A, 610 aa, A26, 9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















73
TVLEQTLSP
15


31
SVPSWSSSC
14


70
YTGTVLEQT
13


28
LVRSVPSWS
12


71
TGTVLEQTL
12


72
GTVLEQTLS
12


62
PQPGCPPAY
11


10
RSSWQNCAF
9


12
SWQNCAFWL
9


20
LRRHPGQPL
9


27
PLVRSVPSW
9


44
ASKPRPAPL
9


67
PPAYTGTVL
9
















TABLE XXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1


610 aa, Part A, B0702-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















67
PPAYTGTVL
24


63
QPGCPPAYT
21


61
SPQPGCPPA
19


66
CPPAYTGTV
18


20
LRRHPGQPL
17


23
HPGQPLVRS
16


46
KPRPAPLSC
16


44
ASKPRPAPL
15


48
RPAPLSCPR
14


26
QPLVRSVPS
13


54
CPRICCPSP
13


59
CPSPQPGCP
13


8
WPRSSWQNC
12


32
VPSWSSSCG
12


50
APLSCPRIC
12
















TABLE XXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1


610 aa, Part A, B08-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















44
ASKPRPAPL
28


1
MCKRRWHWP
18


67
PPAYTGTVL
17


20
LRRHPGQPL
16


26
QPLVRSVPS
16
















TABLE XXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1,


610 aa, Part A, B1510-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















22
RHPGQPLVR
14


44
ASKPRPAPL
13


67
PPAYTGTVL
13


71
TGTVLEQTL
13


6
WHWPRSSWQ
12


20
LRRHPGQPL
11


12
SWQNCAFWL
10


10
RSSWQNCAF
8
















TABLE XXX





158P3D2 v.18, ORF: 2932-4764, Frame +1,


610 aa, Part A, B2705-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















20
LRRHPGQPL
21


21
RRHPGQPLV
20


22
RHPGQPLVR
18


48
RPAPLSCPR
17


3
KRRWHWPRS
15


4
RRWHWPRSS
15


10
RSSWQNCAF
15


29
VRSVPSWSS
14


40
GWAWASKPR
14


9
PRSSWQNCA
13


14
QNCAFWLRR
13


44
ASKPRPAPL
13


47
PRPAPLSCP
13


55
PRICCPSPQ
13


67
PPAYTGTVL
13


38
SCGWAWASK
12


71
TGTVLEQTL
12


2
CKRRWHWPR
11


12
SWQNCAFWL
11


13
WQNCAFWLR
11


15
NCAFWLRRH
11


49
PAPLSCPRI
11


62
PQPGCPPAY
11
















TABLE XXXI





158P3D2-v.18, ORF: 2932-4764, Frame +1


610 aa, Part A, B2709-9-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















21
RRHPGQPLV
24


20
LRRHPGQPL
20


3
KRRWHWPRS
14


4
RRWHWPRSS
14


10
RSSWQNCAF
12


44
ASKPRPAPL
12
















TABLE XXXII





158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa,


Part A, B4402 9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















44
ASKPRPAPL
18


62
PQPGCPPAY
15


10
RSSWQNCAF
13


12
SWQNCAFWL
13


35
WSSSCGWAW
13


11
SSWQNCAFW
12


27
PLVRSVPSW
12


5
RWHWPRSSW
11


67
PPAYTGTVL
11


71
TGTVLEQTL
11


20
LRRHPGQPL
10


33
PSWSSSCGW
10


49
PAPLSCPRI
10
















TABLE XXXIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


B5101-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















49
PAPLSCPRI
23


66
CPPAYTGTV
23


67
PPAYTGTVL
21


24
PGQPLVRSV
17


68
PAYTGTVLE
16


71
TGTVLEQTL
15


41
WAWASKPRP
14


50
APLSCPRIC
14


16
CAFWLRRHP
13


23
HPGQPLVRS
13


26
QPLVRSVPS
13


32
VPSWSSSCG
12


46
KPRPAPLSC
12
















TABLE XXXIV





158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa,


Part A, A1-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















61
SPQPGCPPAY
21


72
GTVLEQTLSP
12


21
RRHPGQPLVR
11


44
ASKPRPAPLS
11
















TABLE XXXV





158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa,


Part A, A0201 10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















73
TVLEQTLSPL
22


19
WLRRHPGQPL
20


70
YTGTVLEQTL
18


23
HPGQPLVRSV
16


43
WASKPRPAPL
16


65
GCPPAYTGTV
15


11
SSWQNCAFWL
13


20
LRRHPGQPLV
13


48
RPAPLSCPRI
13


51
PLSCPRICCP
11


69
AYTGTVLEQT
11
















TABLE XXXVI





158P3D2-v.18, ORF: 2932-4764, Frame + 1


610 aa, Part A, A020310-mers







No Results Found.
















TABLE XXXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, A3-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















19
WLRRHPGQPL
19


21
RRHPGQPLVR
19


27
PLVRSVPSWS
18


37
SSCGWAWASK
18


73
TVLEQTLSPL
16


28
LVRSVPSWSS
15


31
SVPSWSSSCG
14


56
RICCPSPQPG
13


30
RSVPSWSSSC
12


46
KPRPAPLSCP
12


61
SPQPGCPPAY
12


24
PGQPLVRSVP
11


4
RRWHWPRSSW
10


18
FWLRRHPGQP
10


22
RHPGQPLVRS
10


44
ASKPRPAPLS
10


45
SKPRPAPLSC
10


51
PLSCPRICCP
10


66
CPPAYTGTVL
10
















TABLE XXXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa,


Part A, A26-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















73
TVLEQTLSPL
26


70
YTGTVLEQTL
17


72
GTVLEQTLSP
14


31
SVPSWSSSCG
13


61
SPQPGCPPAY
13
















TABLE XXXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, B0702 10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















66
CPPAYTGTVL
23


48
RPAPLSCPRI
20


8
WPRSSWQNCA
18


23
HPGQPLVRSV
18


43
WASKPRPAPL
15


46
KPRPAPLSCP
14


59
CPSPQPGCPP
14


67
PPAYTGTVLE
14


19
WLRRHPGQPL
13


50
APLSCPRICC
13


54
CPRICCPSPQ
13


61
SPQPGCPPAY
13


26
QPLVRSVPSW
12


32
VPSWSSSCGW
12
















TABLE XL





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, B08-10-mers







No Results Found.
















TABLE XLI





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, B1510-10-mers







No Results Found.
















TABLE XLII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, B2705-10-mers







No Results Found.
















TABLE XLIII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, B2709-10-mers







No Results Found.
















TABLE XLIV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, B4402-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















61
SPQPGCPPAY
17


34
SWSSSCGWAW
14


9
PRSSWQNCAF
13


11
SSWQNCAFWL
12


26
QPLVRSVPSW
12


43
WASKPRPAPL
12


66
CPPAYTGTVL
12


70
YTGTVLEQTL
12


73
TVLEQTLSPL
12


4
RRWHWPRSSW
11


10
RSSWQNCAFW
11


19
WLRRHPGQPL
11


32
VPSWSSSCGW
11


48
RPAPLSCPRI
11


44
ASKPRPAPLS
8
















TABLE XLV





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part A, B5101-10-mers







No Results Found.
















TABLE XLVI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, DRB1 0101-15-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the


start position plus fouteen.

















26
QPLVRSVPSWSSSCG
30


17
AFWLRRHPGQPLVRS
26


16
CAFWLRRHPGQPLVR
24


25
GQPLVRSVPSWSSSC
23


57
ICCPSPQPGCPPAYT
22


71
TGTVLEQTLSPLWDE
22


23
HPGQPLVRSVPSWSS
21


69
AYTGTVLEQTLSPLW
19


32
VPSWSSSCGWAWASK
18


38
SCGWAWASKPRPAPL
18


40
GWAWASKPRPAPLSC
18


1
MCKRRWHWPRSSWQN
17


36
SSSCGWAWASKPRPA
17


42
AWASKPRPAPLSCPR
17


64
PGCPPAYTGTVLEQT
17


3
KRRWHWPRSSWQNCA
16


9
PRSSWQNCAFWLRRH
16


22
RHPGQPLVRSVPSWS
16


34
SWSSSCGWAWASKPR
16


41
WAWASKPRPAPLSCP
16


46
KPRPAPLSCPRICCP
16


49
PAPLSCPRICCPSPQ
16


70
YTGTVLEQTLSPLWD
16


39
CGWAWASKPRPAPLS
15
















TABLE XLVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, DRB1 0301-15-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the start


position plus fourteen.

















17
AFWLRRHPGQPLVRS
21


71
TGTVLEQTLSPLWDE
21


7
HWPRSSWQNCAFWLR
16


68
PAYTGTVLEQTLSPL
16


26
QPLVRSVPSWSSSCG
13


29
VRSVPSWSSSCGWAW
12


54
CPRICCPSPQPGCPP
12


72
GTVLEQTLSPLWDEL
12


25
GQPLVRSVPSWSSSC
11
















TABLE XLVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, DRB1 0401-15-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the


start position plus fourteen.

















26
QPLVRSVPSWSSSCG
26


3
KRRWHWPRSSWQNCA
22


23
HPGQPLVRSVPSWSS
18


63
QPGCPPAYTGTVLEQ
18


69
AYTGTVLEQTLSPLW
18


15
NCAFWLRRHPGQPLV
17


38
SCGWAWASKPRPAPL
16


67
PPAYTGTVLEQTLSP
16


17
AFWLRRHPGQPLVRS
14


25
GQPLVRSVPSWSSSC
14


29
VRSVPSWSSSCGWAW
14


71
TGTVLEQTLSPLWDE
14


72
GTVLEQTLSPLWDEL
14
















TABLE XLIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part A, DRB1 1101-15-mers


Each peptide is a portion of SEQ ID NO: 31;


each start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is


the start position plus fourteen.

















