# Patent

## Title
Nucleic acids and corresponding proteins entitled 254P1D6B useful in treatment and detection of cancer

## Abstract

A novel gene 254P1D6B and its encoded protein, and variants thereof, are described wherein 254P1D6B exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 254P1D6B provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 254P1D6B 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 254P1D6B can be used in active or passive immunization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a non-provisional utility patent application that claims priority from U.S. provisional patent application U.S. Ser. No. 60/442,526, filed Jan. 24, 2003. 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.
FIELD OF THE INVENTION
The invention described herein relates to genes and their encoded proteins, termed 254P1D6B and variants thereof, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 254P1D6B.
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 Sep. 2, 1996 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci U S A. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (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 malignandes. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 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 heeded 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 issue 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 254P1D6B, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 254P1 D6B 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 254P1D6B are provided. The tissue-related profile of 254P1D6B in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 254P1 D6B 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 254P1D6B genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 254P1D6B-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 254P1D6B-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 254P1D6B genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 254P1D6B genes, mRNAs, or to 254P1D6B-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 254P1D6B. Recombinant DNA molecules containing 254P1D6B polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 254P1D6B gene products are also provided. The invention further provides antibodies that bind to 254P1D6B 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 254P1D6B polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 254P1D6B. A typical embodiment of this invention provides methods for monitoring 254P1D6B 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 254P1D6B such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 254P1D6B as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 254P1D6B 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 254P1D6B. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 254P1D6B 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 254P1D6B 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 254P1D6B as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 254P1D6B. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 254P1D6B (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 254P1D6B production) or a ribozyme effective to lyse 254P1D6B 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.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 254P1D6B SSH sequence of 284 nucleotides.
FIG. 2. A) The cDNA and amino acid sequence of 254P1D6B variant 1 (also called “254P1D6B v.1” or “254P1D6B variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic add 512-3730 including the stop codon.
B) The cDNA and amino acid sequence of 254P1D6B variant 2 (also called “254P1D6B v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 512-3730 including the stop codon.
C) The cDNA and amino acid sequence of 254P1D6B variant 3 (also called “254P1D6B v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 739-3930 including the stop codon.
D) 254P1D6B v.4 through v.20, SNP variants of 254P1D6B v.1. The 254P1D6B v.4 through v.20 (also called “254P1D6B variant 4 through variant 20”) proteins have 1072 amino acids. Variants 254P1D6B v.4 through v.20 are variants with single nucleotide difference from 254P1D6B v.1. 254P1 D6B v.5 and v.6 proteins differ from 254P1D6B v.1 by one amino acid. 254P1D6B v.4 and v.7 through v.20 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 FIG. 2A, FIG. 2B, and FIG. 2C.
A) The amino acid sequence of 254P1D6B v.1 clone LCP-3 is shown in FIG. 3A; it has 1072 amino acids.
B) The amino acid sequence of 254P1D6B v.2 is shown in FIG. 3B; it has 1072 amino acids.
C) The amino acid sequence of 254P1D6B v.3 is shown in FIG. 3C; it has 1063 amino acids.
D) The amino acid sequence of 254P1D6B v.5 is shown in FIG. 3D; it has 1072 amino acids.
E) The amino acid sequence of 254P1D6B v.6 is shown in FIG. 3E; it has 1072 amino acids.
As used herein, a reference to 254P1D6B includes all variants thereof, including those shown in FIGS. 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.
FIG. 4. Expression of 254P1D6b in 293T cells. FIG. 4A. 293T cells were transfected with either an empty pCDNA 3.1 vector plasmid or pCDNA 3.1 plasmid encoding the full length cDNA of 254P1D6b. 2 days post- transfection, lysates were prepared from the transfected cells and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using an anti-His pAb (Santa Cruz Biotechnology, Santa Cruz, California) to detect the C-terminal epitope tag on the protein. An arrow indicates the band corresponding to the full length 254P1D6b protein product. An additional verified lysate containing an epitope tagged AGSX protein served as a positive control. FIG. 4B. 293T cells were transfected with either an empty vector or the Tag5 expression vector encoding the extracellular domain (ECD) of 254P1D6 (amino acids 26-953) and subjected to SDS-PAGE and Western blotting as described above. An arrow indicates the band conesponding to the 254P1D6b ECD present in the lysates and the media from transfected cells.
FIG. 5. Hydrophilicity amino acid profile of 254P1D6B v.1 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. Hydropathicity amino acid profile of 254P1D6B v.1 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. Percent accessible residues amino acid profile of 254P1D6B v.1 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. Average flexibility amino acid profile of 254P1D6B v.1 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. Beta-turn amino acid profile of 254P1D6B v.1 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.chlcgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 10. Structures of transcript variants of 254P1D6B. Variant 254P1D6B v.3 was identified as a transcript variant of 254P1D6B v.1. Variant 254P1D6B v.3 extended exon 1 by 109 bp as compared to v.1 and added an exon in between exons 2 and 3 of variant v.1. Poly A tails and SNP are not shown here. Numbers in “( )” underneath the boxes correspond to those of 254P1D6B v.1. Lengths of introns and exons are not proportional.
FIG. 11. Schematic alignment of protein variants of 254P1D6B. Protein variants correspond to nucleotide variants. Nucleotide variants 254P1D6B v.4 and v.7 through v.20 coded for the same protein as v.1. Variant v.2 coded the same protein as variant v.6. 254P1 D6Bv.5 coded for a protein that differed by one amino acid from v.1. Nucleotide variant 254P1D6B v.3 was a transcript variant of v.1, as shown in FIG. 10, and coded a protein that differed from v.1 in the N-terminal. SNP in v.1 could also appear in v.3. Single amino acid differences were indicated above the boxes. Black boxes represent the same sequence as 254P1D6B v.1. Numbers underneath the box correspond to 254P1D6B.
FIG. 12. Schematic alignment of SNP variants of 254P1D6B. Variants 254P1D6B v.4 through v.20 were variants with single nucleotide differences as compared to variant v.1 (ORF: 512-3730). Though these SNP variants were shown separately, they could also occur in any combinations, (e.g., occur with 254P1D6Bv.2, and in any transcript variants that contained the base pairs, such as v.3 shown in FIG. 10. Numbers correspond to those of 254P1D6B v.1. Black box shows the same sequence as 254P1D6B v.1. SNPs are indicated above the box.
FIG. 13. Secondary structure and transmembrane domains prediction for 254PI D6b protein variant 1. FIG. 13A: The secondary structures of 254P1D6b protein variant was predicted using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at .expasy.ch/tools/. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein variant in a given secondary structure is also listed. FIG. 13B: Schematic representation of the probability of existence of transmembrane regions of 254P1D6b variant 1 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. 13C: Schematic representation of the probability of the existence of transmembrane regions of 254P1D6b variant 1 based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at .expasy.ch/tools/.
FIG. 14. Expression of 254P1D6B by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal lung, ovary cancer pool, lung cancer pool (FIG. 14A), as well as from normal stomach, brain, heart, liver, spleen, skeletal muscle, testis, prostate, bladder, kidney, colon, lung and ovary cancer pool (FIG. 14B). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 254P1D6B, was performed at 26 and 30 cycles of amplification. Results show strong expression of 254P1D6B in lung cancer pool and ovary cancer pool but not in normal lung nor in vital pool 1. Low expression was detected in vital pool 2.
FIG. 15. Expression of 254P1D6B in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/lane were probed with the 254P1D6B sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of two 254P1D6B transcript, 4.4 kb and 7.5 kb primarily in brain and testis, and only the 4.4 kb transcript in placenta, but not in any other normal tissue tested.
FIG. 16. Expression of 254P1D6B in lung cancer patient specimens. First strand cDNA was prepared from normal lung lung cancer cell line A427 and a panel of lung cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semiquantitative PCR, using primers to 254P1D6B, was performed at 26 and 30 cycles of amplification. Results show expression of 254P1D6B in 13 out of 30 tumor specimens tested but not in normal lung. Expression was also detected in the A427 cell line.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 254P1D6B Polynucleotides

    
    
        II.A.) Uses of 254P1D6B 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 254P1D6B-Encoding Nucleic Acid Molecules
            II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
        
        
    
    


III.) 254P1D6B-related Proteins

    
    
        III.A.) Motif-bearing Protein Embodiments
        III.B.) Expression of 254P1D6B-related Proteins
        III.C.) Modifications of 254P1D6B-related Proteins
        III.D.) Uses of 254P1D6B-related Proteins
    
    


IV.) 254P1D6B Antibodies
V.) 254P1D6B Cellular Immune Responses
VI.) 254P1D6B Transgenic Animals
VII.) Methods for the Detection of 254P1D6B
VIII.) Methods for Monitoring the Status of 254P1D6B-related Genes and Their Products
IX.) Identification of Molecules That Interact With 254P1D6B
X.) Therapeutic Methods and Compositions

    
    
        X.A.) Anti-Cancer Vaccines
        X.B.) 254P1D6B as a Target for Antibody-Based Therapy
        X.C.) 254P1D6B 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 254P1D6B.
XII.) Inhibition of 254P1D6B Protein Function

    
    
        XII.A.) Inhibition of 254P1D6B With Intracellular Antibodies
        XII.B.) Inhibition of 254P1D6B with Recombinant Proteins
        XII.C.) Inhibition of 254P1D6B Transcription or Translation
        XII.D.) General Considerations for Therapeutic Strategies
    
    


XII.) Identification, Characterization and Use of Modulators of 254P1D6B
XIV.) RNAi and Therapeutic use of small interfering RNA
XV.) 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 cancers”, “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 prostate 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 254P1D6B (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 254P1D6B. 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 254P1D6B-related protein). For example, an analog of a 254P1D6B protein can be specifically bound by an antibody or T cell that specifically binds to 254P1D6B.
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-254P1D6B 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 ant-254P1D6B antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-254P1D6B 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)), oligocarbarnates (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, Jan 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, Re186, 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 254P1D6B genes or that encode polypeptides other than 254P1D6B gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 254P1D6B polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 254P1D6B proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 254P1D6B 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 heterocydic 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 254P D6B-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 treat metastases in the


(AC-227)
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 for sterilization of


(Co-60)
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 diagnostic studies (i.e., breast


(Cu-67)
and colon cancers, and lymphoma)


Dysprosium-166
Cancer radioimmunotherapy


(Dy-166)


Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and


(Er-169)
toes


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


(Eu-152)


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


(Eu-154)


Gadolinium-153
Osteoporosis detection and nuclear medical quality assurance devices


(Gd-153)


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


(Au-198)


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


(Ho-166)
marrow ablation, and rheumatoid arthritis treatment


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


(I-125)
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 thyroid cancer as well as


(I-131)
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 blocked arteries (i.e.,


(Ir-192)
arteriosclerosis and restenosis), and implants for breast and prostate tumors


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


(Lu-177)
restenosis)


Molybdenum-99
Parent of Technetium-99 m (Tc-99 m) which is used for imaging the brain, liver, lungs, heart,


(Mo-99)
and other organs. Currently, Tc-99 m 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-195 m
Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug


(Pt-195 m)


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


(P-32)
diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids



for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e.,



arteriosclerosis and restenosis), and intracavity therapy


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


(P-33)
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 and treatment of


(Re-186)
lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy


Rhenium-188
Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief,


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


Rhodium-105
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 by gamma-scintigraphy, lateral


(Se-75)
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 therapy


(Sr-89)


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


(Tc-99 m)


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


(Th-228)


Thorium-229
Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha


(Th-229)
emitters used in cancer radioimmunotherapy


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


(Tm-170)


Tin-117 m
Cancer immunotherapy and bone cancer pain relief


(Sn-117 m)


Tungsten-188
Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone


(W-188)
cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e.,



arteriosclerosis and restenosis)


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


(Xe-127)
and cerebral blood flow studies


Ytterbium-175
Cancer radioimmunotherapy


(Yb-175)


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


(Y-90)


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


(Y-91)
(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 254P1D6B, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 254P1D6B 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, 254P1D6B 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 NaCI, 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 supetypes are as follows:
A2: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207
A3: A3. A11, 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 254P1D6B 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 “254P1D6B-related proteins” of the invention include those specifically identified herein, as well as allelic variant 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 254P1D6B proteins or fragments thereof, as well as fusion proteins of a 254P1D6B protein and a heterologous polypeptide are also included. Such 254P1D6B proteins are collectively referred to as the 254P1D6B-related proteins, the proteins of the invention, or 254P1D6B. The term “254P1D6B-related protein” refers to a polypeptide fragment or a 254P1D6B 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.) 254P1D6B Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 254P1D6B gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 254P1D6B-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 254P1D6B gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 254P1D6B gene, mRNA, or to a 254P1D6B encoding polynucleotide (collectively, “254P1D6B polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 254P1D6B polynucleotide include: a 254P1D6B polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 254P1D6B 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 254P1D6B 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 512 through nucleotide residue number 3730, 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 512 through nucleotide residue number 3730, 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 739 through nucleotide residue number 3930, 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 512 through nucleotide residue number 3730, including the stop codon, wherein T can also be U;
        (VI) a polynucleotide that encodes a 254P1D6B-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-D;
        (VII) a polynucleotide that encodes a 254P1D6B-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-D;
        (VIII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
        (IX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 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, 3B, 3D, and 3E in any whole number increment up to 1072 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 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, 3B, 3D, and 3E in any whole number increment up to 1072 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;
        (XI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 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, 3B, 3D, and 3E in any whole number increment up to 1072 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;
        (XII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A, 3B, 3D, and 3E in any whole number increment up to 1072 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;
        (XIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A, 3B, 3D, and 3E in any whole number increment up to 1072 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;
        (XIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 1063 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 1063 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;
        (XVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 1063 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;
        (XVII) 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. 3C in any whole number increment up to 1063 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;
        (XVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 1063 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;
        (XIX) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XVIII);
        (XX) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XIX);
        (XXI) a peptide that is encoded by any of (I) to (XX); and;
        (XXII) a composition comprising a polynucleotide of any of (I)-(XX) or peptide of (XXI) together with a pharmaceutical excipient and/or in a human unit dose form;
        (XXIII) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to modulate a cell expressing 254P1D6B;
        (XXIV) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 254P1D6B;
        (XXV) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 254P1D6B, said cell from a cancer of a tissue listed in Table I;
        (XXVI) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
        (XXVII) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and;
        (XXVIII) a method of using a polynucleotide of any (I)-(XX) or peptide of (XXI) or a composition of (XXII) in a method to identify or characterize a modulator of a cell expressing 254P1D6B.
    