38
SCGWAWASKPRPAPL
26


40
GWAWASKPRPAPLSC
24


22
RHPGQPLVRSVPSWS
23


14
QNCAFWLRRHPGQPL
21


15
NCAFWLRRHPGQPLV
18


26
QPLVRSVPSWSSSCG
18


16
CAFWLRRHPGQPLVR
16


69
AYTGTVLEQTLSPLW
16


13
WQNCAFWLRRHPGQP
15


25
GQPLVRSVPSWSSSC
14


67
PPAYTGTVLEQTLSP
14
















TABLE XXII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A1-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















1
GISRQLLKH
10
















TABLE XXIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A0201-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















1
GISRQLLKH
16


6
LLKHNFDED
13


5
QLLKHNFDE
12
















TABLE XXIV





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, A0203-9-mers







No Results Found.
















TABLE XXV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A3-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















1
GISRQLLKH
18


5
QLLKHNFDE
14


6
LLKHNFDED
11
















TABLE XXVI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A26-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















1
GISRQLLKH
10


3
SRQLLKHNF
10


2
ISRQLLKHN
5
















TABLE XXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B0702-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















8
KHNFDEDEM
7


3
SRQLLKHNF
6


1
GISRQLLKH
4


6
L L K H N F D E D
17


4
R Q L L K H N F D
11


3
S R Q L L K H N F
9
















TABLE XXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B08-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















6
LLKHNFDED
17


4
RQLLKHNFD
11


3
SRQLLKHNF
9
















TABLE XXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B1510-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















8
KHNFDEDEM
17
















TABLE XXX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B2705-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















3
SRQLLKHNF
25


1
GISRQLLKH
15
















TABLE XXXI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B2709-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















3
SRQLLKHNF
19


8
KHNFDEDEM
11
















TABLE XXXII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B4402-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















3
SRQLLKHNF
13
















TABLE XXXIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B5101 9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















2
ISRQLLKHN
5


1
GISRQLLKH
4


4
RQLLKHNFD
4


5
QLLKHNFDE
4


6
LLKHNFDED
3
















TABLE XXXIV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A1-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















1
AGISRQLLKH
9
















TABLE XXXV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A0201-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















6
QLLKHNFDED
14


2
GISRQLLKHN
12


1
AGISRQLLKH
11


7
LLKHNFDEDE
11


8
LKHNFDEDEM
8
















TABLE XXXVI





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, A0203-10-mers







No Results Found.
















TABLE XXXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A3-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















6
QLLKHNFDED
14


1
AGISRQLLKH
13


2
GISRQLLKHN
11


7
LLKHNFDEDE
11


3
ISRQLLKHNF
9


5
RQLLKHNFDE
7
















TABLE XXXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, A26-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















1
AGISRQLLKH
11


2
GISRQLLKHN
8


3
ISRQLLKHNF
8
















TABLE XXXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B0702-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















3
ISRQLLKHNF
9


8
LKHNFDEDEM
6
















TABLE XL





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, B08-10-mers







No Results Found.
















TABLE XLI







158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, B1510-10-mers
















TABLE XLVI





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, DRB1 0101-15-mers







No Results Found.
















TABLE XLII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, B2705-10-mers







No Results Found.
















TABLE XLIII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, B2709-10-mers







No Results Found.
















TABLE XLIV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, B4402-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















3
ISRQLLKHNF
11


1
AGISRQLLKH
9
















TABLE XLV





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part B, B5101-10-mers







No Results Found.
















TABLE XLVI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, DRB1 0101-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end


position for each peptide is the start


position plus fourteen.

















14
KHNFDEDEMDDPGDS
19


6
AGISRQLLKHNFDED
18


1
PFLAEAGISRQLLKH
15


5
EAGISRQLLKHNFDE
15


2
FLAEAGISRQLLKHN
14


9
SRQLLKHNFDEDEMD
10
















TABLE XLVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, DRB1 0301-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length


of the peptide is 15 amino acids, and the


end position for each peptide is the


start position plus fourteen.

















14
KHNFDEDEMDDPGDS
20


2
FLAEAGISRQLLKHN
17


6
AGISRQLLKHNFDED
17


9
SRQLLKHNFDEDEMD
12


5
EAGISRQLLKHNFDE
11


10
RQLLKHNFDEDEMDD
11


12
LLKHNFDEDEMDDPG
11
















TABLE XLVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, DRB1 0401-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end


position for each peptide is the start


position plus fourteen.

















2
FLAEAGISRQLLKHN
18


14
KHNFDEDEMDDPGDS
16


5
EAGISRQLLKHNFDE
14


1
PFLAEAGISRQLLKH
12


3
LAEAGISRQLLKHNF
12


7
GISRQLLKHNFDEDE
12


11
QLLKHNFDEDEMDDP
12
















TABLE XLIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part B, DRB1 1101-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end


position for each peptide is the start


position plus fourteen.

















14
KHNFDEDEMDDPGDS
16


2
FLAEAGISRQLLKHN
15


6
AGISRQLLKHNFDED
14


7
GISRQLLKHNFDEDE
14
















TABLE XXII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A1-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















5
QYEVWVQQG
11


3
ASQYEVWVQ
7
















TABLE XXIII





158P3D2-v.18 ORF: 2932-4764, Frame +1, 610 aa,


Part C, A0201-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















2
LASQYEVWV
19


1
GLASQYEVW
13
















TABLE XXIV





158P3D2-v.18 ORF: 2932-4764, Frame + 1,


610 aa, Part C, A0203-9-mers







No Results Found.
















TABLE XXV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A3-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















1
GLASQYEVW
15


4
SQYEVWVQQ
14


9
WVQQGPQEP
13


7
EVWVQQGPQ
11
















TABLE XXVI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A26-9mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















7
EVWVQQGPQ
21


9
WVQQGPQEP
10
















TABLE XXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B0702-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















10
VQQGPQEPF
12


2
LASQYEVWV
10
















TABLE XXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B08-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















1
GLASQYEVW
7


10
VQQGPQEPF
7


2
LASQYEVWV
5


4
SQYEVWVQQ
5
















TABLE XXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B1510-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















10
VQQGPQEPF
8


1
GLASQYEVW
4


2
LASQYEVWV
4


9
WVQQGPQEP
4
















TABLE XXX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B2705-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















10
VQQGPQEPF
15
















TABLE XXXI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B2709-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.

















2
LASQYEVWV
9


10
VQQGPQEPF
8
















TABLE XXXII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B4402-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















1
GLASQYEVW
11


10
VQQGPQEPF
11


6
YEVWVQQGP
10
















TABLE XXXIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B5101-9-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.

















2
LASQYEVWV
22
















TABLE XXXIV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A1-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















6
QYEVWVQQGP
10


4
ASQYEVWVQQ
5
















TABLE XXXV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A0201-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















2
GLASQYEVWV
25
















TABLE XXXVI





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, A0203-10-mers







No Results Found.
















TABLE XXXVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A3-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















2
GLASQYEVWV
16


8
EVWVQQGPQE
16


10
WVQQGPQEPF
14


4
ASQYEVWVQQ
10


5
SQYEVWVQQG
9


1
CGLASQYEVW
8
















TABLE XXXVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, A26-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















8
EVWVQQGPQE
22


10
WVQQGPQEPF
18
















TABLE XXXIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B0702-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















2
GLASQYEVWV
9


10
WVQQGPQEPF
7
















TABLE XL





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, B08-10-mers







No Results Found.
















TABLE XLI





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, B1510-10-mers







No Results Found.
















TABLE XLII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, B2705-10-mers







No Results Found.
















TABLE XLIII





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, B2709-10-mers







No Results Found.
















TABLE XLIV





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, B4402-10-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.

















1
CGLASQYEVW
12


7
YEVWVQQGPQ
10


10
WVQQGPQEPF
10
















TABLE XLV





158P3D2-v.18, ORF: 2932-4764, Frame + 1,


610 aa, Part C, B5101-10-mers







No Results Found.
















TABLE XLVI





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, DRB1 0101-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end position


for each peptide is the start position plus


fourteen.

















5
NCGLASQYEVWVQQG
22


9
ASQYEVWVQQGPQEP
20


2
HRANCGLASQYEVWV
14
















TABLE XLVII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, DRB1 0301-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end position


for each peptide is the start position plus


fourteen.

















5
NCGLASQYEVWVQQG
18


1
HHRANCGLASQYEVW
9
















TABLE XLVIII





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, DRB1 0401-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end position


for each peptide is the start position plus


fourteen.

















3
RANCGLASQYEVWVQ
18


10
SQYEVWVQQGPQEPF
18


9
ASQYEVWVQQGPQEP
16


5
NCGLASQYEVWVQQG
14


2
HRANCGLASQYEVWV
12


7
GLASQYEVWVQQGPQ
12
















TABLE XLIX





158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,


Part C, DRB1 1101-15-mers


Each peptide is a portion of SEQ ID NO: 31; each


start position is specified, the length of the


peptide is 15 amino acids, and the end position


for each peptide is the start position plus


fourteen.

