    


As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 254P1D6B polynucleotides that encode specific portions of 254P1D6B 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, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1060. 1065, 1070, and 1072 or more contiguous amino acids of 254P1D6B variant 1; the maximal lengths relevant for other variants are: variant 2, 1072 amino acids; variant 3, 1063 amino acids, variant 5, 1072 amino acids, variant 6, 1072 amino acids, and variants 4, 7-20, 1072 amino acids.
For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 254P1D6B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 254P1D6B 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 add sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include 254P1D6B polynucleotide fragments encoding one or more of the biological motifs contained within a 254P1D6B protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 254P1D6B gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 254P1D6B.” For example, because the 254P1D6B gene maps to this chromosome, polynucleotides that encode different regions of the 254P1D6B 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(34): 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 254P1D6B proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 254P1D6B 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 254P1D6B was shown to be highly expressed in prostate and other cancers, 254P1D6B polynucleotides are used in methods assessing the status of 254P1D6B gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 254P1D6B 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 254P1D6B 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 254P1D6B. 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 254P1D6B 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 ate complementary to their intracellular targets, e.g., 254P1D6B. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 254P1D6B 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., lyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and lyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 254P1D6B antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 254P1D6B mRNA. Optionally, 254P1D6B 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 254P1D6B. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 254P1D6B 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 254P1D6B polynucleotide in a sample and as a means for detecting a cell expressing a 254P1D6B protein.
Examples of such probes include polypeptides comprising all or part of the human 254P1D6B cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 254P1D6B 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 254P1D6B mRNA.
The 254P1D6B 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 254P1D6B 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 254P1D6B polypeptides; as tools for modulating or inhibiting the expression of the 254P1D6B gene(s) and/or translation of the 254P1D6B transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 254P1D6B or 254P1D6B 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 254P1D6B-Encoding Nucleic Acid Molecules
The 254P1D6B cDNA sequences described herein enable the isolation of other polynucleotides encoding 254P1D6B gene product(s), as well as the isolation of polynucleotides encoding 254P1D6B gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 254P1D6B gene product as well as polynucleotides that encode analogs of 254P1D6B-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 254P1D6B 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 254P1D6B gene cDNAs can be identified by probing with a labeled 254P1D6B cDNA or a fragment thereof. For example, in one embodiment, a 254P1D6B 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 254P1D6B gene. A 254P1D6B gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B or a fragment, analog or homolog thereof can be used to generate 254P1D6B 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 254P1D6B 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, 254P1D6B 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 254P1D6B protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 254P1D6B and 254P1D6B mutations or analogs.
Recombinant human 254P1D6B protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 254P1D6B-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 254P1D6B or fragment, analog or homolog thereof, a 254P1D6B-related protein is expressed in the 293T cells, and the recombinant 254P1D6B protein is isolated using standard purification methods (e.g., affinity purification using anti-254P1D6B antibodies). In another embodiment, a 254P1D6B coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 254P1D6B 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 254P1D6B coding sequence can be used for the generation of a secreted form of recombinant 254P1D6B protein.
As discussed herein, redundancy in the genetic code permits variation in 254P1D6B 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.) 254P1D6B-related Proteins
Another aspect of the present invention provides 254P1D6B-related proteins. Specific embodiments of 254P1D6B proteins comprise a polypeptide having all or part of the amino acid sequence of human 254P1D6B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 254P1D6B proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 254P1D6B shown in FIG. 2 or FIG. 3.
Embodiments of a 254P1D6B polypeptide include: a 254P1D6B polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 254P1D6B 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 254P1D6B peptides comprise, without limitation:

    
    
        (I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-D or FIG. 3A-E;
        (II) a 254P1D6B-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 FIGS. 2A-D or 3A-E;
        (III) a 254P1D6B-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 FIGS. 2A-D or 3A-E;
        (IV) a protein that comprises at least one peptide set forth in Tables VII 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 FIGS. 3A, 3B, 3D, and 3E in any whole number increment up to 1072 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 FIGS. 3A, 3B, 3D, and 3E, in any whole number increment up to 1072 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 FIGS. 3A, 3B, 3D, and 3E, in any whole number increment up to 1072 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 FIGS. 3A, 3B, 3D, and 3E, in any whole number increment up to 1072 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 FIGS. 3A, 3B, 3D, and 3E in any whole number increment up to 1072 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 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. 3C, in any whole number increment up to 1063 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;
        (XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 1063 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;
        (XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 1063 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;
        (XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 1063 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;
        (XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3C in any whole number increment up to 1063 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;
        (XIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
        (XX) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XXI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XXII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XXIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;
        (XXIV) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
        (XXV) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
        (XXVI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
        (XXVII) a peptide which comprises one two, three, four, or five of the following characteristics, or an 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;
        
        
        (XXVIII) a composition comprising a peptide of (I)-(XXVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
        (XXIX) a method of using a peptide of (I)-(XXVII), or an antibody or binding region thereof or a composition of (XXVIII) in a method to modulate a cell expressing 254P1D6B;
        (XXX) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 254P1D6B;
        (XXXI) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 254P1D6B, said cell from a cancer of a tissue listed in Table I;
        (XXXII) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
        (XXXIII) a method of using a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a issue listed in Table I; and;
        (XXXIV) a method of using a a peptide of (I)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to identify or characterize a modulator of a cell expressing 254P1D6B
    
    


As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 254P1D6B polynucleotides that encode specific portions of 254P1D6B 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, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1060, 1070 and 1072 or more contiguous amino acids of 254P1D6B variant 1; the maximal lengths relevant for other variants are: variant 2, 1072 amino acids; variant 3, 1063 amino acids, variant 5, 1072 amino acids, variant 6, 1072 amino acids, and variants 4, 7-20, 1072 amino acids.
In general, naturally occurring allelic variants of human 254P1D6B share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 254P1D6B protein contain conservative amino acid substitutions within the 254P1D6B sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 254P1D6B. One class of 254P1D6B allelic variants are proteins that share a high degree of homology with at least a small region of a particular 254P1D6B 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, glydine (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 pKs 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 May 19, 1995; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 254P1D6B proteins such as polypeptides having amino acid insertions, deletions and substitutions. 254P1D6B 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 254P1D6B 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, 254P1D6B variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 254P1D6B 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 254P1D6B variant also specifically binds to a 254P1D6B 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 254P1D6B 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 254P1D6B-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 254P1D6B protein variants or analogs comprises one or more of the 254P1D6B biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 254P1D6B protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 254P1D6B 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 254P1D6B 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.
254P1D6B-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 add molecules that encode a 254P1D6B-related protein. In one embodiment, nucleic add molecules provide a means to generate defined fragments of a 254P1D6B protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 254P1D6B polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 254P1D6B 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.edulseq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; EpimatixTM and EpimerTM, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrtnih.gov/.).
Motif bearing subsequences of all 254P1D6B variant proteins are set forth and identified in Tables VI II-XXI and XXII-XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wusb.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 254P1D6B motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 254P1D6B motifs discussed above are associated with growth dysregulation and because 254P1D6B 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 254P1D6B protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, URL brown.edu/ResearchrTB-HIV_Lab/epimatrix/epimabix.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(34): 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., Immuno. 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.
254P1D6B-related proteins are embodied in many forms, preferably in isolated form. A purified 254P1D6B protein molecule will be substantially free of other proteins or molecules that impair the binding of 254P1D6B to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 254P1D6B-related proteins include purified 254P1D6B-related proteins and functional, soluble 254P1D6B-related proteins. In one embodiment, a functional, soluble 254P1D6B protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 254P1D6B proteins comprising biologically active fragments of a 254P1D6B amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 254P1D6B protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 254P1D6B 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.
254P1D6B-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-254P1D6B antibodies or T cells or in identifying cellular factors that bind to 254P1D6B. 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 254P1D6B 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 254P1D6B that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 254P1D6B 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 254P1D6B 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.dcrlnih.gov/) are to be “applied” to a 254P1D6B protein in accordance with the invention. As used in this context “applied” means that a 254P1D6B 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 254P1D6B 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 254P1D6B-related Proteins
In an embodiment described in the examples that follow, 254P1D6B 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 254P1D6B with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 254P1D6B protein in transfected cells. The secreted HIS-tagged 254P1D6B in the culture media can be purified e.g., using a nickel column using standard techniques.
III.C.) Modifications of 254P1D6B-related Proteins
Modifications of 254P1D6B-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 254P1D6B polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C terminal residues of a 254P1D6B protein. Another type of covalent modification of a 254P1D6B 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 254P1D6B comprises linking a 254P1D6B 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. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 254P1D6B-related proteins of the present invention can also be modified to form a chimeric molecule comprising 254P1D6B 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 254P1D6B 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 254P1D6B. A chimeric molecule can comprise a fusion of a 254P1D6B-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 254P1D6B protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 254P1D6B-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 254P1D6B 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 254P1D6B-related Proteins
The proteins of the invention have a number of different specific uses. As 254P1D6B is highly expressed in prostate and other cancers, 254P1D6B-related proteins are used in methods that assess the status of 254P1D6B gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 254P1D6B 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 254P1D6B-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 254P1D6B 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, 254P1D6B-related proteins that contain the amino acid residues of one or more of the biological motifs in a 254P1D6B protein are used to screen for factors that interact with that region of 254P1D6B.
254P1D6B protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 254P1D6B protein), for identifying agents or cellular factors that bind to 254P1D6B 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 254P1D6B 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 254P1D6B gene product Antibodies raised against a 254P1D6B protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 254P1D6B protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 254P1D6B-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 254P1D6B proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzymelinked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 254P1 D6B-expressing cells (e.g., in radioscintigraphic imaging methods). 254P1D6B proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 254P1D6B Antibodies
Another aspect of the invention provides antibodies that bind to 254P0D6B-related proteins. Preferred antibodies specifically bind to a 254P1D6B-related protein and do not bind (or bind weakly) to peptides or proteins that are not 254P1D6B-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Trisbuffered 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 254P1D6B can bind 254P1D6B-related proteins such as the homologs or analogs thereof.
254P1D6B 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 254P1D6B 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 254P1D6B is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 254P1D6B and mutant 254P1D6B-related proteins. Such assays can comprise one or more 254P1D6B antibodies capable of recognizing and binding a 254P1D6B-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), enzymelinked 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 histocompatability complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 254P1D6B are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 254P1D6B antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 254P1D6B expressing cancers such as prostate cancer.
254P1D6B antibodies are also used in methods for purifying a 254P1D6B-related protein and for isolating 254P1D6B homologues and related molecules. For example, a method of purifying a 254P D6B-related protein comprises incubating a 254P1D68 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 254P1D6B-related protein under conditions that permit the 254P1D6B antibody to bind to the 254P1D6B-related protein; washing the solid matrix to eliminate impurities; and eluting the 254P1D6B-related protein from the coupled antibody. Other uses of 254P1D6B antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 254P1D6B 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 254P1D6B-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 254P1D6B can also be used, such as a 254P1D6B 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 254P1D6B-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 254P1D6B-related protein or 254P1D6 B 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 254P1D6B protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 254P1D6B protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 254P1D6B amino acid sequence are used to identify hydrophilic regions in the 254P1D6B structure. Regions of a 254P1D6B protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, 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 254P1D6B 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 254P1D6B 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.
254P1D6B 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 254P1D6B-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 254P1D6B protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 254P1D6B 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 254P1D6B 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 254P1D6B monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kuchedapat 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 Dec. 19, 2000; U.S. Pat. No. 6,150,584 issued Nov. 12, 2000; and U.S. Pat. No. 6,114,598 issued Sep. 5, 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 254P1D6B antibodies with a 254P1D6B-related protein can be established by a number of well known means, including Western blot, immunoprecipitaton, ELISA, and FACS analyses using, as appropriate, 254P1D6B-related proteins, 254P1D6B-expressing cells or extracts thereof. A 254P1D6B 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 254P1D6B 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.) 254P1D6B Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the worldwide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. 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 Nov; 50(3-4):201-12 Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, 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; Stem et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. 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.) 254P1D6B Transgenic Animals
Nucleic acids that encode a 254P1D6B-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 254P1D6B can be used to clone genomic DNA that encodes 254P1D6B. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 254P1D6B. 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 254P1D1878 6B transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 254P1D6B can be used to examine the effect of increased expression of DNA that encodes 254P1D6B. 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 254P1D6B can be used to construct a 254P1D6B “knock out” animal that has a defective or altered gene encoding 254P1D6B as a result of homologous recombination between the endogenous gene encoding 254P1D6B and altered genomic DNA encoding 254P1D6B introduced into an embryonic cell of the animal. For example, cDNA that encodes 254P1D6B can be used to clone genomic DNA encoding 254P1D6B in accordance with established techniques. A portion of the genomic DNA encoding 254P1D6B 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 254P1D6B polypeptide.
VII.) Methods for the Detection of 254P1D6B
Another aspect of the present invention relates to methods for detecting 254P1D6B polynucleotides and 254P1D6B-related proteins, as well as methods for identifying a cell that expresses 254P1D6B. The expression profile of 254P1D6B makes it a diagnostic marker for metastasized disease. Accordingly, the status of 254P1D6B 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 254P1D6B 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 254P1D6B polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 254P1D6B polynucleotides include, for example, a 254P1D6B gene or fragment thereof, 254P1D6B mRNA, alternative splice variant 254P1D6B mRNAs, and recombinant DNA or RNA molecules that contain a 254P1D6B polynucleotide. A number of methods for amplifying and/or detecting the presence of 254P1D6B 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 254P1D6B 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 254P1D6B polynucleotides as sense and antisense primers to amplify 254P1D6B cDNAs therein; and detecting the presence of the amplified 254P1D6B cDNA. Optionally, the sequence of the amplified 254P1D6B cDNA can be determined.
In another embodiment, a method of detecting a 254P1D6B gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 254P1D6B polynucleotides as sense and antisense primers; and detecting the presence of the amplified 254P1D6B gene. Any number of appropriate sense and antisense probe combinations can be designed from a 254P1D6B nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 254P1D6B protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 254P1D6B-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 254P1D6B-related protein in a biological sample comprises first contacting the sample with a 254P1D6B antibody, a 254P1D6B-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 254P1D6B antibody; and then detecting the binding of 254P1D6B-related protein in the sample.
Methods for identifying a cell that expresses 254P1D6B are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 254P1D6B gene comprises detecting the presence of 254P1D6B 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 254P1D6B riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 254P1D6B, 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 254P1D6B gene comprises detecting the presence of 254P1D6B-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 254P1D6B-related proteins and cells that express 254P1D6B-related proteins.
254P1D6B expression analysis is also useful as a tool for identifying and evaluating agents that modulate 254P1D6B gene expression. For example, 254P1D6B 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 254P1D6B 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 254P1D6B expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 254P1D6B-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 254P1D6B 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 254P1D6B in a biological sample of interest can be compared, for example, to the status of 254P1D6B 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 254P1D6B 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., Grever et al., J. Comp. Neurol. Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 254P1D6B 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 254P1D6B expressing cells) as well as the level, and biological activity of expressed gene products (such as 254P1D6B mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 254P1D6B comprises a change in the location of 254P1D6B and/or 254P1D6B expressing cells and/or an increase in 254P1D6B mRNA and/or protein expression.
254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B gene), Northern analysis and/or PCR analysis of 254P1D6B mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 254P1D6B 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 254P1D6B proteins and/or associations of 254P1D6B proteins with polypeptide binding partners). Detectable 254P1D6B polynucleotides include, for example, a 254P1D6B gene or fragment thereof, 254P1D6B mRNA, alternative splice variants, 254P1D6B mRNAs, and recombinant DNA or RNA molecules containing a 254P1D6B polynucleotide.
The expression profile of 254P1D6B 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 254P1D6B provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 254P1D6B status and diagnosing cancers that express 254P1D6B, such as cancers of the tissues listed in Table I. For example, because 254P1D6B mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 254P1D6B mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 254P1D6B dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 254P1D6B 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 254P1D6B 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 254P1D6B in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 254P1D6B 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 254P1D6B expressing cells (e.g. those that express 254P1D6B mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 254P1D6B-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 254P1D6B 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 Aug. 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 254P1D6B gene products by determining the status of 254P1D6B 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 254P1D6B gene products in a corresponding normal sample. The presence of aberrant 254P1D6B 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 254P1D6B 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 254P1D6B mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 254P1D6B 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 254P1D6B mRNA or express it at lower levels.
In a related embodiment, 254P1D6B status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 254P1D6B protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 254P1D6B expressed in a corresponding normal sample. In one embodiment, the presence of 254P1D6B protein is evaluated, for example, using immunohistochemical methods. 254P1D6B antibodies or binding partners capable of detecting 254P1D6B 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 254P1D6B 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 254P1D6B may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 254P1D6B 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 254P1D6B 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 Sep. 7, 1999, and U.S. Pat. No. 5,952,170 issued Jan. 17, 1995).
Additionally, one can examine the methylation status of a 254P1D6B gene in a biological sample. Aberrant demethylation and/or hypermethyation 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 prostate intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in 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 methyation 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 254P1D6B. 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 254P1D6B expression. The presence of RT-PCR amplifiable 254P1D6B 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 254P1D6B mRNA or 254P1D6B protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 254P1D6B mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 254P1D6B in prostate or other tissue is examined, with the presence of 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B mRNA or 254P1D6B protein expressed by tumor cells, comparing the level so determined to the level of 254P1D6B mRNA or 254P1D6B protein expressed in a corresponding normal tissue taken from the same individual or a normal issue reference sample, wherein the degree of 254P1D6B mRNA or 254P1D6B 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 254P1D6B is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 254P1D6B 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 254P1D6B mRNA or 254P1D6B protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 254P1D6B mRNA or 254P1D6B protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 254P1D6B mRNA or 254P1D6B 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 254P1D6B expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 254P1D6B 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 254P1D6B gene and 254P1D6B gene products (or perturbations in 254P1D6B gene and 254P1D6B gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a issue 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 254P1D6B gene and 254P1D6B gene products (or perturbations in 254P1D6B gene and 254P1D6B 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 254P1D6B gene and 254P1D6B gene products (or perturbations in 254P1D6B gene and 254P1D6B gene products) and another factor associated with malignancy entails detecting the overexpression of 254P1D6B mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a issue sample (or PSCA or PSM expression), and observing a coincidence of 254P1D6B mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 254P1D6B and PSA mRNA in prostate tissue is examined, where the coincidence of 254P1D6B 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 254P1D6B 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 254P1D6B mRNA include in situ hybridization using labeled 254P1D6B riboprobes, Northern blot and related techniques using 254P1068 polynucleotide probes, RT-PCR analysis using primers specific for 254P1D6B, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitatve RT-PCR is used to detect and quantify 254P1D6B mRNA expression. Any number of primers capable of amplifying 254P1D6B 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 254P1D6B protein can be used in an immunohistochemical assay of biopsied issue.
IX.) Identification of Molecules that Interact with 254P1D6B
The 254P1D6B protein and nucleic add sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 254P1D6B, as well as pathways activated by 254P1D6B 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 assays”). 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 Sep. 21, 1999, U.S. Pat. No. 5,925,523 issued Jul. 20, 1999, U.S. Pat. No. 5,846,722 issued Dec. 8, 1998 and U.S. Pat. No. 6,004,746 issued Dec. 21, 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: Nov. 4, 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 254P1D6B protein sequences. In such methods, peptides that bind to 254P1D6B 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 254P1D6B 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 254P1D6B protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued Mar. 3, 1998 and U.S. Pat. No. 5,733,731 issued Mar. 31, 1998.
Alternatively, cell lines that express 254P1D6B are used to identify protein-protein interactions mediated by 254P1D6B. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 254P1D6B protein can be immunoprecipitated from 254P1D6B-expressing cell lines using anti-254P1D6B antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 254P1D6B 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 254P1D6B 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 254P1D6B'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 254P1D6B-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 254P1D6B (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 254P1D6B function can be identified based on their ability to bind 254P1D6B and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued Jul. 27, 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 254P1D6B 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 254P1D6B.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 254P1D6B amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 254P1D6B amino acid sequence, allowing the population of molecules and the 254P1D6B amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 254P1D6B amino acid sequence, and then separating molecules that do not interact with the 254P1D6B 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 254P1D6B amino acid sequence. The identified molecule can be used to modulate a function performed by 254P1D6B. In a preferred embodiment, the 254P1D6B amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 254P1D6B 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 254P1D6B protein are useful for patients suffering from a cancer that expresses 254P1D6B. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 254P1D6B protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 254P1D6B gene or translation of 254P1D6B mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 254P1D6B-related protein or 254P1D6B-related nucleic acid. In view of the expression of 254P1D6B, cancer vaccines prevent and/or treat 254P1D6B-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 254P1D6B-related protein, or a 254P1D6B-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 254P1D6B 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 Feb 31(1 ):66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral 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 254P1D6B 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 254P1D6B immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 254P1D6B indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire 254P1D6B protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitielio, 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. Imnmunol. 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 254P1D6B-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.
Cellular Vaccines
CTL epitopes can be determined using specific algorithms to identify peptides within 254P1D6B protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University (URL brown.edu/Research/TB-HIV_Lablepimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 254P1D6B 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.
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 254P1D6B protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 254P1D6B in a host, by contacting the host with a sufficient amount of at least one 254P1D6B B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 254P1D6B B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 254P1D6B-related protein or a man-made multiepitopic peptide comprising: administering 254P1D6B immunogen (e.g. a 254P1D6B protein or a peptide fragment thereof, a 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B, in order to generate a response to the target antigen.
Nucleic Acid Vaccines
Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 254P1D6B. Constructs comprising DNA encoding a 254P1D6B-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 254P1D6B proteinfimmunogen. Alternatively, a vaccine comprises a 254P1D6B-related protein. Expression of the 254P1D6B-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 254P1D6B 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 254P1D6B-related protein into the patient (e.g., intramuscularly or intradermally) to induce an ant-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 254P1D6B-related nucleic acid molecule. In one embodiment, the full-length human 254P1D6B cDNA is employed. In another embodiment, 254P1D6B nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
Ex Vivo Vaccines
Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCS) such as dendritic cells (DC) to present 254P1D6B 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 254P1D6B peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 254P1D6B peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 254P1D6B protein. Yet another embodiment involves engineering the overexpression of a 254P1D6B 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 254P1D6B can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 254P1D6B as a Target for Antibody-Based Therapy
254P1D6B 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 254P1D6B is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 254P1D6B-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 254P1D6B are useful to treat 254P1D6B-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
254P1D6B antibodies can be introduced into a patient such that the antibody binds to 254P1D6B 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 254P1D6B, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor antiogenesis 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 254P1D6B 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., Slevers 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. 254P1D6B), 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-254P1D6B antibody) that binds to a marker (e.g. 254P1D6B) 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 254P1D6B, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 254P1D6B 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-254P1D6B 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, 254P1D6B 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 maytahsinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).
Although 254P1D6B 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 254P1D6B 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 254P1D6B expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 254P1D6B imaging, or other techniques that reliably indicate the presence and degree of 254P1D6B 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-254P1D6B 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-254P1D6B 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-254P1D6B mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 254P1D6B. 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-254P1D6B 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 254P1D6B antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-254P1D6B 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-254P1D6B 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-254P1D6B mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-254P1D6B 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-254P1D6B 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-254P1D6B 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 254P1D6B expression in the patient, the extent of circulating shed 254P1D6B 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 254P1D6B in a given sample (e.g. the levels of circulating 254P1D6B antigen and/or 254P1D6B 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-254P1D6B antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 254P1D6B-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-254P1D6B antibodies that mimic an epitope on a 254P1D6B-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.) 254P1D6B 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 L-lysine, poly L-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 254P1D6B 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 TM 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 254P1D6B, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 254P1D6B (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, Bio Techniques 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: 13), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNVVNS; (SEQ ID NO: 14), and Streptococcus 18 kD protein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 15). 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: 16), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response 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 254P1D6B. 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 254P1D6B.
X.D. Adoptive Immunotherapy
Antigenic 254P1D6B-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 254P1D6B. 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 254P1D6B. 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 254P1D6B-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 TM-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the parent's health status. For example, in a patient with a tumor that expresses 254P1D6B, a vaccine comprising 254P1D6B-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 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 34 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-254P1 D6B 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 patent body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-254P1D6B 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 254P1D6B expression in the patient, the extent of circulating shed 254P1D6B 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 patent 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 254P1D6B
As disclosed herein, 254P1D6B 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 254P1D6B in normal tissues, and patient specimens”).
254P1D6B 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. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med Jul. 4, 1999(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 254P1D6B polynucleotides and polypeptides (as well as 254P1D6B polynucleotide probes and anti-254P1D6B 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 254P1D6B 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 254P1D6B polynucleotides described herein can be utilized in the same way to detect 254P1D6B 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 254P1D6B polypeptides described herein can be utilized to generate antibodies for use in detecting 254P1D6B 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 254P1D6B 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 254P1D6B-expressing cells (lymph node) is found to contain 254P1D6B-expressing cells such as the 254P1D6B expression seen in LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 254P1D6B 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 254P1D6B or express 254P1D6B at a different level are found to express 254P1D6B or have an increased expression of 254P1D6B (see, e.g., the 254P1D6B 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 254P1D6B) 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 254P1D6B 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 254P1D6B 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 localizabon/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 254P1D6B, the 254P1D6B 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 254P1D6B protein and immune responses related thereto very useful. Use of the 254P1D6B compositions allows those skilled in the art to make important diagnostic and therapeutic decisions. Immunohistochemical reagents specific to 254P1D6B are also useful to detect metastases of tumors expressing 254P1D6B when the polypeptide appears in tissues where 254P1D6B is not normally produced.
Thus, 254P1D6B 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, 254P1D6B 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 254P1D6B in normal tissues, and patient specimens,” where a 254P1D6B polynucleotide fragment is used as a probe to show the expression of 254P1D6B 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. Nov.-Dec. 11, 1996(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 254P1D6B 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. 254P1D6B 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 254P1D6B 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 254P1D6B polypeptide shown in FIG. 3);
As shown herein, the 254P1D6B polynucleotides and polypeptides (as well as the 254P1D6B polynucleotide probes and anti-254P1D6B 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 254P1D6B 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 254P1D6B polynucleotides and polypeptides (as well as the 254P1D6B polynucleotide probes and anti-254P1D6B 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 254P1D6B 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 254P1D6B gene maps (see the Example entitled “Chromosomal Mapping of 254P1D6B” below). Moreover, in addition to their use in diagnostic assays, the 254P1D6B-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 Jun. 28, 1996;80(1-2): 63-9).
Additionally, 254P1D6B-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 254P1D6B. 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 254P1D6B antigen. Antibodies or other molecules that react with 254P1D6B can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 254P1D6B Protein Function
The invention includes various methods and compositions for inhibiting the binding of 254P1D6B to its binding partner or its association with other protein(s) as well as methods for inhibiting 254P1D6B function.
XII.A.) Inhibition of 254P1D6B with Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 254P1D6B are introduced into 254P1D6B expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-254P1D6B antibody is expressed intracellularly, binds to 254P1D6B 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 add 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 254P1D6B in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 254P1D6B intrabodies in order to achieve the desired targeting. Such 254P1D6B intrabodies are designed to bind specifically to a particular 254P1D6B domain. In another embodiment, cytosolic intrabodies that specifically bind to a 254P1D6B protein are used to prevent 254P1D6B from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 254P1D6B 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 254P1D6B with Recombinant Proteins
In another approach, recombinant molecules bind to 254P1D6B and thereby inhibit 254P1D6B function. For example, these recombinant molecules prevent or inhibit 254P1D6B 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 254P1D6B specific antibody molecule. In a particular embodiment, the 254P1D6B binding domain of a 254P1D6B binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 254P1D6B 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 254P1D6B, whereby the dimeric fusion protein specifically binds to 254P1D6B and blocks 254P1D6B interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 254P1D6B Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 254P1D6B gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 254P1D6B mRNA into protein.
In one approach, a method of inhibiting the transcription of the 254P1D6B gene comprises contacting the 254P1D6B gene with a 254P1D6B antisense polynucleotide. In another approach, a method of inhibiting 254P1D6B mRNA translation comprises contacting a 254P1D6B mRNA with an antisense polynucleotide. In another approach, a 254P1D6B specific ribozyme is used to cleave a 254P1D6B message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 254P1D6B gene, such as 254P1D6B promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 254P1D6B gene transcription factor are used to inhibit 254P1D6B 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 254P1D6B by interfering with 254P1D6B transcriptional activation are also useful to treat cancers expressing 254P1D6B. Similarly, factors that interfere with 254P1D6B processing are useful to treat cancers that express 254P1D6B. 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 254P1D6B (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 254P1D6B inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 254P1D6B antisense polynucleotides, ribozymes, factors capable of interfering with 254P1D6B 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 patents 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 254P1D6B to a binding partner, etc.
In vivo, the effect of a 254P1D6B 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 254P1D6B
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.
Modulator-Related Identification and Screening Assays
Gene Expression-Related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high 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 Zlokamik, 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.
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.
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.
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.
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.
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 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.
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.
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.
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.
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 Stern 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.
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.
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.
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.
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.
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)).
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)).
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.
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.
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.) RNAi and Therapeutic Use of Small Interfering RNA (siRNAs)
The present invention is also directed towards siRNA oligonucleotides, particularly double stranded RNAs encompassing at least a fragment of the 254P1D6B coding region or 5′′ UTR regions, or complement, or any antisense oligonucleotide specific to the 254P1D6B sequence. In one embodiment such oligonucleotides are used to elucidate a function of 254P1D6B, or are used to screen for or evaluate modulators of 254P1D6B function or expression embodiment, gene expression of 254P1D6B is reduced by using siRNA transfection and results in significantly diminished proliferative capacity of transformed cancer cells that endogenously express the antigen; cells treated with specific 254P1D6B siRNAs show reduced survival as measured, e.g., by a metabolic readout of cell viability, correlating to the reduced proliferative capacity. Thus, 254P1D6B siRNA compositions comprise siRNA (double stranded RNA) that correspond to the nucleic acid ORF sequence of the 254P1D6B protein or subsequences thereof; these subsequences 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 and contain sequences that 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.
RNA interference is a novel approach to silencing genes in vitro and in vivo, thus small double stranded RNAs (siRNAs) are valuable therapeutic agents. The power of siRNAs to silence specific gene activities has now been brought to animal models of disease and is used in humans as well. For example, hydrodynamic infusion of a solution of siRNA into a mouse with a siRNA against a particular target has been proven to be therapeutically effective.
The pioneering work by Song et al indicates that one type of entirely natural nucleic acid, small interfering RNAs (siRNAs), served as therapeutic agents even without further chemical modification (Song, E., et al. “RNA interference targeting Fas protects mice from fulminant hepatitis” Nat. Med. 9(3): 347-51(2003)). This work provided the first in vivo evidence that infusion of siRNAs into an animal could alleviate disease. In that case, the authors gave mice injections of siRNA designed to silence the FAS protein (a cell death receptor that when over-activated during inflammatory response induces hepatocytes and other cells to die). The next day, the animals were given an antibody specific to Fas. Control mice died of acute liver failure within a few days, while over 80% of the siRNA-treated mice remained free from serious disease and survived. About 80% to 90% of their liver cells incorporated the naked siRNA oligonucleotides. Furthermore, the RNA molecules functioned for 10 days before losing effect after 3 weeks.
For use in human therapy, siRNA is delivered by efficient systems that induce long-lasting RNAi activity. A major caveat for clinical use is delivering siRNAs to the appropriate cells. Hepatocytes seem to be particularly receptive to exogenous RNA. Today, targets located in the liver are attractive because liver is an organ that can be readily targeted by nucleic acid molecules and viral vectors. However, other tissue and organs targets are preferred as well.
Formulations of siRNAs with compounds that promote transit across cell membranes are used to improve administration of siRNAs in therapy. Chemically modified synthetic siRNA, that are resistant to nucleases and have serum stability have concomitant enhanced duration of RNAi effects, are an additional embodiment.
Thus, siRNA technology is a therapeutic for human malignancy by delivery of siRNA molecules directed to 254P1D6B to individuals with the cancers, such as those listed in Table 1. Such administration of siRNAs leads to reduced growth of cancer cells expressing 254P1D6B, and provides an anti-tumor therapy, lessening the morbidity and/or mortality associated with malignancy.
The effectiveness of this modality of gene product knockdown is significant when measured in vitro or in vivo. Effectiveness in vitro is readily demonstrable through application of siRNAs to cells in culture (as described above) or to aliquots of cancer patient biopsies when in vitro methods are used to detect the reduced expression of 254P1D6B protein.
XV.) 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 254P1D6B 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 254P1D6B and modulating the function of 254P1D6B.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the 254P1D6B Gene
To isolate genes that are over-expressed in prostate cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from prostate cancer xenograft tissues. LAPC-9AD xenograft was obtained from Dr. Charles Sawyers (UCLA) and was generated as described (Klein et al., 1997, Nature Med. 3:402408; Craft et al.,. 1999, Cancer Res. 59:5030-5036). LAPC-9AD2 was generated from LAPC-9AD xenograft by growing LAPC-9AD xenograft tissues within a piece of human bone implanted in SCID mice. Tumors were then harvested and subsequently passaged subcutaneously into other SCID animals to generate LAPC-9AD2.
The 254P1D6B SSH cDNA of 284 bp is listed in FIG. 1. The full length 254P1D6B variant 1 and variants 2-20, cDNAs and ORFs are described in FIG. 2 with the protein sequences listed in FIG. 3.
Materials and Methods
RNA Isolation
Tumor tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/108 cells to 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):