9
ASQYEVWVQQGPQEP
19


8
LASQYEVWVQQGPQE
12
















TABLE L







Protein Characteristics of 158P3D2 v.17











Bioinformatic




158P3D2 var. 17
Program
URL on World Wide Web
Outcome





Protein length


2036 aa


Transmembrane
TM Pred
.ch.embnet.org/
1 TM helix 2003-2020; N-terminus intracellular


region


(type II)



HMMTop
.enzim.hu/hmmtop/
1 TM helix 2003-2022; N-terminus intracellular





(type II)



Sosui
.genome.ad.jp/SOSui/
1 TM helix 1999-2021



TMHMM
.cbs.dtu.dk/services/TMHMM
1 TM helix 2000-2022; N-terminus extracellular





(type I)


Signal Peptide
Signal P
.cbs.dtu.dk/services/SignalP/
None


pI
pI/MW tool
.expasy.ch/tools/
5.64


Molecular weight
pI/MW tool
.expasy.ch/tools/
227.6 kDa


Localization
PSORT II
psort.nibb.ac.jp/
26% cytoplasmic, 17% vesicles of secretory





system, 13% nuclear, 13% mitochondrial, 8%





plasma membrane


Motifs
Pfam
.sanger.ac.uk/Pfam/
C2 domain; amino acid transporter; 7TM





chemoreceptor; bradykinin; glycosyl hydrolase



Prints
.biochem.ucl.ac.uk/
C2 domain; endolaptase



Blocks
.blocks.fhcrc.org/
C2 domain
















TABLE LI







Exon composition of transcript 158P3D2 v.1









Exon Number
Start
End












Exon 1
1
836


Exon 2
837
922


Exon 3
923
1021


Exon 4
1022
1263


Exon 5
1264
1547


Exon 6
1548
1648


Exon 7
1649
1961
















TABLE LII(a)





Nucleotide sequence of transcript variant 158P3D2 v.2 (SEQ ID NO: 335)
















atcaaggccc tgggctggag gaagacatcc cagatccaga ggagctcgac tgggggtcca
60


 


agtactatgc gtcgctgcag gagctccagg ggcagcacaa ctttgatgaa gatgaaatgg
120


 


atgatcctgg agattcagat ggggtcaacc tcatttctat ggttggggag atccaagacc
180


 


agggtgaggc tgaagtcaaa ggcactgtgt ccccaaaaaa agcagttgcc accctgaaga
240


 


tctacaacag gtccctggag gaagaattta accactttga agactggctg aatgtgtttc
300


 


ctctgtaccg agggcaaggg ggccaggatg gaggtggaga agaggaagga tctggacacc
360


 


ttgtgggcaa gttcaagggc tccttcctca tttaccctga atcagaggca gtgttgttct
420


 


ctgagcccca gatctctcgg gggatcccac agaaccggcc catcaagctc ctggtcagag
480


 


tgtatgttgt aaaggctacc aacctggctc ctgcagaccc caatggcaaa gcagaccctt
540


 


acgtggtggt gagcgctggc cgggagcggc aggacaccaa ggaacgctac atccccaagc
600


 


agctcaaccc catctttgga gagatcctgg agctaagcat ctctctccca gctgagacgg
660


 


agctgacggt cgccgtattt gaacatgacc tcgtgggttc tgacgacctc atcggggaga
720


 


cccacattga tctggaaaac cgattctata gccaccacag agcaaactgt gggctggcct
780


 


cccagtatga agtgtgggtc cagcagggcc cacaggagcc attctgagtt tctggccaaa
840


 


cacattcaag ctcacattcc cttttgtgtc tccagatcct atgatttcat ggaaggggac
900


 


cctcccaccc accgccactg ccaaccaaga catagctcag tggtcaagac ttgggcttgg
960


 


gagtcgggat cctgtaacga atgtcacttg accgctttct ttttttatga aacagtctcg
1020


 


ctctgtctcc caggttggag tgcagtggca cgatctcggc tgactgcaac ctccacctcc
1080


 


tgggttcaag cgattctcct gcctcagcct ccccagtagc tgggattaca ggcgtgggcc
1140


 


cccatgtcca gctaattttt atattttcgc tctgtctccc aggttggagt gcagtggcac
1200


 


gatctcggct gactgcaacc tccacctcct gggttcaagc gattctcctg cctcagcctc
1260


 


cccagtagct gggattacag gcgtgggccc ccatgtccag ctaattttta tatttttagt
1320


 


agagacaggg tttcaccatg ttgtccaggc tggtcttgaa cccctgacct caagtgatcc
1380


 


acccacctct gcctcccaaa gtgctgggat tacaggtgtg agccaccatg ccaggccctc
1440


 


ttaacctctt caagtctgtt ttctcatctg caaaacagag gtaataagat cagtatcttc
1500


 


ttaatggaag cacctgggct acattttttt cattcattgt tatcataaat gaggactaac
1560


 


ctgtctcccg ttgggagttt tgaacctaga cctcatgtct tcatgacgtc atcactgccc
1620


 


caggcccagc tgtgtcccta caccagcccc agctgacgca tcttcttttt ctgcctgtag
1680


 


agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg
1740


 


ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa
1800


 


agtcttcctg acaccaccgg agaccctgcc cccagggatc tcttcacatg tggattgaca
1860


 


tctttcctca agatgtgcct gctccacccc cagttgacat caagcctcgg cagccaatca
1920


 


gctatgagct cagagttgtc atctggaaca cggaggatgt ggttctggat gacgagaatc
1980


 


cactcaccgg agagatgtcg agtgacatct atgtgaagag ctgggtgaag gggttggagc
2040


 


atgacaagca ggagacagac gttcacttca actccctgac tggggagggg aacttcaatt
2100


 


ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc gtctggcgca
2160


 


ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg ctggtcctgc
2220


 


aggatccctg gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg
2280


 


ctctgtgcag ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg
2340


 


cctgaggggc tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc
2400


 


gcaggaggct caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga
2460


 


agacctggag ttcacagaca tgggtggcaa tgtgtacatc ctcacgggca aggtggaggc
2520


 


agagtttgag ctgctgactg tggaggaggc cgagaaacgg ccagtgggga aggggcggaa
2580


 


gcagccagag cctctggaga aacccagccg ccccaaaact tccttcaact ggtttgtgaa
2640


 


cccgctgaag acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct
2700


 


gctactggtg ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat
2760


 


cagccaggtc atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc
2820


 


agggtgccaa cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc
2880


 


agagtctagt gcttcctctg aataaaccta tcacagcc
2918
















TABLE LIII (a)





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ


ID NO: 336) and 158P3D2 v.2 (SEQ ID NO: 337)















Score = 2348 bits (1221), Expect = 0.0 Identities = 1223/1224 (99%) Strand = Plus/Plus
































































































































Score = 1340 bits (697), Expect = 0.0 Identities = 697/697 (100%) Strand = Plus/Plus
























































































TABLE LIV(a)





Peptide sequences of protein coded by 158P3D2 v.2
















>158P3D2 v.2A (SEQ ID NO: 338)



MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL KIYNRSLEEE FNHFEDWLNV
60


 


FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVL FSEPQISRGI PQNRPIKLLV
120


 


RVYVVKATNL APADPNGKAD PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE
180


 


TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF
236


 


>158P3D2 v.2B (SEQ ID NO: 339)



MVRGARGPEL CSVQLARNGA GPRCNLFRCR RLRGWWPVVK LKEAEDVERE AQEAQAGKKK
60


 


RKQRRRKGRP EDLEFTDMGG NVYILTGKVE AEFELLTVEE AEKRPVGKGR KQPEPLEKPS
120


 


RPKTSFNWFV NPLKTFVFFI WRRYWRTLVL LLLVLLTVFL LLVFYTIPGQ ISQVIFRPLH
180


 


K
181
















TABLE LV(a)





Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 340) and


158P3D2 v.2 (SEQ ID NO: 341)


Score = 372 bits (956), Expect = e−103Identities = 181/181 (100%),


Positives = 181/181 (100%)


















V.1:
148
MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK
207




MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK



V.14:
1
MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK
60


 


V.1:
208
RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS
267




RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS



V.14:
61
RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS
120


 


V.1:
268
RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH
327




RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH



V.14:
121
RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH
180


 


V.1:
328
K
328




K



V.14:
181
K
181





Note:


Protein variant 158P3D2 v.2A does not share common sequence with protein 158P3D2 v.1.













TABLE LII(b)





Nucleotide sequence of transcript variant 158P3D2 v.14


(SEQ ID NO: 342)
















caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg
60


 


gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg
120


 


acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc
180


 


agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga
240


 


gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac
300


 


cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct
360


 


agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc
420


 


cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag
480


 


cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct
540


 


ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga
600


 


tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga
660


 


actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga
720


 


tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca
780


 


gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg
840


 


tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga
900


 


atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt
960


 


aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc
1020


 


ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt
1080


 


caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt
1140


 


tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt
1200


 


gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg
1260


 


ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc
1320


 


gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct
1380


 


gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt
1440


 


gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg
1500


 


gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca
1560


 


gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag
1620


 


gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt
1680


 


gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga
1740


 


aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt
1800


 


gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg
1860


 


gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca
1920


 


gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga
1980


 


ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct
2040


 


tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag
2100


 


cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag
2160


 


ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc
2220


 


agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga
2280


 


cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg
2340


 


gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc
2400


 


cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg
2460


 


cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg
2520


 


ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa
2580


 


actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct
2640


 


gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg
2700


 


ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg
2760


 


gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga
2820


 


ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc
2880


 


agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca
2940


 


agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct
3000


 


gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca
3060


 


ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct
3120


 


catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga
3180


 


tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga
3240


 


gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct
3300


 


gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga
3360


 


gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt
3420


 


tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga
3480


 


gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa
3540


 


cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact
3600


 


tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca
3660


 


aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt
3720


 


caggcatctg acagtggact tgccggagca gccttacttg cagcctccac tcagcatctt
3780


 


ggtgattgag cgccgggcct ttggccacac agtccttgtg ggttcccaca ttgtccccca
3840


 


catgctgcga ttcacatttc ggggtcatga ggatcctcct gaggaggaag gagagatgga
3900


 


ggagacaggg gatatgatgc ccaagggacc tcaaggacag aagtccctgg atcccttctt
3960


 


ggctgaagcg ggtatatcca gacagctcct gaagcctcct ctgaagaagc tcccactagg
4020


 


aggcctccta aatcaaggcc ctgggctgga ggaagacatc ccagatccag aggagctcga
4080


 


ctgggggtcc aagtactatg cgtcgctgca ggagctccag gggcagcaca actttgatga
4140


 


agatgaaatg gatgatcctg gagattcaga tggggtcaac ctcatttcta tggttgggga
4200


 


gatccaagac caggatctac aacaggtccc tgaaggaaga atttaaccac tttgaagact
4260


 


ggctgaatgt gtttcctctg taccgagggc aagggggcca ggatggaggt ggagaagagg
4320


 


aaggatctgg acaccttgtg ggcaagttca agggctcctt cctcatttac cctgaatcag
4380


 


aggcagtgtt gttctctgag ccccagatct cccgggggat cccacagaac cggcccatca
4440


 


agctcctggt cagagtgtat gttgtaaagg ctaccaacct ggctcctgca gaccccaatg
4500


 


gcaaagcaga cccttacgtg gtggtgagcg ctggccggga gcggcaggac accaaggaac
4560


 


gctacatccc caagcagctc aaccccatct ttggagagat cctggagcta agcatctctc
4620


 


tcccagctga gacggagctg acggtcgccg tatttgatca tgacctcgtg ggttctgacg
4680


 


acctcatcgg ggagacccac attgatctgg aaaaccgatt ctatagccac cacagagcaa
4740


 


actgtgggct ggcctcccag tatgaagtgt gggtccagca gggcccacag gagccattct
4800


 


gagtttctgg ccaaacacat tcaagctcac attccctttt gtgtctccag atcctatgat
4860


 


ttcatggaag gggaccctcc cacccaccgc cactgccaac caagacatag ctcagtggtc
4920


 


aagacttggg cttgggagtc gggatcctgt aacgaatgtc acttgaccgc tttctttttt
4980


 


tatgaaacag tctcgctctg tctcccaggt tggagtgcag tggcacgatc tcggctgact
5040


 


gcaacctcca cctcctgggt tcaagcgatt ctcctgcctc agcctcccca gtagctggga
5100


 


ttacaggcgt gggcccccat gtccagctaa tttttatatt ttcgctctgt ctcccaggtt
5160


 


ggagtgcagt ggcacgatct cggctgactg caacctccac ctcctgggtt caagcgattc
5220


 


tcctgcctca gcctccccag tagctgggat tacaggcgtg ggcccccatg tccagctaat
5280


 


ttttatattt ttagtagaga cagggtttca ccatgttgtc caggctggtc ttgaacccct
5340


 


gacctcaagt gatccaccca cctctgcctc ccaaagtgct gggattacag gtgtgagcca
5400


 


ccatgccagg ccctcttaac ctcttcaagt ctgttttctc atctgcaaaa cagaggtaat
5460


 


aagatcagta tcttcttaat ggaagcacct ggactacatt tttttcattc attgttatca
5520


 


taaatgagga ctaacctgtc tcccgttggg agttttgaac ctagacctca tgtcttcatg
5580


 


acgtcatcac tgccccaggc ccagctgtgt ccctacacca gccccagctg acgcatcttc
5640


 


tttttctgcc tgtagagatg gttacaatgc ctggcgtgat gcattctggc cttcgcagat
5700


 


cctggcgggg ctgtgccaac gctgtggcct ccctgcccct gaataccgag ccggtgctgt
5760


 


caaggtgggc agcaaagtct tcctgacacc accggagacc ctgcccccag ggatctcttc
5820


 


acatgtggat tgacatcttt cctcaagatg tgcctgctcc acccccagtt gacatcaagc
5880


 


ctcggcagcc aatcagctat gagctcagag ttgtcatctg gaacacggag gatgtggttc
5940


 


tggatgacga gaatccactc accggagaga tgtcgagtga catctatgtg aagaggtagg
6000


 


ctgctggccg ggcggggcaa cggcggtgca ctagggggat tgcaaatggg tgtgggccct
6060


 


cgggctgagt ccagagcccc gaccccaggc cctccgtggt gctgagagcg gggtgaggag
6120


 


tgggttctcc atgtagctcc agccctgacg ctcacccacc ccggccccag ctgggtgaag
6180


 


gggttggagc atgacaagca ggagacagac gttcacttca actccctgac tggggagggg
6240


 


aacttcaatt ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc
6300


 


gtctggcgca ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg
6360


 


ctggtcctgc aggtctggga ctatgaccgc atctctgcca atgacttcct tggatccctg
6420


 


gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg ctctgtgcag
6480


 


ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg cctgaggggc
6540


 


tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc gcaggaggct
6600


 


caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga agacctggag
6660


 


ttcacagaca tgggtggcaa tgtgtacatc ctcacgggca aggtggaggc agagtttgag
6720


 


ctgctgactg tggaggaggc cgagaaacgg ccagtgggga aggggcggaa gcagccagag
6780


 


cctctggaga aacccagccg ccccaaaact tccttcaact ggtttgtgaa cccgctgaag
6840


 


acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct gctactggtg
6900


 


ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat cagccaggtc
6960


 


atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc agggtgccaa
7020


 


cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc agagtctagt
7080


 


gcttcctctg aataaaccta tcacagcc
7108
















TABLE LIII (b) 





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ


ID NO: 343) and 158P3D2 v.14 (SEQ ID NO: 344)















Score = 1946 bits (1012), Expect = 0.0 Identities = 1018/1021 (99%) Strand = Plus/Plus














































































































Score = 1796 bits (934), Expect = 0.0 Identities = 938/940 (99%) Strand = Plus/Plus
















































































































TABLE LIV(b)





Peptide sequences of protein coded by 158P3D2 v.14 (SEQ ID NO: 345)
















MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC
60


 


LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP
120


 


EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE
180


 


ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK
240


 


LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG
300


 


VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP
360


 


LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR
420


 


LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN
480


 


EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP
540


 


LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL
600


 


TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF
660


 


GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE
720


 


EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE
780


 


RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL
840


 


LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ
900


 


RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA
960


 


KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI
1020


 


STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG
1080


 


RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP
1140


 


QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG
1200


 


VESEVLASYR ESPNFTELVR HLTVDLPEQP YLQPPLSILV IERRAFGHTV LVGSHIVPHM
1260


 


LRFTFRGHED PPEEEGEMEE TGDMMPKGPQ GQKSLDPFLA EAGISRQLLK PPLKKLPLGG
1320


 


LLNQGPGLEE DIPDPEELDW GSKYYASLQE LQGQHNFDED EMDDPGDSDG VNLISMVGEI
1380


 


QDQDLQQVPE GRI
1393
















TABLE LV(b)





Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.14







No significant similarity
















TABLE LII(c)





Nucleotide sequence of transcript variant 158P3D2 v.15


(SEQ ID NO: 346)
















caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg
60


 


gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg
120


 


acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc
180


 


agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga
240


 


gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac
300


 


cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct
360


 


agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc
420


 


cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag
480


 


cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct
540


 


ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga
600


 


tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga
660


 


actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga
720


 


tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca
780


 


gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg
840


 


tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga
900


 


atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt
960


 


aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc
1020


 


ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt
1080


 


caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt
1140


 


tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt
1200


 


gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg
1260


 


ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc
1320


 


gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct
1380


 


gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt
1440


 


gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg
1500


 


gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca
1560


 


gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag
1620


 


gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt
1680


 


gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga
1740


 


aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt
1800


 


gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg
1860


 


gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca
1920


 


gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga
1980


 


ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct
2040


 


tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag
2100


 


cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag
2160


 


ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc
2220


 


agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga
2280


 


cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg
2340


 


gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc
2400


 


cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg
2460


 


cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg
2520


 


ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa
2580


 


actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct
2640


 


gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg
2700


 


ctttctggct gaggagcaca actttgatga agatgaaatg gatgatcctg gagattcaga
2760


 


tggggtcaac ctcatttcta tggttgggga gatccaagac cagggtgagg ctgaagtcaa
2820


 


aggcactgtg tccccaaaaa aagcagttgc caccctgaag atctacaaca ggtccctgaa
2880


 


ggaagaattt aaccactttg aagactggct gaatgtgttt cctctgtacc gagggcaagg
2940


 


gggccaggat ggaggtggag aagaggaagg atctggacac cttgtgggca agttcaaggg
3000


 


ctccttcctc atttaccctg aatcagaggc agtgttgttc tctgagcccc agatctcccg
3060


 


ggggatccca cagaaccggc ccatcaagct cctggtcaga gtgtatgttg taaagctaag
3120


 


aaacctttgc aaaatccaag gtcatgaaga cttttgcctg ttttctgctg ctaccaacct
3180


 


ggctcctgca gaccccaatg gcaaagcaga cccttacgtg gtggtgagcg ctggccggga
3240


 


gcggcaggac accaaggaac gctacatccc caagcagctc aaccccatct ttggagagat
3300


 


cctggagcta agcatctctc tcccagctga gacggagctg acggtcgccg tatttgatca
3360


 


tgacctcgtg ggttctgacg acctcatcgg ggagacccac attgatctgg aaaaccgatt
3420


 


ctatagccac cacagagcaa actgtgggct ggcctcccag tatgaagtgt gggtccagca
3480


 


gggcccacag gagccattct gagtttctgg ccaaacacat tcaagctcac attccctttt
3540


 


gtgtctccag atcctatgat ttcatggaag gggaccctcc cacccaccgc cactgccaac
3600


 


caagacatag ctcagtggtc aagacttggg cttgggagtc gggatcctgt aacgaatgtc
3660


 