 


5′TTTTGATCAAGCTT303′
(SEQ ID NO: 17)


 


Adaptor 1:


 


5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′
(SEQ ID NO: 18)


 


3′GGCCCGTCCTAG5′
(SEQ ID NO: 19)


 


Adaptor 2:


 


5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′
(SEQ ID NO: 20)


 


3′CGGCTCCTAG5′
(SEQ ID NO: 21)


 


PCR primer 1:


 


5′CTAATACGACTCACTATAGGGC3′
(SEQ ID NO: 22)


 


Nested primer (NP)1:


 


5′TCGAGCGGCCGCCCGGGCAGGA3′
(SEQ ID NO: 23)


 


Nested primer (NP)2:


 


5′AGCGTGGTCGCGGCCGAGGA3′
(SEQ ID NO: 24)






Suppression Subtractive Hybridization
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from prostate cancer xenograft LAPC-9AD2. The gene 254P1D6B was derived from a prostate cancer xenograft LAPC-9AD2 minus prostate cancer xenograft LAPC-9AD tissues. The SSH DNA sequence (FIG. 1), was identified.
The cDNA derived from prostate cancer xenograft LAPC-9AD tissue was used as the source of the “driver” cDNA, while the cDNA from prostate cancer xenograft LAPC-9AD2 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 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.
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 Adapt 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 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles the 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 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 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. 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: 25) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 26) 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 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 bp 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 254P1D6B 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.
A typical RT-PCR expression analysis is shown in FIGS. 14(a) and 14(b). First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal lung ovary cancer pool, lung cancer pool (FIG. 14A), as well as from normal stomach, brain, heart, liver, spleen, skeletal muscle, testis, prostate, bladder, kidney, colon, lung and ovary cancer pool (FIG. 14B). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 254P1D6B, was performed at 26 and 30 cycles of amplification. Results show strong expression of 254P1D6B in lung cancer pool and ovary cancer pool but not in normal lung nor in vital pool 1. Low expression was detected in vital pool 2.
Example 2
Isolation of Full Length 254P1D6B Encoding DNA
To isolate genes that are involved in prostate cancer, an experiment was conducted using the prostate cancer xenograft LAPC-9AD2. The gene 254P1D6B was derived from a subtraction consisting of a prostate cancer xenograft LAPC-9AD2 minus prostate cancer xenograft LAPC-9AD. The SSH DNA sequence (FIG. 1) was designated 254P1D6B. Variants of 254P1D6B were identified (FIGS. 2 and 3).
Example 3
Chromosomal Mapping of 254P1D6B
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 Cornell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).
254P1D6B maps to chromosome 6p22 using 254P1D6B 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 254P1D6B in Normal Tissues and Patient Specimens
FIGS. 14(a) and 14(b) shows expression of 254P1D6B by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal lung ovary cancer pool, lung cancer pool. (FIG. 14A), as well as from normal stomach, brain, heart, liver, spleen, skeletal muscle, testis, prostate, bladder, kidney, colon, lung and ovary cancer pool (FIG. 14B). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 254P1D6B, was performed at 26 and 30 cycles of amplification. Results show strong expression of 254P1D6B in lung cancer pool and ovary cancer pool but not in normal lung nor in vital pool 1. Low expression was detected in vital pool 2.
FIG. 15 shows expression of 254P1D6B in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 μg of mRNA/lane were probed with the 254P1D6B sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of two 254P1D6B transcript, 4.4 kb and 7.5 kb primarily in brain and testis, and only the 4.4 kb transcript in placenta, but not in any other normal tissue tested.
FIG. 16 shows expression of 254P1D6B 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. Semiquantitative PCR, using primers to 254P1D6B, was performed at 26 and 30 cycles of amplification. Results show expression of 254P1D6B in 13 out of 30 tumor specimens tested but not in normal lung. Expression was also detected in the A427 cell line.
Example 5
Splice Variants of 254P1D6B
As used herein, the term variant or comprises Transcript variants and Single Nucleotide Polymorphisms (SNPs). Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for the same, similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different subcellular or extracellular localizations, 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 experiments, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not yet a full-length clone, that portion of the variant is very useful as a research tool, e.g., for antigen generation and for further cloning of the full-length splice variant, using techniques known to those skilled in the art.
Moreover, computer programs are available to those skilled in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. April 2000; 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. Jun. 8, 2001; 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 U S A. Nov. 7, 2000; 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. Aug. 199917; 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 254P1D6B has a particular expression profile related to cancer (See, e.g., Table I). Alternative transcripts and splice variants of 254P1D6B are also be involved in cancers in the same or different tissues, thus serving a tumor-associated markers/antigens.
Using the full-length gene and EST sequences, one additional transcript variant was identified, designated as 254P1D6B v.3. The boundaries of exons in the original transcript, 254P1D6B v.1 are shown in Table LI. The structure of the transcript variants are shown in FIG. 10. Variant 254P1D6B v.3 extended exon 1 of v.1 by 109 base pairs and added an exon in between exons 2 and 3 of v.1.
Table LII shows nucleotide sequence of the transcript variant. Table LIII shows the alignment of the transcript variant with nucleic acid sequence of 254P1D6B v.1. Table LIV lays out amino acid translation of the transcript variant for the identified reading frame orientation. Table LV displays alignments of the amino acid sequence encoded by the splice variant with that of 254P1D6B v.1.
Example 6
Single Nucleotide Polymorphisms of 254P1D6B
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (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).
SNPs are identified by, a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. October-November 2001; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. July 1998; 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, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (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, seventeen SNPs were identified in the original transcript, 254P1D6B v.1, at positions 286 (C/G), 935 (C/A), 980 (T/G), 2347 (G/A), 3762 (C/T), 3772 (A/G), 3955 (C/T), 4096 (C/T), 4415 (G/A), 4519 (G/A), 4539 (A/G), 4614 (G/T), 5184 (G/C), 5528 (T/G), 5641 (G/A), 6221 (T/C) and 6223 (G/A). The transcripts or proteins with alternative alleles were designated as variants 254P1D6B v.4 through v.20, 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 variant 1 are not shown in FIG. 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes, such as v.2) and in any one of the transcript variants (such as 254P1D6B v.3) that contains the sequence context of the SNPs.
Example 7
Production of Recombinant 254P1D6B in Prokaryotic Systems
To express recombinant 254P1D6b and 254P1D6b variants in prokaryotic cells, the full or partial length 254P1D6B and 254P1D6B variant cDNA sequences are cloned into anyone of a variety of expression vectors known in the art. One or more of the following regions of 254P1D6B variants are expressed: the full length sequence presented in FIGS. 2 and 3, 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 254P1D6B, variants, or analogs thereof.
A. In Vitro Transcription and Translation Constructs
pCRII: To generate 254P1D6B 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 254P1D6B cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 254P1D6B RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 254P1D6B at the RNA level. Transcribed 254P1D6B RNA representing the cDNA amino acid coding region of the 254P1D6B gene is used in vitro translation systems such as the TnTM. Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 254P1D6B protein.
B. Bacterial Constructs
pGEX Constructs: To generate recombinant 254P1D6B proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 254P1D6B 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 254P1D6B 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 254P1D6B-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 254P1D6B proteins that are fused to maltose-binding protein (MBP), all or parts of the 254P1D6B 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 254P1D6B 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 254P1D6B. 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 254P1D6B in bacterial cells, all or parts of the 254P1D6B cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 254P1D6B 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 254P1D6B protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs
pESC Constructs: To express 254P1D6B in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 254P1D6B 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 254P1D6B. 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 254P1D6B in the yeast species Saccharomyces pombe, all or parts of the 254P1D6B cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 254P1D6B 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 254P1D6B in Higher Eukaryotic Systems
A. Mammalian Constructs
To express recombinant 254P1D6B in eukaryotic cells, the full or partial length 254P1D6B cDNA sequence cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 254P1D6B were expressed in these constructs, amino acids 1 to 1072, 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 254P1D6B v.1, v.2, v.5, and v.6; amino acids 1 to 1063 of v.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 254P1D6B 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-254P1D6B polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express 254P1D6B in mammalian cells, a 254P1D6B ORF, or portions thereof, of 254P1D6B 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 254P1D6B in mammalian cells, a 254P1D6B ORF, or portions thereof, of 254P1D6B with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression was 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. 
The complete ORF of 254P1D6B v.2 was cloned into the pcDNA3.1/MycHis construct to generate 254P1D6B.pcDNA3.1/MycHis. FIG. 4A shows expression of 254P1D6B.pcDNA3.1/MycHis following transfection into 293T cells. 293T cells were transfected with either 254P1D6B.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control. Forty hours later, cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression of 254P1D6B from the 254P1D6B.pcDNA3.1/MycHis construct in the lysates of transfected cells.
pcDNA3.1/CT-GFP-TOPO Construct: To express 254P1D6B in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 254P1D6B ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, Calif.). 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 pcDNA3.1 CT-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 254P1D6B protein.
PAPtag: A 254P1D6B 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 254P1D6B 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 254P1D6B protein. The resulting recombinant 254P1D6B 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 254P1D6B 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 254P1D6B ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 254P1D6B 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 254P1D6B 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 254P1D6B 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. 
The extracellular domain, amino acids 26-953, of 254P1D6B v.1 was cloned into the pTag5 construct to generate 254P1D6B.pTag5. FIG. 4B shows expression and secretion of the extracellular domain of 254P1D6B following 254P1D6B.pTag5 vector transfection into 293T cells. 293T cells were transfected with 254P1D6B.pTag5 construct. Forty hours later, supernatant as well as cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression and secretion of 254P1D6B from the 254P1D6B.pTag5 transfected cells.
PsecFc: A 254P1D6B 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, Calif.). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 254P1D6B proteins, while fusing the IgGK signal sequence to N-terminus. 254P1D6B fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 254P1D6B 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 254P1D6B 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 254P1D6B constitutively, 254P1D6B ORF, or portions thereof, of 254P1D6B 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, 254P1D6B, 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 FLAGTM tag to the carboxyl-terminus of 254P1D6B sequences to allow detection using anti-Flag antibodies. For example, the FLAGTM sequence 5′ gattacaaggat gacgacgataag 3′ (SEQ ID NO: 27) 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 254P1D6B proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 254P1D6B. High virus titer leading to high level expression of 254P1D6B is achieved in viral delivery system such as adenoviral vectors and herpes amplicon vectors. A 254P1D6B 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, 254P1D6B 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 254P1D6B in mammalian cells, coding sequences of 254P1D6B, 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 254P1D6B. These vectors are thereafter used to control expression of 254P1D6B in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 254P1D6B proteins in a baculovirus expression system, 254P1D6B 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-254P1D6B 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 254P1D6B protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 254P1D6B protein can be detected using anti-254P1D6B or anti-His-tag antibody. 254P1D6B protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 254P1D6B.
Example 9
Antigenicity Profiles and Secondary Structure
FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five amino acid profiles of 254P1D6B variant 1, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) 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., Doolitte 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 254P1D6B variant proteins. Each of the above amino acid profiles of 254P1D6B 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 254P1D6B 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-254P1D6B 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 254P1D6B 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 FIGS. 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 structure of 254P1D6B protein variant 1, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). The analysis indicates that 254P1D6B variant 1 is composed of 18.19% alpha helix, 24.81% extended strand, and 57.00% random coil (FIG. 13A).
Analysis for the potential presence of transmembrane domains in the 254P1D6B variant protein 1 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Shown graphically in FIG. 13B is the result of analysis of variant 1 using the TMpred program and in FIG. 13C results using the TMHMM program. Both the TMpred program and the TMHMM program predict the presence of 1 transmembrane domain. Analyses of the variants using other structural prediction programs are summarized in Table VI.
Example 10
Generation of 254P1D6B 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 254P1D6B protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structures”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 254P1D6B protein variant 1).
For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 254P1D6B protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in the Example entitled “Generation of 254P1D6B Monoclonal Antibodies (mAbs)”. For example, in 254P1D6B variant 1, such regions include, but are not limited to, amino acids 21-32, amino amino acids 82-96, amino acids 147-182, amino acids 242-270, amino acids 618-638, amino acids 791-818, and amino acids 980-1072. 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 147-182 of 254P1D6B variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 254P1D6B variant proteins, analogs or fusion proteins thereof. For example, the 254P1D6B variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. In another embodiment, amino acids 980-1072 of 254P1D6B variant 1. is fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 254P1D6B 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 Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 254P1D6B in Eukaryotic Systems”), and retains post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 26-953 of 254P1D6B variant 1 was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells (FIG. 4). The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 254P1D6B 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 as the rabbit serum derived from immunization with the GST-fusion of 254P1D6B variant 1 protein, the full-length 254P1D6B variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 254P1D6B in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-254P1D6B serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 254P1D6B protein using the Western blot technique (FIG. 4). In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 254P1D6B-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 254P1D6B are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 254P1D6B 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-254P1D6B 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-254P1D6B 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 254P1D6B Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 254P1D6B 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 254P1D6B 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 254P1D6B protein variant sequence, regions predicted to contain functional motifs, and regions of the 254P1D6B 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. Structures”). 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 254P1D6B variant, such as 293T-254P1D6B variant 1 or 300.19-254P1D6B variant 1murine Pre-B cells, are used to immunize mice.
To generate mAbs to a 254P1D6B variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 254P1D6B-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 24 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 254P1D6B variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 26-953 of 254P1D6B of variant 1 is cloned into the Tag5 mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 254P1D6B variant 1 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 254P1D6B variant.
Alternatively, mice may be immunized directly into their footpads. In this case, 10-50 μg of protein immunogen or 107 254P1D6B-expressing cells are injected sub-cutaneously into the footpad of each hind leg. The first immunization is given with Titermax (SigmaTM) as an adjuvant and subsequent injections are given with Alum-gel in conjunction with CpG oligonucleotide sequences with the exception of the final injection which is given with PBS. Injections are given twice weekly (every three to four days) for a period of 4 weeks and mice are sacrificed 3-4 days after the final injection, at which point lymph nodes immediately draining from the footpad are harvested and the B-cells are collected for use as antibody producing fusion partners.
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 254P1D6B monoclonal antibodies, a GST-fusion of variant 1 antigen encoding amino acids 21-182 is expressed and purified from bacteria. Balb C mice are initially immunized intraperitoneally with 25 μg of the GST-254P1D6B variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the GST-fusion antigen and a cleavage product from which the GST portion is removed determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 254P1D6B variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 254P1D6B variant 1 cDNA (see e.g., the Example entitled “Production of Recombinant 254P1D6B in Eukaryotic Systems” and FIG. 4). Other recombinant 254P1D6B variant 1-expressing cells or cells endogenously expressing 254P1D6B variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 254P1D6B specific antibody-producing clones.
The binding affinity of 254P1D6B variant specific monoclonal antibodies is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 254P1D6B 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.
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 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B 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 254P1D6B protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14
Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening
The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -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 50 IU/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 20 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 254P1D6B. 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 254P1D6B-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 254P1D6B-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 254P1D6B-derived, HLA class II HTL epitopes, a 254P1D6B 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 254P1D6B-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. 254P1D6B-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 254P1D6B 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 254P1D6B-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 254P1D6B-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., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, 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 254P1D6B expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 254P1D6B 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 254P1D6B-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 254P1D6B-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: [(1/50,000)−(1/500,000)]×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 254P1D6B-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 254P1D6B clearance. The number of epitopes used depends on observations of patients who spontaneously clear 254P1D6B. For example, if it has been observed that patients who spontaneously clear 254P1D6B-expressing cells generate an immune response to at least three (3) epitopes from 254P1D6B 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 254P1D6B, 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 254P1D6B.
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 254P1D6B, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 254P1D6B to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the li protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the li protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
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 (1x=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 254P1D6B 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 254P1D6B-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 254P1D6B-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 254P1D6B Sequences
A native 254P1D6B 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 254P1D6B 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 254P1D6B, 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 254P1D6B 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 254P1D6B and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 254P1D6B as well as tumor-associated antigens that are often expressed with a target cancer associated with, 254P1D6B 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 254P1D6B. 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, 254P1D6B HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 254P1D6B 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 254P1D6B epitope, and thus the status of exposure to 254P1D6B, 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 254P1D6B-associated disease or who have been vaccinated with a 254P1D6B vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 254P1D6B 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 μ/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 254P1D6B or a 254P1D6B 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 254P1D6B 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 254P1D6B
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 254P1D6B. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 254P1D6B, 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 254P1D6B.
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 254P1D6B-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 254P1D6B 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 254P1D6B protein from which 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 patent 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 254P1D6B antigens can be induced by incubating, in tissue culture, the patients, 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. 254P1D6B. 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 254P1D6B to isolate peptides corresponding to 254P1D6B that have been presented 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 254P1D6B-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 254P1D6B. 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 254P1D6B. 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 254P1D6B-encoding transcript.
Example 35
Purification of Naturally-occurring or Recombinant 254P1D6B Using 254P1D6B-Specific Antibodies
Naturally occurring or recombinant 254P1D6B is substantially purified by immunoaffinity chromatography using antibodies specific for 254P1D6B. An immunoaffinity column is constructed by covalently coupling anti-254P1D6B antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturers instructions.
Media containing 254P1D6B are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 254P1D6B (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/254P1D6B 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 254P1D6B
254P1D6B, or biologically active fragments thereof, are labeled with 121 1 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 254P1D6B, washed, and any wells with labeled 254P1D6B complex are assayed. Data obtained using different concentrations of 254P1D6B are used to calculate values for the number, affinity, and association of 254P1D6B with the candidate molecules.
Example 37
In Vivo Assay for 254P1D6B Tumor Growth Promotion
The effect of a 254P1D6B protein on tumor cell growth can be confirmed in vivo by gene overexpression in a variety of cancer cells such as those in Table I. For example, as appropriate, SCID mice can be injected SQ on each flank with 1×106 prostate, kidney, colon or bladder cancer cells (such as PC3, LNCaP, SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki, A498 and SW839 cells) containing tkNeo empty vector or 254P1D6B.
At least two strategies can be used:
(1) Constitutive 254P1D6B expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems.
(2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors or by following serum markers such as PSA. Tumor development is followed over time to validate that 254P1D6B-expressing cells grow at a faster rate and/or that tumors produced by 254P1D6B-expressing cells to demonstrate characteristics of altered aggressiveness (e.g., enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs). Tumor volume is evaluated by caliper measurements. Additionally, mice can be implanted with the same cells orthotopically in the prostate, bladder, colon or kidney to determine if 254P1D6B has an effect on local growth, e.g., in the prostate, bladder, colon or kidney or on the ability of the cells to metastasize, specifically to lungs or lymph nodes (Saffran et al., Proc Natl Acad Sci U S A. 2001, 98: 2658; Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939; Chang, S., et al., Anticancer Res., 1997, 17: 3239-3242; Peralta, E. A., et al., J. Urol., 1999. 162: 1806-1811). For instance, the orthotopic growth of PC3 and PC3-254P1D6B can be compared in the prostate of SCID mice. Such experiments reveal the effect of 254P1D6B on orthotopic tumor growth, metastasis and/or angiogenic potential.
Furthermore, this assay is useful to confirm the inhibitory effect of candidate therapeutic compositions, such as 254P1D6B antibodies or intrabodies, and 254P1D6B antisense molecules or ribozymes, or 254P1D6B directed small molecules, on cells that express a 254P1D6B protein.
Example 38
254P1D6B Monoclonal Antibody-Mediated Inhibition of Tumors in Vivo
The significant expression of 254P1D6B, in cancer tissues, together with its restricted expression in normal tissues makes 254P1D6B an excellent target for antibody therapy. Similarly, 254P1D6B is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-254P1D6B mAbs is evaluated, e.g., in human prostate cancer xenograft mouse models using androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al. Cancer Res, 1999. 59(19): p. 5030-5036), kidney cancer xenografts (AGS-K3, AGS-K6), kidney cancer metastases to lymph node (AGS-K6 met) xenografts, and kidney cancer cell lines transfected with 254P1D6B, such as 769P-254P1D6B, A498-254P1D6B.
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in mouse orthotopic prostate cancer xenograft models and mouse kidney xenograft models. The antibodies can be unconjugated, as discussed in this example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-254P1D6B mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-254P1D6B tumor xenografts. Anti-254P1D6B mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-254P1D6B mAbs in the treatment of local and advanced stages of, e.g., prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or located on the World Wide Web at (.pnas.org/cgi/doi/10.1073/pnas.051624698). Similarly, anti-254P1D6B mAbs inhibit formation of AGS-K3 and AGS-K6 tumors in SCID mice, and prevent or retard the growth A498-254P1D6B tumor xenografts. These results indicate the use of anti-254P1D6B mAbs in the treatment of prostate and/or kidney cancer.
Administration of the anti-254P1D6B mAbs leads to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 254P1D6B is an attractive target for immunotherapy and demonstrate the therapeutic use of anti-254P1D6B mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 254P1D6B monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice.
Tumor Inhibition using Multiple Unconjugated 254P1D6B mAbs
Materials and Methods
254P1D6B Monoclonal Antibodies
Monoclonal antibodies are obtained against 254P1D6B, as described in Example 11 entitled: Generation of 254P1D6B Monoclonal Antibodies (mAbs), or may be obtained commercially. The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 254P1D6B. Epitope mapping data for the anti-254P1D6B mAbs, as determined by ELISA and Western analysis, recognize epitopes on a 254P1D6B protein. Immunohistochemical analysis of cancer tissues and cells is performed with these antibodies.
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, e.g., LAPC-9 prostate tumor xenografts.
Cancer Xenografts and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA) is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by subcutaneous (s.c.) trocar implant (Craft, N., et al., 1999, Cancer Res. 59:5030-5036). The AGS-K3 and AGS-K6 kidney xenografts are also passaged by subcutaneous implants in 6- to 8-week old SCID mice. Single-cell suspensions of tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS, and the kidney carcinoma line A498 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% FBS.
PC3-254P1D6B and A498-254P1D6B cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: A Prostate-specific Cell-surface Antigen Highly Expressed in Human Prostate Tumors, Proc Natl. Acad. Sci. U S A, 1999. 96(25): p. 14523-14528. Anti-254P1D6B staining is detected by using, e.g. an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
Xenograft Mouse Models
Subcutaneous (s.c.) tumors are generated by injection of 1×106 LAPC-9, AGS-K3, AGS-K6, PC3, PC3-254P1D6B, A498 or A498-254P1D6B cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-254P1D6B mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or on the world wide web as pnas.org/cgi/doi/10.1073/pnas.051624698)
Orthotopic prostate injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5×105 ) mixed with Matrigel are injected into each dorsal lobe in a 10 μl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. For kidney orthotopic models, an incision is made through the abdominal muscles to expose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel are injected under the kidney capsule. The mice are segregated into groups for appropriate treatments, with anti-254P1D6B or control mAbs being injected i.p.
Anti-254P1D6B mAbs Inhibit Growth of 254P1D6B-Expressing Xenograft-Cancer Tumors
The effect of anti-254P1D6B mAbs on tumor formation is tested by using, e.g., LAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, 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 allow for tracking of the therapeutic effect of mAbs on clinically relevant end points.
Accordingly, tumor cells are injected into the mouse prostate or kidney, and the mice are segregated into two groups and treated with either: a) 200-500 μg, of anti-254P1D6B Ab, or b) PBS for two to five weeks.
As noted, a major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et al., Proc Natl. Acad. Sci. U S A, 1999. 96(25): p. 14523-14528) or anti-G250 antibody for kidney cancer models. G250 is a clinically relevant marker for renal clear cell carcinoma, which is selectively expressed on tumor but not normal kidney cells (Grabmaier K et al, Int J Cancer. 2000, 85: 865).
Mice bearing established orthotopic LAPC-9 tumors are administered 500-1000 μg injections of either anti-254P1D6B mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate/kidney and lungs are analyzed for the presence of tumor cells by IHC analysis.
These studies demonstrate a broad anti-tumor efficacy of anti-254P1D6B antibodies on initiation and/or progression of prostate and kidney cancer in xenograft mouse models. Anti-254P1D6B antibodies inhibit tumor formation of both androgen-dependent and androgen-independent prostate tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-254P1D6B mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Similar therapeutic effects are seen in the kidney cancer model. Thus, anti-254P1D6B mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic use of Anti-254P1D6B Antibodies in Humans
Anti-254P1D6B 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-254P1D6B mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 254P1D6B in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-254P1D6B 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-254P1D6B mAb specifically binds to carcinoma cells. Thus, anti-254P1D6B antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintography 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 254P1D6B. Shedding or release of an extracellular domain of 254P1D6B into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 254P1D6B by anti-254P1D6B antibodies in serum and/or urine samples from suspect patients.
Anti-254P1D6B antibodies that specifically bind 254P1D6B are used in therapeutic applications for the treatment of cancers that express 254P1D6B. Anti-254P1D6B 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-254P1D6B 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 “254P1D6B Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”. Either conjugated and unconjugated anti-254P1D6B 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-254P1D6B Antibodies in Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 254P1D6B, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 254P1D6B 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.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-254P1D6B 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-254P1D6B 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-254P1D6B antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-254P1D6B antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-254P1D6B 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 254P1D6B. In connection with the use of the anti-254P1D6B 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)-254P1D6B antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 254P1D6B (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-254P1D6B 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-254P1D6B 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-254P1D6B antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-254P1D6B 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-254P1D6B 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-254P1D6B 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-254P1D6B antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 254P1D6B 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 254P1D6B. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-254P1D6B antibodies are found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-254P1D6B Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-254P1D6B 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-254P1D6B 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-254P1D6B 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 254P1D6B. 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-254P1D6B 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-254P1D6B Antibody
Anti-254P1D6B 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-254P1D6B antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-254P1D6B 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-254P1D6B 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
Involvement in Tumor Progression
The 254P1D6B gene contributes to the growth of cancer cells. The role of 254P1D6B in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, colon, bladder and kidney cell lines, as well as NIH 3T3 cells engineered to stably express 254P1D6B. Parental cells lacking 254P1D6B and cells expressing 254P1D6B are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, et al., Prostate 2000;44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288). The effect of 254P1D6B can also be observed on cell cycle progression. Control and 254P1D6B-expressing cells are grown in low serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide incorporation by FACS analysis.
To confirm the role of 254P1D6B in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 254P1D6B are compared to NIH-3T3 cells expressing 254P1D6B, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000;60:6730).
To confirm the role of 254P1D6B in invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive (e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Control cells, including prostate, and bladder cell lines lacking 254P1D6B are compared to cells expressing 254P1D6B. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane analog (e.g. the Transwell insert) and used in the assay. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
254P1D6B also plays a role in cell cycle and apoptosis. Parental cells and cells expressing 254P1D6B are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 254P1D6B, including normal and tumor prostate, and kidney cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 254P1D6B can play a critical role in regulating tumor progression and tumor load.
When 254P1D6B plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 45
Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). 254P1D6B plays a role in angiogenesis. Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 254P1D6B in angiogenesis, enhancement or inhibition, is confirmed. For example, endothelial cells engineered to express 254P1D6B are evaluated using tube formation and proliferation assays. The effect of 254P1D6B is also confirmed in animal models in vivo. For example, cells either expressing or lacking 254P1D6B are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 254P1D6B affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46
Involvement in Cell Adhesion
Cell adhesion plays a critical role in tissue colonization and metastasis. 254P1D6B participates in cellular organization, and as a consequence cell adhesion and motility. To confirm that 254P1D6B regulates cell adhesion, control cells lacking 254P1D6B are compared to cells expressing 254P1D6B, using techniques previously described (see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. In another embodiment, cells lacking or expressing 254P1D6B are analyzed for their ability to mediate cell-cell adhesion using similar experimental techniques as described above. Both of these experimental systems are used to identify proteins, antibodies and/or small molecules that modulate cell adhesion to extracellular matrix and cell-cell interaction. Cell adhesion plays a critical role in tumor growth, progression, and, colonization, and 254P1D6B is involved in these processes. Thus, it serves as a diagnostic, prognostic, preventative and/or therapeutic modality.
Example 47
In Vitro Biologic Target Validation: Target Activation/Inactivation: RNA Interference (RNAi)
Systematic alteration of 254P1D6B gene activity in relevant cell assays or in animal models is an approach for understanding gene function. There are two complementary platforms to alter gene function: Target activation and target inactivation. 254P1D6B target gene activation induces a disease phenotype (i.e. tumurogenesis) by mimicking the differential gene activity that occurs in several tumors. Conversely, 254P1D6B target inactivation reverses a phenotype found in a particular disease and mimics the inhibition of the target with a putative lead compound/agent.
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, dsRNAs (>30 bp) can activate the interferon pathway which induces non-specific mRNA degradation and protein translation inhibition. When transfecting small synthetic dsRNA (21-23 nucleotides in length), the activation of the interferon pathway is no longer observed, however these dsRNAs 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):498 (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 254P1D6B specific target genes in cell survival/proliferation assays is relevant. RNAi technology is implemented to the cell survival (cellular metabolic activity as measured by MTS) and proliferation (DNA synthesis as measured by 3H-thymidine uptake) assays as a first filter to assess 254P1D6B target validation (TV). Tetrazolium-based colorimetric assays (i.e. MTT and MTS) detect viable cells exclusively. Living cells are metabolically active and can reduce tetrazolium salts to colored formazan compounds. Dead cells do not reduce the salts.
An alternative method to analyze 254P1D6B cell proliferation is the measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (i.e. 3H-Thymidine) are used and their incorporation to DNA is quantified. Incorporation of the labeled precursor into DNA is directly proportional to the amount of cell division occurring in the culture.
Correlating 254P1D6B cellular phenotype with gene knockdown is critical following RNAi treatments to draw valid conclusions and rule out toxicity or other non-specific effects of these reagents. Assays to measure the levels of expression of both protein and mRNA for the 254P1D6B target after RNAi treatments are important. Specific antibodies against the 254P1D6B target permit this question to be addressed by performing Western blotting with whole cell lysates.
An alternative method is the use of a tagged full length 254P1D6B target cDNA inserted in a mammalian expression vector (i.e. pcDNA3 series) providing a tag for which commercial Abs are available (Myc, His, V5 etc) is transiently co-transfected with individual siRNAs for 254P1D6B gene target, for instance in COS cells. Transgene expression permits the evaluation of which siRNA is efficiently silencing target gene expression, thus providing the necessary information to correlate gene function with protein knockdown. Both endogenous and transgene expression approaches show similar results.
A further alternative method for 254P1D6B target gene expression is measurement of mRNA levels by RT-PCR or by Taqman/Cybergreen. These methods are applied in a high throughput manner and are used in cases where neither Abs nor full length cDNAs are available. Using this method, poly-A mRNA purification and a careful design of primers/probes (should be 5′ to the siRNA targeted sequence) is needed for the Taqman approach. Some considerations apply to the primer design if pursuing RT-PCR from total RNA (primers should flank the siRNA targeted sequence). However, in some instances, the correlation between mRNA/protein is not complete (i.e., protein a with long half life) and the results could be misleading.
Several siRNAs per 254P1D6B target gene are selected and tested in parallel in numerous cell lines (usually with different tissue origin) in the survival and proliferation assays. Any phenotypic effect of the siRNAs in these assays is correlated with the protein and/or mRNA knockdown levels in the same cell lines. To further correlate cell phenotype and specific gene knockdown by RNAi, serial siRNA titrations are performed and are tested in parallel cell phenotype and gene knockdown. When 254P1D6B is responsible for the phenotype, a similar IC50 value in both assays is obtained.
Another method used to measure 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 254P1D6B 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 apoptotis. Assays for determination of caspase 3 activation detect early events of apoptotis. 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 bebbling, apoptotic bodies help to further support cell death as apoptotic.
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 differentiate 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 254P1D6B gene, RNAi studies facilitate 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. When 254P1D6B plays a role in cell survival, cell proliferation, tumorogenesis, or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 48
RNA Interference (RNAi)
Various protocols for achieving RNA interference are available.
Exemplary Protocol 1
RNA interference (RNAi) makes use of sequence specific double stranded RNA to prevent gene expression. Small interfering RNA (siRNA) is transfected into mammalian cells and thereby induce sequence specific mRNA degradation (Elbashir, et al, Nature, 2001; vol. 411: 494-498).
The sense strand of 254P1D6B is labeled at 3′ with fluorescein, 6-FAM (ABS 494 nm, EMM 525 nm, green). The siRNA is dissolved in RNA-free sterile buffer (100 mM KOAc, 30 mM HEPES KOH, 2 mM MOAc, at pH. 7.4) to make 20 μM stock (200-fold concentration). The siRNA is transfected into cells seeded on 6-well plates with oligofectamine reagent (GIBCO/Invitrogen, Carlsbad, Calif.). The final concentration of siRNA is determined.
254P1D6B protein expression is detected 24 hours after transfection by immunostaining followed by flow cytometry. In addition, confirmation of altered gene expression is performed by Western blotting. Expression reduction is confirmed by Western blot analysis where 254P1D6B protein is substantially reduced in 254P1D6B RNAi treated cells relative to control and untreated cells.
Exemplary Protocol 2
In one embodiment, the day before siRNA transfection, cells are plated in media (e.g., RPMI 1640 (GIBCO/Invitrogen, Carlsbad, Calif.) with 10% FBS without antibiotics) at 2×103 cells/well in 80 μl (96 well plate format) for the survival, proliferation and apoptosis assays. In another embodiment, the day before siRNA transfection, cells are plated in media (e.g., RPMI 1640 with 10% FBS without antibiotics) at 5×104 cells/well in 800 μl (12 well plate format) for the cell cycle analysis by flow cytometry, gene silencing by Western blot and/or PCR analysis. In parallel with the 254P1D6B siRNA sequences, the following sequences are included in every experiment as controls. Mock transfected cells with Lipofectamine 2000 (GIBCO/Invitrogen, Carlsbad, Calif.) and annealing buffer (no siRNA), non-specific siRNA (targeted sequence not represented in the human genome 5′ AATTCTCCGAACGTGTCACGTTT 3′; commercial control from Xeragon/Qiagen, Valencia, Calif.) (SEQ ID NO: 275); Luciferase specific siRNA (targeted sequence: 5′ AAGGGACGAAGACGAACACUUCTT 3′) (SEQ ID NO: 276) and Eg5 specific siRNA (targeted sequence: 5′ AACTGAAGACCTGAAGACAATAA 3′) (SEQ ID NO: 277). The siRNAs are used at various concentrations (ranging from 200 μM to 100 nM) and 1 μg/ml Lipofectamine 2000.
The procedure is as follows: First siRNAs are diluted in OPTIMEM (serum-free transfection media, Invitrogen) at suitable μM (10-fold concentrated) and incubated 5-10 min at room temperature (RT). Lipofectamine 2000 was diluted at 10 μg/ml (10-fold concentrated ) for the total number transfections and incubated 5-10 min RT. Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 are mixed 1:1 with diluted 10-fold concentrated siRNA and incubated at RT for 20-30 minutes (5-fold concentrated transfection solution). 20 or 200 μl of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37° C. for 48 to 96 hours (depending upon the assay employed, such as proliferation, apoptosis, survival, cell cycle analysis, migration or Western blot).
Reduced gene expression of 254P1D6B using siRNA transfection results in significantly diminished proliferation of transformed cancer cells that endogenously express the antigen. Cells treated with specific siRNAs show reduced survival as measured, e.g., by a metabolic readout of cell viability, corresponding to the reduced proliferative capacity. Further, such cells undergo apoptosis in response to RNAi as measured, e.g., by a nucleosome-release assay (Roche Applied Science, Indianapolis, Ind.) or detection of sub-G1 populations during cell cycle analysis by propidium iodide staining and flow cytometry. These results demonstrate that siRNA treatment provides an effective therapeutic for the elimination of cancer cells that specifically express the 254P1D6B antigen.
Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within 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.