acttgaccgc tttctttttt tatgaaacag tctcgctctg tctcccaggt tggagtgcag
3720


 


tggcacgatc tcggctgact gcaacctcca cctcctgggt tcaagcgatt ctcctgcctc
3780


 


agcctcccca gtagctggga ttacaggcgt gggcccccat gtccagctaa tttttatatt
3840


 


ttcgctctgt ctcccaggtt ggagtgcagt ggcacgatct cggctgactg caacctccac
3900


 


ctcctgggtt caagcgattc tcctgcctca gcctccccag tagctgggat tacaggcgtg
3960


 


ggcccccatg tccagctaat ttttatattt ttagtagaga cagggtttca ccatgttgtc
4020


 


caggctggtc ttgaacccct gacctcaagt gatccaccca cctctgcctc ccaaagtgct
4080


 


gggattacag gtgtgagcca ccatgccagg ccctcttaac ctcttcaagt ctgttttctc
4140


 


atctgcaaaa cagaggtaat aagatcagta tcttcttaat ggaagcacct ggactacatt
4200


 


tttttcattc attgttatca taaatgagga ctaacctgtc tcccgttggg agttttgaac
4260


 


ctagacctca tgtcttcatg acgtcatcac tgccccaggc ccagctgtgt ccctacacca
4320


 


gccccagctg acgcatcttc tttttctgcc tgtagagatg gttacaatgc ctggcgtgat
4380


 


gcattctggc cttcgcagat cctggcgggg ctgtgccaac gctgtggcct ccctgcccct
4440


 


gaataccgag ccggtgctgt caaggtgggc agcaaagtct tcctgacacc accggagacc
4500


 


ctgcccccag ggatctcttc acatgtggat tgacatcttt cctcaagatg tgcctgctcc
4560


 


acccccagtt gacatcaagc ctcggcagcc aatcagctat gagctcagag ttgtcatctg
4620


 


gaacacggag gatgtggttc tggatgacga gaatccactc accggagaga tgtcgagtga
4680


 


catctatgtg aagaggtagg ctgctggccg ggcggggcaa cggcggtgca ctagggggat
4740


 


tgcaaatggg tgtgggccct cgggctgagt ccagagcccc gaccccaggc cctccgtggt
4800


 


gctgagagcg gggtgaggag tgggttctcc atgtagctcc agccctgacg ctcacccacc
4860


 


ccggccccag ctgggtgaag gggttggagc atgacaagca ggagacagac gttcacttca
4920


 


actccctgac tggggagggg aacttcaatt ggcgctttgt gttccgcttt gactacctgc
4980


 


ccacggagcg ggaggtgagc gtctggcgca ggtctggacc ctttgccctg gaggaggcgg
5040


 


agttccggca gcctgcagtg ctggtcctgc aggtctggga ctatgaccgc atctctgcca
5100


 


atgacttcct tggtattaca atgcttagcc ttccccaccc tcagcccctg cctccagccc
5160


 


tcacctccgc ccctgcctcc agccctcact tccgtccccc agttccctac tctgacccaa
5220


 


ccttgaatct tgggattttg gacccgaggt gtgaaacctt tgctttctgg cctaattact
5280


 


gagttaatta ggcctagacc acagtaacct ccattcccac ccagagtctc tgattcaact
5340


 


ctgatttgac cctagcttgt caccctgaca ccgactccac agcctttggt ccttggcact
5400


 


ctgatcccga cccttggccc tcttccactg ggaagtagca atgggtggac cgctgggctg
5460


 


tggtctgggt ggtctatagc tgtggcctga ccgcacactg caacaacttt caatgcccca
5520


 


atttacaacc ttggtgtgtt gcctcctcac ccctggcaca atgagacttt gatcccatgc
5580


 


ctaatctggt gtgctctgga cttgcaggat ccctggagtt gcagctacca gacatggtgc
5640


 


gtggggcccg gggccccgag ctctgctctg tgcagctggc ccgcaatggg gccgggccga
5700


 


ggtgcaatct gtttcgctgc cgccgcctga ggggctggtg gccggtagtg aagctgaagg
5760


 


aggcagagga cgtggagcgg gaggcgcagg aggctcaggc tggcaagaag aagcgaaagc
5820


 


agaggaggag gaagggccgg ccagaagacc tggagttcac agacatgggt ggcaatgtgt
5880


 


acatcctcac gggcaaggtg gaggcagagt ttgagctgct gactgtggag gaggccgaga
5940


 


aacggccagt ggggaagggg cggaagcagc cagagcctct ggagaaaccc agccgcccca
6000


 


aaacttcctt caactggttt gtgaacccgc tgaagacctt tgtcttcttc atctggcgcc
6060


 


ggtactggcg caccctggtg ctgctgctac tggtgctgct caccgtcttc ctcctcctgg
6120


 


tcttctacac catccctggc cagatcagcc aggtcatctt ccgtcccctc cacaagtgac
6180


 


tctcgctgac cttggacact cacccagggt gccaaccctt caatgcctgc tcctggaagt
6240


 


ctttcttacc catgtgagct accccagagt ctagtgcttc ctctgaataa acctatcaca
6300


 


gcc
6303
















TABLE LIII (c) 





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ


ID NO: 347) and 158P3D2 v.15 (SEQ ID NO: 348)















Score = 1963 bits (1021), Expect = 0.0 Identities = 1021/1021 (100%) Strand = Plus/Plus














































































































Score = 1340 bits (697), Expect = 0.0 Identities = 697/697 (100%) Strand = Plus/Plus










































































Score = 471 bits (245), Expect = e-129 Identities = 245/245 (100%) Strand = Plus/Plus














































TABLE LIV(c)





Peptide sequences of protein coded by 158P3D2 v.15


(SEQ ID NO: 349)
















MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC
60


 


LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP
120


 


EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE
180


 


ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK
240


 


LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG
300


 


VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP
360


 


LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR
420


 


LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN
480


 


EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP
540


 


LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL
600


 


TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF
660


 


GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE
720


 


EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE
780


 


RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL
840


 


LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEHNFDED EMDDPGDSDG
900


 


VNLISMVGEI QDQGEAEVKG TVSPKKAVAT LKIYNRSLKE EFNHFEDWLN VFPLYRGQGG
960


 


QDGGGEEEGS GHLVGKFKGS FLIYPESEAV LFSEPQISRG IPQNRPIKLL VRVYVVKLRN
1020


 


LCKIQGHEDF CLFSAATNLA PADPNGKADP YVVVSAGRER QDTKERYIPK QLNPIFGEIL
1080


 


ELSISLPAET ELTVAVFDHD LVGSDDLIGE THIDLENRFY SHHRANCGLA SQYEVWVQQG
1140


 


PQEPF
1145
















TABLE LV(c)





Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.15







No significant similarity
















TABLE LII(d)





Nucleotide sequence of transcript variant 158P3D2


v.16 (SEQ ID NO: 350)
















caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg
60


 


gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg
120


 


acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc
180


 


agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga
240


 


gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac
300


 


cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct
360


 


agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc
420


 


cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag
480


 


cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct
540


 


ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga
600


 


tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga
660


 


actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga
720


 


tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca
780


 


gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg
840


 


tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga
900


 


atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt
960


 


aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc
1020


 


ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt
1080


 


caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt
1140


 


tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt
1200


 


gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg
1260


 


ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc
1320


 


gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct
1380


 


gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt
1440


 


gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg
1500


 


gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca
1560


 


gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag
1620


 


gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt
1680


 


gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga
1740


 


aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt
1800


 


gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg
1860


 


gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca
1920


 


gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga
1980


 


ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct
2040


 


tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag
2100


 


cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag
2160


 


ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc
2220


 


agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga
2280


 


cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg
2340


 


gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc
2400


 


cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg
2460


 


cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg
2520


 


ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa
2580


 


actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct
2640


 


gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg
2700


 


ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg
2760


 


gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga
2820


 


ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc
2880


 


agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca
2940


 


agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct
3000


 


gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca
3060


 


ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct
3120


 


catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga
3180


 


tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga
3240


 


gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct
3300


 


gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga
3360


 


gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt
3420


 


tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga
3480


 


gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa
3540


 


cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact
3600


 


tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca
3660


 


aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt
3720


 


caggcatctg acagtggtct tcaaagacac agctcctctc ttccaccccc aggacttgcc
3780


 


ggagcagcct tacttgcagc ctccactcag catcttggtg attgagcgcc gggcctttgg
3840


 


ccacacagtc cttgtgggtt cccacattgt cccccacatg ctgcgattca catttcgggg
3900


 


tcatgaggat cctcctgagg aggaaggaga gatggaggag acaggggata tgatgcccaa
3960


 


gggacctcaa ggacagaagt ccctggatcc cttcttggct gaagcgggta tatccagaca
4020


 


gctcctgaag cctcctctga agaagctccc actaggaggc ctcctaaatc aaggccctgg
4080


 


gctggaggaa gacatcccag atccagagga gctcgactgg gggtccaagt actatgcgtc
4140


 


gctgcaggag ctccaggggc agcacaactt tgatgaagat gaaatggatg atcctggaga
4200


 


ttcagatggg gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga
4260


 


agtcaaaggc actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc
4320


 


cctgaaggaa gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg
4380


 


gcaagggggc caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt
4440


 


caagggctcc ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat
4500


 


ctcccggggg atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa
4560


 


ggctaccaac ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag
4620


 


cgctggccgg gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat
4680


 


ctttggagag atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc
4740


 


cgtatttgat catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct
4800


 


ggaaaaccga ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt
4860


 


agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg
4920


 


ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa
4980


 


agtcttcctg acaccaccgg agaccctgcc cccagtggcg agcggggacc ctgaagaggc
5040


 


ccaggcattg cttgtgctgc ggcgctggca ggaaatgccg ggttttggga tccagctggt
5100


 


acccgagcat gtagaaacca ggcctctcta ccatccccac agcccagggc tgctacaggg
5160


 


atctcttcac atgtggattg acatctttcc tcaagatgtg cctgctccac ccccagttga
5220


 


catcaagcct cggcagccaa tcagctatga gctcagagtt gtcatctgga acacggagga
5280


 


tgtggttctg gatgacgaga atccactcac cggagagatg tcgagtgaca tctatgtgaa
5340


 


gagctgggtg aaggggttgg agcatgacaa gcaggagaca gacgttcact tcaactccct
5400


 


gactggggag gggaacttca attggcgctt tgtgttccgc tttgactacc tgcccacgga
5460


 


gcgggaggtg agcgtctggc gcaggtctgg accctttgcc ctggaggagg cggagttccg
5520


 


gcagcctgca gtgctggtcc tgcaggtctg ggactatgac cgcatctctg ccaatgactt
5580


 


ccttggatcc ctggagttgc agctaccaga catggtgcgt ggggcccggg gccccgagct
5640


 


ctgctctgtg cagctggccc gcaatggggc cgggccgagg tgcaatctgt ttcgctgccg
5700


 


ccgcctgagg ggctggtggc cggtagtgaa gctgaaggag gcagaggacg gcaaggtgga
5760


 


ggcagagttt gagctgctga ctgtggagga ggccgagaaa cggccagtgg ggaaggggcg
5820


 


gaagcagcca gagcctctgg agaaacccag ccgccccaaa acttccttca actggtttgt
5880


 


gaacccgctg aagacctttg tcttcttcat ctggcgccgg tactggcgca ccctggtgct
5940


 


gctgctactg gtgctgctca ccgtcttcct cctcctggtc ttctacacca tccctggcca
6000


 


gatcagccag gtcatcttcc gtcccctcca caagtgactc tcgctgacct tggacactca
6060


 


cccagggtgc caacccttca atgcctgctc ctggaagtct ttcttaccca tgtgagctac
6120


 


cccagagtct agtgcttcct ctgaataaac ctatcacagc c
6161
















TABLE LIII(d)





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 351) and


 158P3D2 v.16 (SEQ ID NO: 352)















Score = 1144 bits (595), Expect = 0.0Identities = 595/595 (100%)


Strand = Plus/Plus








 








 








 








 








 








 








 








 








 








 


Score = 792 bits (412), Expect = 0.0Identities = 412/412 (100%)


Strand = Plus/Plus








 








 








 








 








 








 








 


Score = 298 bits (155), Expect = 3e−77Identities = 155/155


(100%) Strand = Plus/Plus








 








 






















TABLE LIV(d)





Peptide sequences of protein coded by 158P3D2 v.16 (SEQ


ID NO: 353)
















MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC
60


 


LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP
120


 


EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE
180


 


ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK
240


 


LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG
300


 


VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP
360


 


LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR
420


 


LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN
480


 


EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP
540


 


LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL
600


 


TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF
660


 


GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE
720


 


EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE
780


 


RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL
840


 


LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ
900


 


RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA
960


 


KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI
1020


 


STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG
1080


 


RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP
1140


 


QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG
1200


 


VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH
1260


 


TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL
1320


 


LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS
1380


 


DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ
1440


 


GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA
1500


 


TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV
1560


 


FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ
1620


 


RCGLPAPEYR AGAVKVGSKV FLTPPETLPP VASGDPEEAQ ALLVLRRWQE MPGFGIQLVP
1680


 


EHVETRPLYH PHSPGLLQGS LHMWIDIFPQ DVPAPPPVDI KPRQPISYEL RVVIWNTEDV
1740


 


VLDDENPLTG EMSSDIYVKS WVKGLEHDKQ ETDVHFNSLT GEGNFNWRFV FRFDYLPTER
1800


 


EVSVWRRSGP FALEEAEFRQ PAVLVLQVWD YDRISANDFL GSLELQLPDM VRGARGPELC
1860


 


SVQLARNGAG PRCNLFRCRR LRGWWPVVKL KEAEDGKVEA EFELLTVEEA EKRPVGKGRK
1920


 


QPEPLEKPSR PKTSFNWFVN PLKTFVFFIW RRYWRTLVLL LLVLLTVFLL LVFYTIPGQI
1980


 


SQVIFRPLHK
1990
















TABLE LV(d)





Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID


NO: 354) and 158P3D2 v.16 (SEQ ID NO: 355)


Score = 580 bits (1496), Expect = e-164Identities = 288/328


(87%), Positives = 288/328 (87%), Gaps = 40/328 (12%)


















V.1:
1
MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV
60




MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV



V.16:
1703
MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV
1762


 


V.1:
61
KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA
120




KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA



V.16:
1763
KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA
1822


 


V.1:
121
VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR
180




VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR



V.16:
1823
VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR
1882


 


V.1:
181
GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF
240




GWWPVVKLKEAED                                        GKVEAEF



V.16
1883
GWWPVVKLKEAED----------------------------------------GKVEAEF
1902


 


V.1:
241
ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL
300




ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL



V.16:
1903
ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL
1962


 


V.1:
301
VLLTVFLLLVFYTIPGQISQVIFRPLHK
328




VLLTVFLLLVFYTIPGQISQVIFRPLHK



V.16:
1963
VLLTVFLLLVFYTIPGQISQVIFRPLHK
1990
















TABLE LII(e)





Nucleotide sequence of transcript variant 158P3D2


v.17 (SEQ ID NO: 356)
















caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg
60


 


gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg
120


 


acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc
180


 


agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga
240


 


gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac
300


 


cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct
360


 


agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc
420


 


cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag
480


 


cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct
540


 


ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga
600


 


tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga
660


 


actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga
720


 


tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca
780


 


gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg
840


 


tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga
900


 


atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt
960


 


aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc
1020


 


ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt
1080


 


caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt
1140


 


tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt
1200


 


gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg
1260


 


ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc
1320


 


gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct
1380


 


gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt
1440


 


gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg
1500


 


gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca
1560


 


gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag
1620


 


gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt
1680


 


gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga
1740


 


aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt
1800


 


gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg
1860


 


gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca
1920


 


gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga
1980


 


ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct
2040


 


tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag
2100


 


cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag
2160


 


ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc
2220


 


agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga
2280


 


cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg
2340


 


gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc
2400


 


cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg
2460


 


cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg
2520


 


ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa
2580


 


actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct
2640


 


gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg
2700


 


ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg
2760


 


gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga
2820


 


ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc
2880


 


agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca
2940


 


agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct
3000


 


gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca
3060


 


ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct
3120


 


catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga
3180


 


tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga
3240


 


gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct
3300


 


gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga
3360


 


gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt
3420


 


tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga
3480


 


gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa
3540


 


cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact
3600


 


tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca
3660


 


aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt
3720


 


caggcatctg acagtggtct tcaaagacac agctcctctc ttccaccccc aggacttgcc
3780


 


ggagcagcct tacttgcagc ctccactcag catcttggtg attgagcgcc gggcctttgg
3840


 


ccacacagtc cttgtgggtt cccacattgt cccccacatg ctgcgattca catttcgggg
3900


 


tcatgaggat cctcctgagg aggaaggaga gatggaggag acaggggata tgatgcccaa
3960


 


gggacctcaa ggacagaagt ccctggatcc cttcttggct gaagcgggta tatccagaca
4020


 


gctcctgaag cctcctctga agaagctccc actaggaggc ctcctaaatc aaggccctgg
4080


 


gctggaggaa gacatcccag atccagagga gctcgactgg gggtccaagt actatgcgtc
4140


 


gctgcaggag ctccaggggc agcacaactt tgatgaagat gaaatggatg atcctggaga
4200


 


ttcagatggg gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga
4260


 


agtcaaaggc actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc
4320


 


cctgaaggaa gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg
4380


 


gcaagggggc caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt
4440


 


caagggctcc ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat
4500


 


ctcccggggg atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa
4560


 


ggctaccaac ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag
4620


 


cgctggccgg gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat
4680


 


ctttggagag atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc
4740


 


cgtatttgat catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct
4800


 


ggaaaaccga ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt
4860


 


agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg
4920


 


ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa
4980


 


agtcttcctg acaccaccgg agaccctgcc cccaggcagc agcagcccca cagtggcgag
5040


 


cggggaccct gaagaggccc aggcattgct tgtgctgcgg cgctggcagg aaatgccggg
5100


 


ttttgggatc cagctggtac ccgagcatgt agaaaccagg cctctctacc atccccacag
5160


 


cccagggctg ctacagggat ctcttcacat gtggattgac atctttcctc aagatgtgcc
5220


 


tgctccaccc ccagttgaca tcaagcctcg gcagccaatc agctatgagc tcagagttgt
5280


 


catctggaac acggaggatg tggttctgga tgacgagaat ccactcaccg gagagatgtc
5340


 


gagtgacatc tatgtgaaga gctgggtgaa ggggttggag catgacaagc aggagacaga
5400


 


cgttcacttc aactccctga ctggggaggg gaacttcaat tggcgctttg tgttccgctt
5460


 


tgactacctg cccacggagc gggaggtgag cgtctggcgc aggtctggac cctttgccct
5520


 


ggaggaggcg gagttccggc agcctgcagt gctggtcctg caggtctggg actatgaccg
5580


 


catctctgcc aatgacttcc ttggatccct ggagttgcag ctaccagaca tggtgcgtgg
5640


 


ggcccggggc cccgagctct gctctgtgca gctggcccgc aatggggccg ggccgaggtg
5700


 


caatctgttt cgctgccgcc gcctgagggg ctggtggccg gtagtgaagc tgaaggaggc
5760


 


agaggacgtg gagcgggagg cgcaggaggc tcaggctggc aagaagaagc gaaagcagag
5820


 


gaggaggaag ggccggccag aagacctgga gttcacagac atgggtggca atgtgtacat
5880


 


cctcacgggc aaggtggagg cagagtttga gctgctgact gtggaggagg ccgagaaacg
5940


 


gccagtgggg aaggggcgga agcagccaga gcctctggag aaacccagcc gccccaaaac
6000


 


ttccttcaac tggtttgtga acccgctgaa gacctttgtc ttcttcatct ggcgccggta
6060


 


ctggcgcacc ctggtgctgc tgctactggt gctgctcacc gtcttcctcc tcctggtctt
6120


 


ctacaccatc cctggccaga tcagccaggt catcttccgt cccctccaca agtgactctc
6180


 


gctgaccttg gacactcacc cagggtgcca acccttcaat gcctgctcct ggaagtcttt
6240


 


cttacccatg tgagctaccc cagagtctag tgcttcctct gaataaacct atcacagcc
6299
















TABLE LIII (e) 