TABLE I





Tissues that Express 254P1D6B when malignant:

















Lung



Ovary



Prostate



Pancreas



Breast

















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









POSITION



POSITION
POSITION
C Terminus



2 (Primary
3 (Primary
(Primary



Anchor)
Anchor)
Anchor)



















SUPERMOTIFS





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













TABLE IV (B)







HLA Class II Supermotif









1
6
9





W, F, Y, V, I, L
A, V, I, L, P, C, S, T
A, V, I, L, C, S, T, M, Y
















TABLE IV (C)







HLA Class II Motifs

















MOTIFS

1° anchor 1
2
3
4
5
1° anchor 6
7
8
9





DR4
preferred
FMYLIVW
M
T

I
VSTCPALIM
MH

MH



deleterious



W


R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious

C
CH
FD
CWD

GDE
D



DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious

C

G


GRD
N
G


DR3
MOTIFS
1° anchor 1
2
3
1° anchor 4
5
1° anchor 6


motif a

LIVMFY


D


preferred


motif b

LIVMFAY


DNQEST

KRH


preferred


DR

MFLIVWY




VMSTACPLI


Supermotif





Italicized residues indicate less preferred or “tolerated” residues.













TABLE IV (D)







HLA Class I Supermotifs












POSITION


















SUPERMOTIFS

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 (4/5)


YFW (3/5)
YFW (4/5)
P (4/5)
1° Anchor





VSMATLI






RK



delete-
DE (3/5);

DE (4/5)



rious
P (5/5)


A24


1° Anchor






1° Anchor





 YFWIVLMT






FIYWLM


B7
preferred
FWY (5/5)
1° Anchor
FWY (4/5)




FWY (3/5)
1° Anchor




LIVM (3/5)
P






 VILFMWYA



delete-
DE (3/5);



DE (3/5)
G (4/5)
QN (4/5)
DE (4/5)



rious
P (5/5);




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 indicates less preferred or “tolerated” residues













TABLE IV (E)







HLA Class I Motifs










POSITION:



















1
2
3
4
5
6
7
8
9
C-terminus
































or













C-terminus


A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor


9-mer


STM






Y



deleterious
DE

RHKLIVMP
A
G
A


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor


9-mer



DEAS





Y



deleterious
A
RHKDEPYFW

DE
PQN
RHK
PG
GP


A1
preferred
YFW
1° Anchor
DEAQN
A
YFWQN

PASTC
GDE
P
1° Anchor


10-mer


STM







Y



deleterious
GP

RHKGLIVM
DE
RHK
QNA
RHKYFW
RHK
A


A1
preferred
YFW
STCLIVM
1° Anchor
A
YFW

PG
G
YFW
1° Anchor


10-mer



DEAS






Y



deleterious
RHK
RHKDEPYFW


P
G

PRHK
QN


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
PRHK
A
YFW

P
1° Anchor





LMVISATF

YFW




KYRHFA



deleterious
DEP
CGD
DE


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





VTLMISAGN






KRYH



deleterious
DEP
CDF




A
G


A24
preferred
YFWRHK
1° Anchor

STC


YFW
YFW
1° Anchor


9-mer


YFWM






FLIW



deleterious
DEG

DE
G
QNP
DERHK
G
AQN


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



Delete-
DEP

DE

ADE
DE
DE
DE



rious


A3301
preferred

1° Anchor
YFW



AYFW

1° Anchor





 MVALFIST






RK



Delete-
GP

DE



rious


A6801
preferred
YFWSTC
1° Anchor


YFW

YFW
P
1° Anchor






AVTMSLI


LIVM



RK



deleterious
GP

DEG

RHK


A


B0702
preferred
RHKFWY
1° Anchor
RHK

RHK
RHK
RHK
PA
1° Anchor





P






LMF












WYAIV



deleterious
DEQNP

DEP
DE
DE
GDE
QN
DE


B3501
preferred
FWY
1° Anchor
FWY



FWY

1° Anchor




LIVM
P






LMF












WYIVA












9












or












C-terminus


A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor


9-mer


STM






Y



deleterious
DE

RHKL
A
G
A






IVMP


A1
preferred
GRHK
ASTC
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor


9-mer


LIVM
DEAS





Y



deleterious
A
RHKDEPYFW

DE
PQN
RHK
PG
GP



deleterious
AGP



G
G


B51
preferred
LIV
1° Anchor
FWY
STC
FWY

G
FWY
1° Anchor




MFWY
P






LIVF












WYAM



deleterious
AGPD



DE
G
DEQN
GDE




ERH




KSTC


B5301
preferred
LIV
1° Anchor
FWY
STC
FWY

LIVM
FWY
1° Anchor




MFWY
P




FWY

IMFW



deleterious
AGPQN




G
RHK
DE
YALV










QN


B5401
preferred
FWY
1° Anchor
FWY

LIVM

ALIVM
FWY
1° Anchor





P
LIVM




AP
ATIV












LMFWY



deleterious
GPQNDE

GDE

RH
DE
QN
DE






STC

KDE

DGE
















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-supertypes
Caucasian
N.A Blacks
Japanese
Chinese
Hispanic
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
99.9
99.6
100.0
99.8
99.9
99.8


and A1


A2, A3, B7,


A24, B44, A1,


B27, B62, and B 58





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













TABLE V







Frequently Occurring Motifs











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




terminal)/b6/petB
superoxide


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




Ubiquinone/plastoquinone
proton translocation across the




(complex I), various chains
membrane


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





residue loop flanked on both sides by a12





residue alpha-helical domain


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




protease
a catalytic aspartyl residue


Collagen
42%
Collagen triple helix repeat
extracellular structural proteins involved




(20 copies)
in 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




(rhodopsin family)
regions, with the N-terminus located





extracellularly while the C-terminus is





cytoplasmic. Signal through G proteins
















TABLE VI





Post-translational modifications of 254P1D6B
















N-Glycosylation site (start position indicated)



 










196
NSSV
(SEQ ID NO: 28)



 


219
NESA
(SEQ ID NO: 29)


 


262
NSSG
(SEQ ID NO: 30)


 


394
NLSQ
(SEQ ID NO: 31)


 


421
NVTV
(SEQ ID NO: 32)


 


498
NYSF
(SEQ ID NO: 33)


 


513
NSTT
(SEQ ID NO: 34)


 


536
NHTI
(SEQ ID NO: 35)


 


551
NQSS
(SEQ ID NO: 36)


 


715
NNSP
(SEQ ID NO: 37)


 


733
NNSI
(SEQ ID NO: 38)


 


1023
NSSL
(SEQ ID NO: 39)


 


1056
NGSI
(SEQ ID NO: 40)


 








Tyrosine sulfation site (Start Position indicated)



 










156
EEMSEYSDDYRE
(SEQ ID NO: 41)



 


160
EYSDDYRELEK
(SEQ ID NO: 42)


 


527
NNAVDYPPVANAGPNH
(SEQ ID NO: 43)


 








Serine predictions (Start Position indicated)



 










9
TGVLSSLLL
(SEQ ID NO: 44)



 


10
GVLSSLLLL
(SEQ ID NO: 45)


 


26
RKQCSEGRT
(SEQ ID NO: 46)


 


32
GRTYSNAVI
(SEQ ID NO: 47)


 


37
NAVISPNLE
(SEQ ID NO: 48)


 


49
IMRVSHTFP
(SEQ ID NO: 49)


 


65
CCDLSSCDL
(SEQ ID NO: 50)


 


66
CDLSSCDLA
(SEQ ID NO: 51)


 


81
CYLVSCPHK
(SEQ ID NO: 52)


 


98
GPIRSYLTF
(SEQ ID NO: 53)


 


125
LNRGSPSGI
(SEQ ID NO: 54)


 


127
RGSPSGIWG
(SEQ ID NO: 55)


 


133
IWGDSPEDI
(SEQ ID NO: 56)


 


154
LEEMSEYSD
(SEQ ID NO: 57)


 


157
MSEYSDDYR
(SEQ ID NO: 58)


 


171
LLQPSGKQE
(SEQ ID NO: 59)


 


179
EPRGSAEYT
(SEQ ID NO: 60)


 


191
LLPGSEGAF
(SEQ ID NO: 61)


 


197
GAFNSSVGD
(SEQ ID NO: 62)


 


198
AFNSSVGDS
(SEQ ID NO: 63)


 


202
SVGDSPAVP
(SEQ ID NO: 64)


 


221
YLNESASTP
(SEQ ID NO: 65)


 


223
NESASTPAP
(SEQ ID NO: 66)


 


233
LPERSVLLP
(SEQ ID NO: 67)


 


243
PTTPSSGEV
(SEQ ID NO: 68)


 


244
TTPSSGEVL
(SEQ ID NO: 69)


 


254
KEKASQLQE
(SEQ ID NO: 70)


 


264
SSNSSGKEV
(SEQ ID NO: 71)


 


272
VLMPSHSLP
(SEQ ID NO: 72)


 


274
MPSHSLPPA
(SEQ ID NO: 73)


 


279
LPPASLELS
(SEQ ID NO: 74)


 


283
SLELSSVTV
(SEQ ID NO: 75)


 


284
LELSSVTVE
(SEQ ID NO: 76)


 


290
TVEKSPVLT
(SEQ ID NO: 77)


 


299
VTPGSTEHS
(SEQ ID NO: 78)


 


303
STEHSIPTP
(SEQ ID NO: 79)


 


310
TPPTSAAPS
(SEQ ID NO: 80)


 


314
SAAPSESTP
(SEQ ID NO: 81)


 


316
APSESTPSE
(SEQ ID NO: 82)


 


319
ESTPSELPI
(SEQ ID NO: 83)


 


324
ELPISPTTA
(SEQ ID NO: 84)


 


338
ELTVSAGDN
(SEQ ID NO: 85)


 


376
WNLISHPTD
(SEQ ID NO: 86)


 


396
TLNLSQLSV
(SEQ ID NO: 87)


 


399
LSQLSVGLY
(SEQ ID NO: 88)


 


410
KVTVSSENA
(SEQ ID NO: 89)


 


411
VTVSSENAF
(SEQ ID NO: 90)


 


439
VAVVSPQLQ
(SEQ ID NO: 91)


 


451
LPLTSALID
(SEQ ID NO: 92)


 


457
LIDGSQSTD
(SEQ ID NO: 93)


 


459
DGSQSTDDT
(SEQ ID NO: 94)


 


467
TEIVSYHWE
(SEQ ID NO: 95)


 


483
EEKTSVDSP
(SEQ ID NO: 96)


 


486
TSVDSPVLR
(SEQ ID NO: 97)


 


492
VLRLSNLDP
(SEQ ID NO: 98)


 


500
PGNYSFRLT
(SEQ ID NO: 99)


 


508
TVTDSDGAT
(SEQ ID NO: 100)


 


514
GATNSTTAA
(SEQ ID NO: 101)


 


545
LPQNSITLN
(SEQ ID NO: 102)


 


553
NGNQSSDDH
(SEQ ID NO: 103)


 


554
GNQSSDDHQ
(SEQ ID NO: 104)


 


565
LYEWSLGPG
(SEQ ID NO: 105)


 


570
LGPGSEGKH
(SEQ ID NO: 106)


 


588
YLHLSAMQE
(SEQ ID NO: 107)


 


604
KVTDSSRQQ
(SEQ ID NO: 108)


 


605
VTDSSRQQS
(SEQ ID NO: 109)


 


609
SRQQSTAVV
(SEQ ID NO: 110)


 


641
FPVESATLD
(SEQ ID NO: 111)


 


647
TLDGSSSSD
(SEQ ID NO: 112)


 


648
LDGSSSSDD
(SEQ ID NO: 113)


 


649
DGSSSSDDH
(SEQ ID NO: 114)


 


650
GSSSSDDHG
(SEQ ID NO: 115)


 


667
VRGPSAVEM
(SEQ ID NO: 116)


 


702
QQGLSSTST
(SEQ ID NO: 117)


 


703
QGLSSTSTL
(SEQ ID NO: 118)


 


705
LSSTSTLTV
(SEQ ID NO: 119)


 


717
KENNSPPRA
(SEQ ID NO: 120)


 


735
LPNNSITLD
(SEQ ID NO: 121)


 


741
TLDGSRSTD
(SEQ ID NO: 122)


 


743
DGSRSTDDQ
(SEQ ID NO: 123)


 


751
QRIVSYLWI
(SEQ ID NO: 124)


 


760
RDGQSPAAG
(SEQ ID NO: 125)


 


770
VIDGSDHSV
(SEQ ID NO: 126)


 


773
GSDHSVALQ
(SEQ ID NO: 127)


 


795
RVTDSQGAS
(SEQ ID NO: 128)


 


799
SQGASDTDT
(SEQ ID NO: 129)


 


815
DPRKSGLVE
(SEQ ID NO: 130)


 


850
NVLDSDIKV
(SEQ ID NO: 131)


 


861
IRAHSDLST
(SEQ ID NO: 132)


 


864
HSDLSTVIV
(SEQ ID NO: 133)


 


873
FYVQSRPPF
(SEQ ID NO: 134)


 


894
HMRLSKEKA
(SEQ ID NO: 135)


 


918
LLKCSGHGH
(SEQ ID NO: 136)


 


933
RCICSHLWM
(SEQ ID NO: 137)


 


950
WDGESNCEW
(SEQ ID NO: 138)


 


955
NCEWSIFYV
(SEQ ID NO: 139)


 


1019
IKHRSTEHN
(SEQ ID NO: 140)


 


1024
TEHNSSLMV
(SEQ ID NO: 141)


 


1025
EHNSSLMVS
(SEQ ID NO: 142)


 


1029
SLMVSESEF
(SEQ ID NO: 143)


 


1031
MVSESEFDS
(SEQ ID NO: 144)


 


1035
SEFDSDQDT
(SEQ ID NO: 145)


 


1042
DTIFSREKM
(SEQ ID NO: 146)


 


1054
NPKVSMNGS
(SEQ ID NO: 147)


 


1058
SMNGSIRNG
(SEQ ID NO: 148)


 


1064
RNGASFSYC
(SEQ ID NO: 149)


 


1066
GASFSYCSK
(SEQ ID NO: 150)


 


1069
FSYCSKDR
(SEQ ID NO: 151)


 








Threonine predictions (Start Position indicated)



 










5
MAPPTGVLS
(SEQ ID NO: 152)



 


16
LLLVTIAGC
(SEQ ID NO: 153)


 


30
SEGRTYSNA
(SEQ ID NO: 154)


 


42
PNLETTRIM
(SEQ ID NO: 155)


 


43
NLETTRIMR
(SEQ ID NO: 156)


 


51
RVSHTFPVV
(SEQ ID NO: 157)


 


58
VVDCTAACC
(SEQ ID NO: 158)


 


101
RSYLTFVLR
(SEQ ID NO: 159)


 


183
SAEYTDWGL
(SEQ ID NO: 160)


 


209
VPAETQQDP
(SEQ ID NO: 161)


 


224
ESASTPAPK
(SEQ ID NO: 162)


 


240
LPLPTTPSS
(SEQ ID NO: 163)


 


241
PLPTTPSSG
(SEQ ID NO: 164)


 


286
LSSVTVEKS
(SEQ ID NO: 165)


 


294
SPVLTVTPG
(SEQ ID NO: 166)


 


296
VLTVTPGST
(SEQ ID NO: 167)


 


300
TPGSTEHSI
(SEQ ID NO: 168)


 


306
HSIPTPPTS
(SEQ ID NO: 169)


 


309
PTPPTSAAP
(SEQ ID NO: 170)


 


317
PSESTPSEL
(SEQ ID NO: 171)


 


326
PISPTTAPR
(SEQ ID NO: 172)


 


327
ISPTTAPRT
(SEQ ID NO: 173)


 


331
TAPRTVKEL
(SEQ ID NO: 174)


 


336
VKELTVSAG
(SEQ ID NO: 175)


 


346
NLIITLPDN
(SEQ ID NO: 176)


 


366
PPVETTYNY
(SEQ ID NO: 177)


 


367
PVETTYNYE
(SEQ ID NO: 178)


 


379
ISHPTDYQG
(SEQ ID NO: 179)


 


392
GHKQTLNLS
(SEQ ID NO: 180)


 


408
VFKVTVSSE
(SEQ ID NO: 181)


 


423
FVNVTVKPA
(SEQ ID NO: 182)


 


446
LQELTLPLT
(SEQ ID NO: 183)


 


450
TLPLTSALI
(SEQ ID NO: 184)


 


460
GSQSTDDTE
(SEQ ID NO: 185)


 


463
STDDTEIVS
(SEQ ID NO: 186)


 


482
IEEKTSVDS
(SEQ ID NO: 187)


 


506
RLTVTDSDG
(SEQ ID NO: 188)


 


512
SDGATNSTT
(SEQ ID NO: 189)


 


515
ATNSTTAAL
(SEQ ID NO: 190)


 


516
TNSTTAALI
(SEQ ID NO: 191)


 


538
GPNHTITLP
(SEQ ID NO: 192)


 


540
NHTITLPQN
(SEQ ID NO: 193)


 


547
QNSITLNGN
(SEQ ID NO: 194)


 


582
QGVQTPYLH
(SEQ ID NO: 195)


 


596
EGDYTFQLK
(SEQ ID NO: 196)


 


602
QLKVTDSSR
(SEQ ID NO: 197)


 


610
RQQSTAVVT
(SEQ ID NO: 198)


 


614
TAVVTVIVQ
(SEQ ID NO: 199)


 


643
VESATLDGS
(SEQ ID NO: 200)


 


680
KAIATVTGL
(SEQ ID NO: 201)


 


682
IATVTGLQV
(SEQ ID NO: 202)


 


688
LQVGTYHFR
(SEQ ID NO: 203)


 