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ


ID NO: 357) and 158P3D2 v.17 (SEQ ID NO: 358)















Score = 2165 bits (1126), Expect = 0.0 Identities = 1126/1126 (100%) Strand = Plus/Plus




















































































































Score = 300 bits (156), Expect = 7e−78 Identities = 156/156 (100%) Strand = Plus/Plus


































TABLE LIV(e)





Peptide sequences of protein coded by 158P3D2 v.17


(SEQ ID NO: 359)
















MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC
60


 


LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP
120


 


EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE
180


 


ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK
240


 


LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG
300


 


VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP
360


 


LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR
420


 


LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN
480


 


EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP
540


 


LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL
600


 


TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF
660


 


GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE
720


 


EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE
780


 


RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL
840


 


LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ
900


 


RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA
960


 


KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI
1020


 


STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG
1080


 


RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP
1140


 


QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG
1200


 


VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH
1260


 


TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL
1320


 


LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS
1380


 


DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ
1440


 


GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA
1500


 


TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV
1560


 


FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ
1620


 


RCGLPAPEYR AGAVKVGSKV FLTPPETLPP GSSSPTVASG DPEEAQALLV LRRWQEMPGF
1680


 


GIQLVPEHVE TRPLYHPHSP GLLQGSLHMW IDIFPQDVPA PPPVDIKPRQ PISYELRVVI
1740


 


WNTEDVVLDD ENPLTGEMSS DIYVKSWVKG LEHDKQETDV HFNSLTGEGN FNWRFVFRFD
1800


 


YLPTEREVSV WRRSGPFALE EAEFRQPAVL VLQVWDYDRI SANDFLGSLE LQLPDMVRGA
1860


 


RGPELCSVQL ARNGAGPRCN LFRCRRLRGW WPVVKLKEAE DVEREAQEAQ AGKKKRKQRR
1920


 


RKGRPEDLEF TDMGGNVYIL TGKVEAEFEL LTVEEAEKRP VGKGRKQPEP LEKPSRPKTS
1980


 


FNWFVNPLKT FVFFIWRRYW RTLVLLLLVL LTVFLLLVFY TIPGQISQVI FRPLHK
2036
















TABLE LV(e)





Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID


NO: 360) and 158P3D2 v.17 (SEQ ID NO: 361)


Score = 679 bits (1751), Expect = 0.0Identities = 328/328


(100%), Positives = 328/328 (100%)


















V.1:
1
MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV
60




MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV



v.17:
1709
MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV
1768


 


V.1:
61
KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA
120




KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA



v.17:
1769
KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA
1828


 


V.1:
121
VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR
180




VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR



v.17:
1829
VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR
1888


 


V.1:
181
GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF
240




GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF



v.17:
1889
GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF
1948


 


V.1:
241
ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL
300




ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL



v.17:
1949
ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL
2008


 


V.1:
301
VLLTVFLLLVFYTIPGQISQVIFRPLHK
328




VLLTVFLLLVFYTIPGQISQVIFRPLHK



v.17:
2009
VLLTVFLLLVFYTIPGQISQVIFRPLHK
2036
















TABLE LII(f)





Nucleotide sequence of transcript variant 158P3D2


v.18 (SEQ ID NO: 362)
















agaagaaagc tggtaggggc tgggagaggg taccacaggg gagtatgatc tacttggggg
60


 


ccagagaagg ttccctgagg aaatagtacc tgaacttaga cttgaaggat aacagatgtt
120


 


aactgggagg agagaatgtt ccaggcagag gaaaaggcat atgcaaaagt ccagcgcctt
180


 


gaaggagcac agctggggtg cctggagtga gatggagctg gaaagatcca ggtggagctt
240


 


gacctgaagt accagccccc agagggcgct actggagcct ggtcagagga ggactttggg
300


 


gcacccatcc aggacagctt cgagttaatc atccccaatg tgggcttcca ggaactggag
360


 


cctggggagg cccagctgga gcggcgggca gtggctctag gccgcaggct agctcgaagt
420


 


ctaggccagc aggacgatga agagaatgag ctggagcttg agctggagca ggacctggat
480


 


gatgagcctg acgtggaact ttctggtgtt atgttcagcc ccctcaagag ccgcgccagg
540


 


gccctggccc atggggatcc cttccaggtg tccagagctc aagacttcca ggtgggagtc
600


 


actgtgctgg aagcccagaa actggtggga gtcaacatta acccctatgt ggccgtgcaa
660


 


gtgggggggc agcgccgtgt gaccgccaca cagcgtggga ccagttgccc cttctacaat
720


 


gagtacttct tgttcgaatt tcatgacacg cggcttcgtc tccaagactt gctgctggag
780


 


atcacggtga gtggggtagg ggtgaccagt gtccttcaga gaagggggga tgagaaagct
840


 


gcaggactaa caccaccttc ccccaaggct ttccattcgc agaccctccc ctttatggcc
900


 


acccggatag gcaccttcag gatggacctg ggcatcatct tggaccagcc aggtatggaa
960


 


tcgtcccctt attgagactc tgcacggaca agggccctag agattgaccc tgcagtgact
1020


 


ccgcatggac ccctatacac tcacttcgga gagggccatc tctggcggag gctgaactct
1080


 


tggcacttcc gcccctccct gctgagccag agaagccctg gccattgtcc gtcactccga
1140


 


tagcctcacg gccaccctgt gcgtcccgcc ggtcgcccct tacccctggc tcgccccttc
1200


 


gcccttagat ggccagttct accaaagatg ggttccgctg catgatcccc gagacacccg
1260


 


cgccgggacc aagggtttca ttaaggtcac cttgtccgtg agggcgcgcg gggacctgcc
1320


 


ccctccaatg ctacccccgg ccccagggca ctgttcggac atcgagaagt gagccggggt
1380


 


gaggtgggga ggaggacatg gatccggggg tggccgtggg gcgcggataa ggggaggggc
1440


 


cgagatccca gtttctcccc ccccgctcgg tgccccctcc cctaggaacc tgctcctgcc
1500


 


gcgcggggtg cccgccgaga ggccatgggc gcggctccgc gtgcgcctgt accgcgccga
1560


 


ggggcttccc gcgctgcgcc tggggctgct gggcagcctg gtccgcgccc tgcacgacca
1620


 


gcgcgtcctg gtggagccct atgtgcgggt gtctttcctg gggcaggagg gcgagacgtc
1680


 


ggtgagcgcc gaggcggcgg cgcccgaatg gaacgagcag ctgagcttcg tggagctctt
1740


 


cccgccgctg acgcgcagcc tccgcctgca gctgcgggac gacgcgcccc tggtcgacgc
1800


 


ggcactcgct acgcacgtgc cggacctgag gcggatctcc catccgggcc gcgcggcggg
1860


 


gtttaaccct accttcggcc cggcctgggt gcccctctat ggctcgcccc ccggcgcggg
1920


 


gctccgggat agtcttcaag gtctcaacga aggcgttggc caaggcattt ggttccgcgg
1980


 


ccgccttctg ctggctgtgt ccatgcaggt gttggaaggg agagctgaac ctgagcctcc
2040


 


ccaggcccag caggggtcca cgttgtcccg gctcacccga aagaagaaaa agaaagccag
2100


 


aagggatcag accccaaagg cggttccgca gcacttggac gccagccccg gtgccgaggg
2160


 


gcctgagatc ccccgtgcca tggaggtgga ggtggaggag ctgctgccgc tgccagagaa
2220


 


tgtcctggcg ccctgtgaag atttcctgct tttcggtgtg ctcttcgagg ccaccatgat
2280


 


cgaccccacc gtggcctccc agcccatcag cttcgagatc tccattggtg tgtggcctag
2340


 


ccgaacccct gagtgccatt tcagacctta gaaccctgga aggggtgttg actttcagtc
2400


 


gcgcaggccg tctggaggag caattgggcc gagggtccag ggctggggag ggaactgagg
2460


 


gtgcagccgt ggaggctcag cctctgctgg gagccaggcc agaggaggag aaagaggagg
2520


 


aagaactggg gacccatgct cagcggcctg agcccatgga cggcagtggg ccatacttct
2580


 


gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg gagttgctgg gaggaccaca
2640


 


cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc cgagaggctg gaccaggggc
2700