694
HFRLTVKDQ
(SEQ ID NO: 204)


 


704
GLSSTSTLT
(SEQ ID NO: 205)


 


706
SSTSTLTVA
(SEQ ID NO: 206)


 


708
TSTLTVAVK
(SEQ ID NO: 207)


 


737
NNSITLDGS
(SEQ ID NO: 208)


 


744
GSRSTDDQR
(SEQ ID NO: 209)


 


779
ALQLTNLVE
(SEQ ID NO: 210)


 


787
EGVYTFHLR
(SEQ ID NO: 211)


 


793
HLRVTDSQG
(SEQ ID NO: 212)


 


801
GASDTDTAT
(SEQ ID NO: 213)


 


803
SDTDTATVE
(SEQ ID NO: 214)


 


805
TDTATVEVQ
(SEQ ID NO: 215)


 


821
LVELTLQVG
(SEQ ID NO: 216)


 


830
VGQLTEQRK
(SEQ ID NO: 217)


 


836
QRKDTLVRQ
(SEQ ID NO: 218)


 


865
SDLSTVIVF
(SEQ ID NO: 219)


 


910
LRVDTAGCL
(SEQ ID NO: 220)


 


927
CDPLTKRCI
(SEQ ID NO: 221)


 


960
IFYVTVLAF
(SEQ ID NO: 222)


 


965
VLAFTLIVL
(SEQ ID NO: 223)


 


970
LIVLTGGFT
(SEQ ID NO: 224)


 


974
TGGFTWLCI
(SEQ ID NO: 225)


 


987
RQKRTKIRK
(SEQ ID NO: 226)


 


993
IRKKTKYTI
(SEQ ID NO: 227)


 


996
KTKYTILDN
(SEQ ID NO: 228)


 


1020
KHRSTEHNS
(SEQ ID NO: 229)


 


1039
SDQDTIFSR
(SEQ ID NO: 230)


 








Tyrosine predictions (Start Position indicated)



 










31
EGRTYSNAV
(SEQ ID NO: 231)



 


78
EGRCYLVSC
(SEQ ID NO: 232)


 


99
PIRSYLTFV
(SEQ ID NO: 233)


 


116
QLLDYGDMM
(SEQ ID NO: 234)


 


156
EMSEYSDDY
(SEQ ID NO: 235)


 


160
YSDDYRELE
(SEQ ID NO: 236)


 


182
GSAEYTDWG
(SEQ ID NO: 237)


 


217
PELHYLNES
(SEQ ID NO: 238)


 


368
VETTYNYEW
(SEQ ID NO: 239)


 


370
TTYNYEWNL
(SEQ ID NO: 240)


 


381
HPTDYQGEI
(SEQ ID NO: 241)


 


403
SVGLYVFKV
(SEQ ID NO: 242)


 


468
EIVSYHWEE
(SEQ ID NO: 243)


 


499
DPGNYSFRL
(SEQ ID NO: 244)


 


527
NAVDYPPVA
(SEQ ID NO: 245)


 


562
QIVLYEWSL
(SEQ ID NO: 246)


 


584
VQTPYLHLS
(SEQ ID NO: 247)


 


595
QEGDYTFQL
(SEQ ID NO: 248)


 


658
GIVFYHWEH
(SEQ ID NO: 249)


 


689
QVGTYHFRL
(SEQ ID NO: 250)


 


752
RIVSYLWIR
(SEQ ID NO: 251)


 


786
VEGVYTFHL
(SEQ ID NO: 252)


 


870
VIVFYVQSR
(SEQ ID NO: 253)


 


944
LIQRYIWDG
(SEQ ID NO: 254)


 


958
WSIFYVTVL
(SEQ ID NO: 255)


 


995
KKTKYTILD
(SEQ ID NO: 256)


 


1013
LRPKYGIKH
(SEQ ID NO: 257)


 


1067
ASFSYCSKD
(SEQ ID NO: 258)
















TABLE VII





Search Peptides
















254P1D6Bv.1



 















1
MAPPTGVLSS
LLLLVTIAGC
ARKQCSEGRT
YSNAVISPNL
ETTRIMRVSH
TFPVVDCTAA
(SEQ ID NO: 259)



 


61
CCDLSSCDLA
WWFEGRCYLV
SCPHKENCEP
KKMGPIRSYL
TFVLRPVQRP
AQLLDYGDMM


 


121
LNRGSPSGIW
GDSPEDIRKD
LPFLGKDWGL
EEMSEYSDDY
RELEKDLLQP
SGKQEPRGSA


 


181
EYTDWGLLPG
SEGAFNSSVG
DSPAVPAETQ
QDPELHYLNE
SASTPAPKLP
ERSVLLPLPT


 


241
TPSSGEVLEK
EKASQLQEQS
SNSSGKEVLM
PSHSLPPASL
ELSSVTVEKS
PVLTVTPGST


 


301
EHSIPTPPTS
AAPSESTPSE
LPISPTTAPR
TVKELTVSAG
DNLIITLPDN
EVELKAFVAP


 


361
APPVETTYNY
EWNLISHPTD
YQGEIKQGHK
QTLNLSQLSV
GLYVFKVTVS
SENAFGEGFV


 


421
NVTVKPARRV
NLPPVAVVSP
QLQELTLPLT
SALIDGSQST
DDTEIVSYHW
EEINGPFIEE


 


481
KTSVDSPVLR
LSNLDPGNYS
FRLTVTDSDG
ATNSTTAALI
VNNAVDYPPV
ANAGPNHTIT


 


541
LPQNSITLNG
NQSSDDHQIV
LYEWSLGPGS
EGKHVVMQGV
QTPYLHLSAM
QEGDYTFQLK


 


601
VTDSSRQQST
AVVTVIVQPE
NNRPPVAVAG
PDKELIFPVE
SATLDGSSSS
DDHGIVFYHW


 


661
EHVRGPSAVE
MENIDKAIAT
VTGLQVGTYH
FRLTVKDQQG
LSSTSTLTVA
VKKENNSPPR


 


721
ARAGGRHVLV
LPNNSITLDG
SRSTDDQRIV
SYLWIRDGQS
PAAGDVIDGS
DHSVALQLTN


 


781
LVEGVYTFHL
RVTDSQGASD
TDTATVEVQP
DPRKSGLVEL
TLQVGVGQLT
EQRKDTLVRQ


 


841
LAVLLNVLDS
DIKVQKIRAH
SDLSTVIVFY
VQSRPPFKVL
KAAEVARNLH
MRLSKEKADF


 


901
LLFKVLRVDT
AGCLLKCSGH
GHCDPLTKRC
ICSHLWMENL
IQRYIWDGES
NCEWSIFYVT


 


961
VLAFTLIVLT
GGFTWLCICC
CKRQKRTKIR
KKTKYTILDN
MDEQERMELR
PKYGIKHRST


 


1021
EHNSSLMVSE
SEFDSDQDTI
FSREKMERGN
PKVSMNGSIR
NGASFSYCSK
DR


 








254P1D6Bv.2



 








9-mers, aa 149-175



GLEEMSEYADDYRELEK (SEQ ID NO: 260)


 


10-mers, aa 148-176


WGLEEMSEYADDYRELEKD (SEQ ID NO: 261)


 


15-mers, aa 143-181


FLGKDWGLEEMSEYADDYRELEKDLLQPS (SEQ ID NO: 262)


 








254P1D6BV.3



 








9-mers, aa 1-18



MTRLGWPSPCCARKQCSE (SEQ ID NO: 263)


 


10-mers, aa 1-19


MTRLGWPSPCCARKQCSEG (SEQ ID NO: 264)


 


15-mers, aa 1-24


MTRLGWPSPCCARKQCSEGRTYSN (SEQ ID NO: 265)


 








254P1D6Bv.5



 








9-mers, aa 134-150



PEDIRKDLTFLGKDWGL (SEQ ID NO: 266)


 


10-mers, aa 133-151


SPEDIRKDLTFLGKDWGLE (SEQ ID NO: 267)


 


15-mers, aa 128-156


GIWGDSPEDIRKDLTFLGKDWGLEEMSEY (SEQ ID NO: 268)
















TABLE VIII







254P1D6B v.1 HLA A1 9-mers


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the length


of peptide is 9 amino acids,


and the end position for each


peptide is the start position


plus eight.









Pos
Subsequence
Score













493
NLDPGNYSF
100.0



 


668
AVEMENIDK
90.000


 


39
NLETTRIMR
45.000


 


649
SSDDHGIVF
37.500


 


936
WMENLIQRY
22.500


 


153
MSEYSDDYR
13.500


 


805
TVEVQPDPR
9.000


 


743
STDDQROVS
6.250


 


182
YTDWGLLPG
6.250


 


459
STDDTEOVS
6.250


 


922
HCDPLTKRC
5.000


 


351
EVELKAFVA
4.500


 


87
NCEPKKMGP
4.500


 


244
SGEVLEKEK
4.500


 


382
QGEIKQGHK
4.500


 


462
DTEIVSYHW
4.500


 


951
NCEWSIFYV
4.500


 


553
SSDDHQIVL
3.750


 


1034
DSDQDTIFS
3.750


 


569
GSEGKHVVM
2.700


 


25
CSEGRTYSN
2.700


 


554
SDDHQIVLY
2.500


 


650
SDDHGIVFY
2.500


 


460
TDDTEIVSY
2.500


 


138
RKDLPFLGK
2.500


 


157
SDDYRELEK
2.500


 


897
KADFLLFKV
2.500


 


378
PTDYQGDIK
2.500


 


800
DTDTATVEV
2.500


 


483
SVDSPVLRL
2.500


 


113
LLDYGDMML
2.500


 


347
LPDNEVELK
2.500


 


505
VTDSDGATN
2.500


 


744
TDDQRIVSY
2.500


 


592
EGDYTFQLK
2.500


 


349
DNEVELKAF
2.250


 


829
LTEQRKDTL
2.250


 


1019
STEHNSSLM
2.250


 


565
SLGPGSEGK
2.000


 


84
HKENCEPKK
1.800


 


279
SLELSSVTV
1.800


 


860
HSDLSTVIV
1.500


 


769
GSDHSVALQ
1.500


 


798
ASDTDTATV
1.500


 


410
SSENAFGEG
1.350


 


190
GSEGAFNSS
1.350


 


778
LTNLVEGVY
1.250


 


130
WGDSPEDIR
1.250


 


809
QPDPRKSGL
1.250


 


681
VTGLQVGTY
1.250


 


601
VTDSSRQQS
1.250


 


519
LIVNNAVDY
1.000


 


705
STLTVAVKK
1.000


 


862
DLSTVIVFY
1.000


 


54
VVDCTAACC
1.000


 


15
VTIAGCARK
1.000


 


524
AVDYPPVAN
1.000


 


179
SAEYTDWGL
0.900


 


712
KKENNSPPR
0.900


 


149
GLEEMSEYS
0.900


 


781
LVEGVYTFH
0.900


 


882
AAEVARNLH
0.900


 


817
LVELTLQVG
0.900


 


210
QQDPELHYL
0.750


 


395
LSQLSVGLY
0.750


 


491
LSNLDPGNY
0.750


 


315
ESTPSELPI
0.750


 


849
DSDIKVQKI
0.750


 


507
DSDGATNST
0.750


 


587
LSAMQEGDY
0.750


 


950
SNCEWSIFY
0.625


 


339
AGDNLIITL
0.625


 


398
LSVGLYVFK
0.600


 


220
ESASTPAPK
0.600


 


704
TSTLTVAVK
0.600


 


224
TPAPKLPER
0.500


 


131
GDSPEDIRK
0.500


 


766
VIDGSDHSV
0.500


 


473
INGPFIEEK
0.500


 


373
NLISHPTDY
0.500


 


274
SLPPASLEL
0.500


 


847
VLDSDIKVQ
0.500


 


360
PAPPVETTY
0.500


 


61
CCDLSSCDL
0.500


 


907
RVDTAGCLL
0.500


 


670
EMENIDKAI
0.450


 


618
QPENNRPPV
0.450


 


299
STEHSIPTP
0.450


 


1006
PMELRPKYG
0.450


 


638
PVESATLDG
0.450


 


469
HWEEINGPF
0.450


 


281
ELSSVTVEK
0.400


 


870
YVQSRPPFK
0.400


 


209
TQQDPELHY
0.375


 


482
TSVDSPVLR
0.300


 


302
HSIPTPPTS
0.300


 


97
RSYLTFVLR
0.300


 


375
ISHPTDYQG
0.300


 


442
LQELTLPLT
0.270


 


576
VMQGVQTPY
0.250










V2-HLA-A1-9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Start
Subsequence
Score












5
MSEYADDYR
13.500


 


9
ADDYRELEK
2.500


 


1
GLEEMSEYA
0.900


 


4
EMSEYADDY
0.250


 


8
YADDYRELE
0.050


 


2
LEEMSEYAD
0.009


 


7
EYADDYREL
0.001


 


6
SEYADDYRE
0.000


 


3
EEMSEYADD
0.000










V3-HLA-A1-9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Start
Subsequence
Score













6
WPSPCCARK
1.000



 


3
TLGWPSPCC
0.020


 


5
GWPSPCCAR
0.005


 


8
SPCCARKQC
0.003


 


4
LGWPSPCCA
0.003


 


7
PSPCCARKQ
0.002


 


9
PCCARKQCS
0.001


 


1
MTRLGWPSP
0.001


 


2
TRLGWPSPC
0.001


 


10
CCARKQCSE
0.000










V5-HLA-A1-9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Start
Subsequence
Score













5
RKDLTFLGK
2.500



 


8
LTFLGKDWG
0.025


 


7
DLTFLGKDW
0.010


 


1
PEDIRKDLT
0.003


 


2
EDIRKDLTF
0.003


 


9
TFLGKDWGL
0.001


 


4
IRKDLTFLG
0.000


 


3
DIRKDLTFL
0.000


 


6
KDLTFLGKD
0.000


















TABLE IX





Start
Subsequence
Score
















HLA-A1-10-



mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.













173
KQEPRGSAEY
135.000



 


743
STDDQRIVSY
125.000


 


459
STDDTEIVSY
125.000


 


649
SSDDHGIVFY
75.000


 


156
YSDDYRELEK
75.000


 


553
SSDDHQIVLY
75.000


 


907
RVDTAGCLLK
50.000


 


493
NLDPGNYSFR
50.000


 


860
HSDLSTVIVF
37.500


 


1034
DSDQDTIFSR
37.500


 


805
TVEVQPDPRK
36.000


 


847
VLDSDIKVQK
20.000


 


410
SSENAFGEGF
13.500


 


130
WGDSPEDIRK
12.500


 


1019
STEHNSSLMV
11.250


 


87
NCEPKKMGPI
9.000


 


849
DSDIKVQKIR
7.500


 


208
ETQQDPELHY
6.250


 


922
HCDPLTKRCI
5.000


 


628
VAGPDKELIF
5.000


 


997
ILDNMDEQER
5.000


 


781
LVEGVYTFHL
4.500


 


39
NLETTRIMRV
4.500


 


882
AAEVARNLHM
4.500


 


949
ESNCEWSIFY
3.750


 


769
GSDHSVALQL
3.750


 


569
GSEGKHVVMQ
2.700


 


66
SCDLAWWFEG
2.500


 


182
YTDWGLLPGS
2.500


 


113
LLDYGDMMLN
2.500


 


829
LTEQRKDTLV
2.250


 


951
NCEWSIFYVT
1.800


 


477
FIEEKTSVDS
1.800


 


817
LVELTLQVGV
1.800


 


210
QQDPELHYLN
1.500


 


1036
DQDTIFSREK
1.500


 


1028
VSESEFDSDQ
1.350


 


25
CSEGRTYSNA
1.350


 


1030
ESEFDSDQDT
1.350


 


190
GSEGAFNSSV
1.350


 


601
VTDSSRQQST
1.250


 


792
VTDSQGASDT
1.250


 


505
VTDSDGATNS
1.250


 


539
ITLPQNSITL
1.250


 


1000
NMDEQERMEL
1.250


 


359
APAPPVETTY
1.250


 


800
DTDTATVEVQ
1.250


 


809
QPDPRKSGLV
1.250


 


35
VISPNLETTR
1.000


 


524
AVDYPPVANA
1.000


 


518
ALIVNNAVDY
1.000


 


186
GLLPGSEGAF
1.000


 


667
SAVEMENIDK
1.000


 


703
STSTLTVAVK
1.000


 


670
EMENIDKAIA
0.900


 


1006
RMELRPKYGI
0.900


 


179
SAEYTDWGLL
0.900


 


668
AVEMENIDKA
0.900


 


648
SSSDDHGIVF
0.750


 


507
DSDGATNSTT
0.750


 


273
HSLPPASLEL
0.750


 


590
MQEGDYTFQL
0.675


 


442
LQELTLPLTS
0.675


 


592
EGDTYFQLKV
0.625


 


378
PTDYQGEIKQ
0.625


 


347
LPDNEVELKA
0.625


 


872
QSRPPFKVLK
0.600


 


704
TSTLTVAVKK
0.600


 


777
QLTNLVEGVY
0.500


 


687
GTYHFRLTVK
0.500


 


897
KADFLLFKVL
0.500


 


766
VIDGSDHSVA
0.500


 


729
LVLPNNSITL
0.500


 


394
NLSQLSVGLY
0.500


 


586
HLSAMQEGDY
0.500


 


445
LTLPLTSALI
0.500


 


61
CCDLSSCDLA
0.500


 


680
TVTGLQVGTY
0.500


 


223
STPAPKLPER
0.500


 


100
LTFVLRPVQR
0.500


 


483
SVDSPVLRLS
0.500


 


1
MAPPTGVLSS
0.500


 


575
VVMQGVQTPY
0.500


 


955
SIFYVTVLAF
0.500


 


345
ITLPDNEVEL
0.500


 


164
EKDLLQPSGK
0.500


 


1039
TIFSREKMER
0.500


 


481
KTSVDSPVLR
0.500


 


490
RLSNLDPGNY
0.500


 


532
NAGPNHTITL
0.500


 


415
FGEGFVNVTV
0.450


 


936
WMENLIQRYI
0.450


 


349
DNEVELKAFV
0.450


 


618
QPENNRPPVA
0.450


 


286
TVEKSPVLTV
0.450


 


1001
MDEQERMELR
0.450


 


76
RCYLVSCPHK
0.400


 


397
QLSVGLYVFK
0.400


 


14
LVTIAGCARK
0.400


 


107
VQRPAQLLDY
0.375











V2-HLA-A1-



10mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
YADDYRELEK
50.000



 


6
MSEYADDYRE
0.270


 


2
GLEEMSEYAD
0.180


 


5
EMSEYADDYR
0.050


 


4
EEMSEYADDY
0.025


 


3
LEEMSEYADD
0.009


 


10
ADDYRELEKD
0.003


 


1
WGLEEMSEYA
0.003


 


7
SEYADDYREL
0.001


 


8
EYADDYRELE
0.000











V3-HLA-A1-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.