 


tgcaggaggt tgagagactg cagcgcaagc cggggcctgg cgcctgtgca cagctcaagc
2760


 


aggcactgga agtactggtg gctgggagca gacagttttg ccacggtgcc gagcgcagga
2820


 


cgatgacccg gcccaatgcc ctggatcgat gccgagggaa actcctggtg cacagcctga
2880


 


accttttggc taagcaagga ctgcgacttc tacgcggcct gagacggcgc aatgtgcaaa
2940


 


agaaggtggc actggccaag aagctcctgg caaaactgcg ctttctggct gaggaggcac
3000


 


ccggggcagc ccctggtgag gtctgtgcca agctggagct cttcctgcgg ctgggcctgg
3060


 


gcaagcaagc caaggcctgc acctctgagc tgcccccgga tttgctgccc gagccctcag
3120


 


ccgggctgcc ctccagccta caccgggacg gtcctggagc agacgctgag ccctctgtgg
3180


 


gatgaactcc tggtatttga gcagttgatc gtggatggga ggagggagca cctgcaggag
3240


 


gagcctccat tagtgatcat caatgtattt gaccacaata agtttggccc ccccgtgttc
3300


 


ctgggcaggg cactggccgc cccaagggta aagctgatgg aggacccata ccaacgccca
3360


 


gagttgcagt tcttccccct gaggaaggga ccctgggcag ccggagagct cattgccgcc
3420


 


tttcaactca ttgaactaga ctacagtggc cgacttgagc cctcagtgcc cagtgaggtg
3480


 


gagccccagg atctggcacc cctggttgag ccccactctg gacgcctgtc ccttccaccc
3540


 


aacgtgtgcc cagtgctcag ggagttccgt gttgaggtgc tgttctgggg tcttagggga
3600


 


cttggtcgtg tgcatctgct cgaggtggag cagccccagg ttgtactgga ggtggctggg
3660


 


caaggtgtgg agtctgaggt cctggccagc taccgtgaga gccccaattt cactgagctt
3720


 


gtcaggcatc tgacagtggt cttcaaagac acagctcctc tcttccaccc ccaggacttg
3780


 


ccggagcagc cttacttgca gcctccactc agcatcttgg tgattgagcg ccgggccttt
3840


 


ggccacacag tccttgtggg ttcccacatt gtcccccaca tgctgcgatt cacatttcgg
3900


 


ggtcatgagg atcctcctga ggaggaagga gagatggagg agacagggga tatgatgccc
3960


 


aagggacctc aaggacagaa gtccctggat cccttcttgg ctgaagcggg tatatccaga
4020


 


cagctcctga agcacaactt tgatgaagat gaaatggatg atcctggaga ttcagatggg
4080


 


gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga agtcaaaggc
4140


 


actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc cctgaaggaa
4200


 


gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg gcaagggggc
4260


 


caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt caagggctcc
4320


 


ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat ctcccggggg
4380


 


atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa ggctaccaac
4440


 


ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag cgctggccgg
4500


 


gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat ctttggagag
4560


 


atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc cgtatttgat
4620


 


catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct ggaaaaccga
4680


 


ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt gtgggtccag
4740


 


cagggcccac aggagccatt ctgagtttct ggccaaacac attcaagctc acattccctt
4800


 


ttgtgtctcc agatcctatg atttcatgga aggggaccct cccacccacc gccactgcca
4860


 


accaagacat agctcagtgg tcaagacttg ggcttgggag tcgggatcct gtaacgaatg
4920


 


tcacttgacc gctttctttt tttatgaaac agtctcgctc tgtctcccag gttggagtgc
4980


 


agtggcacga tctcggctga ctgcaacctc cacctcctgg gttcaagcga ttctcctgcc
5040


 


tcagcctccc cagtagctgg gattacaggc gtgggccccc atgtccagct aatttttata
5100


 


ttttcgctct gtctcccagg ttggagtgca gtggcacgat ctcggctgac tgcaacctcc
5160


 


acctcctggg ttcaagcgat tctcctgcct cagcctcccc agtagctggg attacaggcg
5220


 


tgggccccca tgtccagcta atttttatat ttttagtaga gacagggttt caccatgttg
5280


 


tccaggctgg tcttgaaccc ctgacctcaa gtgatccacc cacctctgcc tcccaaagtg
5340


 


ctgggattac aggtgtgagc caccatgcca ggccctctta acctcttcaa gtctgttttc
5400


 


tcatctgcaa aacagaggta ataagatcag tatcttctta atggaagcac ctggactaca
5460


 


tttttttcat tcattgttat cataaatgag gactaacctg tctcccgttg ggagttttga
5520


 


acctagacct catgtcttca tgacgtcatc actgccccag gcccagctgt gtccctacac
5580


 


cagccccagc tgacgcatct tctttttctg cctgtagaga tggttacaat gcctggcgtg
5640


 


atgcattctg gccttcgcag atcctggcgg ggctgtgcca acgctgtggc ctccctgccc
5700


 


ctgaataccg agccggtgct gtcaaggtgg gcagcaaagt cttcctgaca ccaccggaga
5760


 


ccctgccccc agggatctct tcacatgtgg attgacatct ttcctcaaga tgtgcctgct
5820


 


ccacccccag ttgacatcaa gcctcggcag ccaatcagct atgagctcag agttgtcatc
5880


 


tggaacacgg aggatgtggt tctggatgac gagaatccac tcaccggaga gatgtcgagt
5940


 


gacatctatg tgaagagctg ggtgaagggg ttggagcatg acaagcagga gacagacgtt
6000


 


cacttcaact ccctgactgg ggaggggaac ttcaattggc gctttgtgtt ccgctttgac
6060


 


tacctgccca cggagcggga ggtgagcgtc tggcgcaggt ctggaccctt tgccctggag
6120


 


gaggcggagt tccggcagcc tgcagtgctg gtcctgcagg atccctggag ttgcagctac
6180


 


cagacatggt gcgtggggcc cggggccccg agctctgctc tgtgcagctg gcccgcaatg
6240


 


gggccgggcc gaggtgcaat ctgtttcgct gccgccgcct gaggggctgg tggccggtag
6300


 


tgaagctgaa ggaggcagag gacgtggagc gggaggcgca ggaggctcag gctggcaaga
6360


 


agaagcgaaa gcagaggagg aggaagggcc ggccagaaga cctggagttc acagacatgg
6420


 


gtggcaatgt gtacatcctc acgggcaagg tggaggcaga gtttgagctg ctgactgtgg
6480


 


aggaggccga gaaacggcca gtggggaagg ggcggaagca gccagagcct ctggagaaac
6540


 


ccagccgccc caaaacttcc ttcaactggt ttgtgaaccc gctgaagacc tttgtcttct
6600


 


tcatctggcg ccggtactgg cgcaccctgg tgctgctgct actggtgctg ctcaccgtct
6660


 


tcctcctcct ggtcttctac accatccctg gccagatcag ccaggtcatc ttccgtcccc
6720


 


tccacaagtg actctcgctg accttggaca ctcacccagg gtgccaaccc ttcaatgcct
6780


 


gctcctggaa gtctttctta cccatgtgag ctaccccaga gtctagtgct tcctctgaat
6840


 


aaacctatca cagcc
6855
















TABLE LIII (f) 





Nucleotide sequence alignment of 121P1F1 v.1 (SEQ


ID NO: 363) and 158P3D2 v.18 (SEQ ID NO: 364)















Score = 2354 bits (1224), Expect = 0.0 Identities = 1224/1224 (100%) Strand = Plus/Plus
































































































































Score = 1340 bits (697), Expect = 0.0 Identities = 697/697 (100%) Strand = Plus/Plus
























































































TABLE LIV(f)





Peptide sequences of protein coded by 158P3D2 v.18


(SEQ ID NO: 365)
















MCKRRWHWPR SSWQNCAFWL RRHPGQPLVR SVPSWSSSCG WAWASKPRPA PLSCPRICCP
60


 


SPQPGCPPAY TGTVLEQTLS PLWDELLVFE QLIVDGRREH LQEEPPLVII NVFDHNKFGP
120


 


PVFLGRALAA PRVKLMEDPY QRPELQFFPL RKGPWAAGEL IAAFQLIELD YSGRLEPSVP
180


 


SEVEPQDLAP LVEPHSGRLS LPPNVCPVLR EFRVEVLFWG LRGLGRVHLL EVEQPQVVLE
240


 


VAGQGVESEV LASYRESPNF TELVRHLTVV FKDTAPLFHP QDLPEQPYLQ PPLSILVIER
300


 


RAFGHTVLVG SHIVPHMLRF TFRGHEDPPE EEGEMEETGD MMPKGPQGQK SLDPFLAEAG
360


 


ISRQLLKHNF DEDEMDDPGD SDGVNLISMV GEIQDQGEAE VKGTVSPKKA VATLKIYNRS
420


 


LKEEFNHFED WLNVFPLYRG QGGQDGGGEE EGSGHLVGKF KGSFLIYPES EAVLFSEPQI
480


 


SRGIPQNRPI KLLVRVYVVK ATNLAPADPN GKADPYVVVS AGRERQDTKE RYIPKQLNPI
540


 


FGEILELSIS LPAETELTVA VFDHDLVGSD DLIGETHIDL ENRFYSHHRA NCGLASQYEV
600


 


WVQQGPQEPF
610
















TABLE LV(f)





Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.18







No significant similarity.

## Claims

1. An isolated 158P3D2 protein, wherein the 158P3D2 protein comprises the amino acid sequence of SEQ ID NO:3 wherein S58R, K281N, or T282A.

2. A composition comprising a pharmaceutically acceptable carrier and a protein of claim 1.

3. The isolated 158P3D2 protein of claim 1, wherein the amino acid sequence of SEQ ID NO:3 comprises Arg at position 58.

4. The isolated 158P3D2 protein of claim 1, wherein the amino acid sequence of SEQ ID NO:3 comprises Asn at position 281.

5. The isolated 158P3D2 protein of claim 1, wherein the amino acid sequence of SEQ ID NO:3 comprises Ala at position 282.