4
LGWPSPCCAR
0.025




 



6
WPSPCCARKQ
0.025



 



5
GWPSPCCARK
0.020



 



3
RLGWPSPCCA
0.010



 



8
SPCCARKQCS
0.003



 



1
MTRLGWPSPC
0.003



 



7
PSPCCARKQC
0.002



 



10
CCARKQCSEG
0.001



 



2
TRLGWPSPCC
0.001



 



9
PCCARKQCSE
0.000












V5-HLA-A1-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
SPEDIRKDLT
0.225



 


2
PEDIRKDLTF
0.125


 


9
LTFLGKDWGL
0.025


 


8
DLTFLGKDWG
0.010


 


5
IRKDLTFLGK
0.005


 


6
RKDLTFLGKD
0.003


 


4
DIRKDLTFLG
0.001


 


7
KDLTFLGKDW
0.001


 


10
TFLGKDWGLE
0.000


 


3
EDIRKDLTFL
0.000


















TABLE X





Start
Subsequence
Score
















V1-HLA-A0201-1



HLA-9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













900
FLLFKVLRV
2722.683



 


401
GLYVFKVTV
845.752


 


968
VLTGGFTWL
379.503


 


228
KLPERSVLL
306.550


 


92
KMGPIRSYL
296.997


 


816
GLVELTLQV
285.163


 


7
VLSSLLLLV
271.948


 


99
YLTFVLRPV
147.172


 


396
SQLSVGLYV
143.504


 


944
YIWDGESNC
106.931


 


846
NVLDSDIKV
92.322


 


441
QLQELTLPL
87.586


 


346
TLPDNEVEL
87.586


 


399
SVGLYVFKV
81.185


 


777
QLTNLVEGV
78.385


 


784
GVYTFHLRV
74.003


 


12
LLLVTIAGC
71.872


 


392
TLNLSQLSV
69.552


 


871
VQSRPPFKV
69.531


 


839
RQLAVLLNV
60.011


 


863
LSTVIVFYV
56.629


 


958
YVTVLAFTL
49.871


 


112
QLLDYGDMM
36.929


 


730
VLPNNSITL
36.316


 


960
TVLAFTLIV
35.082


 


961
VLAFTLIVL
34.246


 


655
IVFYHWEHV
31.887


 


828
QLTEQRKDT
30.553


 


452
ALIDGSQST
30.553


 


350
NEVELKAFV
30.497


 


558
QIVLYEWSL
22.030


 


394
NLSQLSVGL
21.362


 


540
TLPQNSITL
21.362


 


274
SLPPASLEL
21.362


 


577
MQGVQTPYL
20.251


 


840
QLAVLLNVL
20.145


 


836
TLVRQLAVL
20.145


 


897
KADFLLFKV
18.041


 


844
LLNVLDSDI
17.736


 


728
VLVLPNNSI
17.736


 


390
KQTLNLSQL
17.436


 


10
SLLLLVTIA
17.334


 


344
IITLPDNEV
16.258


 


607
QQSTAVVTV
16.219


 


6
GVLSSLLLL
159.07


 


113
LLDYGDMML
14.526


 


687
GTYHFRLTV
11.747


 


1045
KMERGNPKV
11.252


 


210
QQDPELHYL
10.960


 


685
QVGTYHFRL
10.841


 


446
TLPLTSALI
10.433


 


591
QEGDYTFQL
9.878


 


186
GLLPGSEGA
9.007


 


673
NIDKAIATV
8.798


 


818
VELTLQVGV
8.507


 


700
GLSSTSTLT
7.452


 


437
VVSPQLQEL
7.309


 


366
TTYNYEWNL
7.121


 


766
VIDGSDHSV
6.503


 


635
LIFPVESAT
6.445


 


821
TLQVGVGQL
6.387


 


429
RVNLPPVAV
6.086


 


284
SVTVEKSPV
6.086


 


774
VALQLTNLV
6.076


 


973
FTWLCICCC
6.059


 


233
SVLLPLPTT
5.549


 


497
GNYSFRLTV
5.521


 


40
LETTRIMRV
5.288


 


191
SEGAFNSSV
5.139


 


47
RVSHTFPVV
4.741


 


419
FVNVTVKPA
4.599


 


279
SLELSSVTV
4.451


 


773
SVALQLTNL
4.299


 


782
VEGVYTFHL
4.096


 


517
AALIVNNAV
3.574


 


969
LTGGFTWLC
3.343


 


669
VEMENIDKA
2.808


 


579
GVQTPYLHL
2.804


 


430
VNLPPVAVV
2.693


 


955
SIFYVTVLA
2.527


 


676
KAIATVTGL
2.388


 


858
RAHSDLSTV
2.222


 


1031
SEFDSDQDT
2.198


 


951
NCEWSIFYV
2.132


 


35
VISPNLETT
1.963


 


627
AVAGPDKEL
1.869


 


445
LTLPLTSAL
1.866


 


483
SVDSPVLRL
1.720


 


729
LVLPNNSIT
1.682


 


292
VLTVTPGST
1.647


 


678
IATVTGLQV
1.642


 


948
GESNCEWSI
1.521


 


988
KIRKKTKYT
1.499


 


962
LAFTLIVLT
1.497


 


538
TITLPQNSI
1.435


 


830
TEQRKDTLV
1.352


 


416
GEGFVNVTV
1.352


 


1020
TEHNSSLMV
1.352


 


465
IVSYHWEEI
1.293


 


822
LQVGVGQLT
1.284











V2-HLA-A0201-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













1
GLEEMSEYA
3.513



 


4
EMSEYADDY
0.008


 


6
SEYADDTYR
0.001


 


8
YADDYRELE
0.001


 


7
EYADDYREL
0.000


 


3
EEMSEYADD
0.000


 


2
LEEMSEYAD
0.000


 


5
MSEYADDYR
0.000


 


9
ADDYRELEK
0.000











V3-HLA-A0201-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
RLGWPSPCC
4.968



 


4
LGWPSPCCA
0.458


 


8
SPCCARKQC
0.032


 


2
TRLGWPSPC
0.003


 


6
WPSPCCARK
0.000


 


10
CCARKQCSE
0.000


 


1
MTRLGWPSP
0.000


 


9
PCCARKQCS
0.000


 


5
GWPSPCCAR
0.000


 


7
PSPCCARKQ
0.000











V5-HLA-A0201-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
TFLGKDWGL
0.412



 


3
DIRKDLTFL
0.212


 


8
LTFLGKDWG
0.018


 


7
DLTFLGKDW
0.006


 


1
PEDIRKDLT
0.001


 


6
KDLTFLGKD
0.000


 


5
RKDLTFLGK
0.000


 


4
IRKDLTFLG
0.000


 


2
EDIRKDLTF
0.000


















TABLE XI





Start
Subsequence
Score
















V1-HLA-A0201-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













862
DLSTVIVFYV
382.727



 


112
QLLDYGDMML
324.068


 


968
VLTGGFTWLC
240.700


 


870
YVQSRPPFK
162.369


 


576
VMQGVQTPYL
144.256


 


950
SNCEWSIFYV
136.577


 


967
IVLTGGFTWL
122.864


 


209
TQQDPELHTL
112.335


 


217
YLNESASTPA
93.696


 


11
LLLLVTIAGC
71.872


 


441
QLQELTLPLT
70.272


 


700
GLSSTSTLTV
69.552


 


843
VLLNVLDSDI
65.622


 


952
CEWSIFYVTV
63.982


 


892
RLSKEKADFL
57.572


 


6
GVLSSLLLLV
51.790


 


776
LQLTNLVEGV
49.989


 


617
VQPENNRPPV
49.151


 


901
LLFKVLRVDT
46.873


 


828
QLTEQRKDTL
42.917


 


45
IMRVSHTFPV
37.642


 


961
VLAFTLIVLT
29.137


 


1000
NMDEQERMEL
25.303


 


692
RLTVKDQQGL
21.362


 


836
TLVRQLAVLL
21.362


 


684
LQVGTYHFRL
21.356


 


92
KMGPIRSYLT
18.837


 


635
LIFPVESATL
18.476


 


120
MLNRGSPSGI
17.736


 


343
LIITLPDNEV
16.258


 


606
RQQSTAVVTV
16.219


 


808
VQPDPRKSGL
15.096


 


269
LMPSHSLPPA
14.029


 


355
KAFVAPAPPV
12.510


 


7
VLSSLLLLVT
11.946


 


729
LVLPNNSITL
11.757


 


400
VGLYVFKVTV
10.852


 


398
LSVGLYVFKV
10.296


 


39
NLETTRIMRV
10.238


 


677
AIATVTGLQV
9.563


 


958
YVTVLAFTLI
7.978


 


654
GIVFYHWEHV
7.966


 


386
KQGHKQTLNL
7.581


 


839
RQLAVLLNVL
7.557


 


821
TLQVGVGQLT
7.452


 


278
ASLELSSVTV
6.887


 


413
NAFGEGFVNV
6.791


 


141
LPFLGKDWGL
6.579


 


960
TVLAFTLIVL
6.522


 


660
WEHVRGPSAV
6.221


 


773
SVALQLTNLV
6.086


 


128
GIWGDSPEDI
5.834


 


94
GPIRSYLTFV
5.743


 


429
RVNLPPVAVV
5.739


 


904
KVLRVDTAGC
5.629


 


370
YEWNLISHPT
5.532


 


965
TLIVLTGGFT
5.328


 


352
VELKAFVAPA
5.311


 


669
VEMENIDKAI
5.232


 


728
VLVLPNNSIT
5.194


 


436
AVVSPQLQEL
4.299


 


178
GSAEYTDWGL
4.288


 


395
LSQLSVGLYV
4.245


 


12
LLLVTIAGCA
4.062


 


797
GASDTDTATV
3.961


 


1054
SMNGSIRNGA
3.588


 


391
QTLNLSQLSV
3.574


 


357
FVAPAPPVET
2.999


 


686
VGTYHFRLTV
2.933


 


551
NQSSDDHQIV
2.891


 


871
VQSRPPFKVL
2.868


 


338
SAGDNLIITL
2.798


 


827
GQLTEQRKDT
2.796


 


959
VTVLAFTLIV
2.559


 


780
NLVEGVYTFH
2.521


 


936
WMENLIQRYI
2.440


 


502
RLTVTDSDGA
2.434


 


630
GPDKELIFPV
2.423


 


247
VLEKEKASQL
2.324


 


698
QQGLSSTSTL
2.166


 


91
KKMGPIRSYL
2.113


 


765
DVIDGSDHSV
1.871


 


539
ITLPQNSITL
1.866


 


345
ITLPDNEVEL
1.866


 


198
SVGDSPAVPA
1.782


 


815
SGLVELTLQV
1.680


 


475
GPFIEEKTSV
1.680


 


444
ELTLPLTSAL
1.602


 


1031
SEFDSDQDTI
1.508


 


102
FVLRPVQRPA
1.480


 


266
KEVLMPSHSL
1.454


 


457
SQSTDDTEIV
1.417


 


633
KELIFPVESA
1.410


 


590
MQEGDYTFQL
1.367


 


26
SEGRTYSNAV
1.352


 


482
TSVDSPVLRL
1.315


 


939
NLIQRYIWDG
1.285


 


421
NVTVKPARRV
1.217


 


781
LVEGVYTFHL
1.180


 


521
VNNAVDYPPV
1.158











V2-HLA-A0201-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
WGLEEMSEYA
6.099



 


7
SEYADDYREL
0.399


 


5
EMSEYADDYR
0.009


 


2
GLEEMSEYAD
0.004


 


9
YADDYRELEK
0.002


 


4
EEMSEYADDY
0.000


 


3
LEEMSEYADD
0.000


 


6
MSEYADDYRE
0.000


 


10
ADDYRELEKD
0.000


 


8
EYADDYRELE
0.000











V3-HLA-A0201-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCCA
4.968



 


1
MTRLGWPSPC
0.009


 


2
TRLGWPSPCC
0.003


 


4
LGWPSPCCAR
0.001


 


7
PSPCCARKQC
0.001


 


10
CCARKQCSEG
0.000


 


8
SPCCARKQCS
0.000


 


6
WPSPCCARKQ
0.000


 


9
PCCARKQCSE
0.000


 


5
GWPSPCCARK
0.000











V5-HLA-A0201-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
LTFLGKDWGL
13.997



 


3
EDIRKDLTFL
0.028


 


8
DLTFLGKDWG
0.015


 


1
SPEDIRKDLT
0.006


 


7
KDLTFLGKDW
0.001


 


4
DIRKDLTFLG
0.000


 


2
PEDIRKDLTF
0.000


 


6
RKDLTFLGKD
0.000


 


10
TFLGKDWGLE
0.000


 


5
IRKDLTFLGK
0.000


















TABLE XII





Start
Subsequence
Score
















V1-HLA-A3-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













780
NLVEGVYTF
40.500



 


565
SLGPGSEGK
30.000


 


683
GLQVGTYHF
18.000


 


68
DLAWWFEGR
10.800


 


576
VMQGVQTPY
9.000


 


397
QLSVGLYVF
9.000


 


589
AMQEGDYTF
9.000


 


281
ELSSVTVEK
9.000


 


401
GLYVFKVTV
9.000


 


493
NLDPGNYSF
9.000


 


748
RIVSYLWIR
9.100


 


39
NLETTRIMR
8.000


 


936
WMENLIQRY
6.000


 


373
NLISHPTDY
6.000


 


866
VIVFYVQSR
5.400


 


152
EMSEYSDDY
5.400


 


92
KMGPIRSYL
4.050


 


879
VLKAAEVAR
4.000


 


668
AVEMENIDK
4.000


 


598
QLKVTDSSD
4.000


 


975
WLCICCCKR
4.000


 


1025
SLMVSESEF
3.000


 


968
VLTGGFTWL
2.700


 


816
GLVELTLQV
2.700


 


228
KLPERSVLL
2.700


 


1008
ELRPKYGIK
2.700


 


862
DLSTVIVFY
2.700


 


705
STLTVAVKK
2.250


 


892
RLSKEKADF
2.000


 


870
YVQSRPPFK
2.000


 


900
FLLFKVLRV
1.800


 


441
QLQELTLPL
1.800


 


961
VLAFTLIVL
1.800


 


784
GVYTFHLRV
1.800


 


274
SLPPASLEL
1.800


 


15
VTIAGCARK
1.500


 


366
TTYNYEWNL
1.350


 


728
VLVLPNNSI
1.350


 


186
GLLPGSEGA
1.350


 


836
TLVRQLAVL
1.350


 


113
LLDYGDMML
1.200


 


825
GVGQLTEQR
1.200


 


730
VLPNNSITL
1.200


 


540
TLPQNSITL
1.200


 


1052
KVSMNGSIR
1.200


 


983
RQKRTKIRK
1.200


 


112
QLLDYGDMM
0.900


 


840
QLAVLLNVL
0.900


 


615
VIVQPENNR
0.900


 


965
TLIVLTGGF
0.900


 


10
SLLLLVTIA
0.900


 


560
VLYEWSLGP
0.900


 


187
LLPGSEGAF
0.900


 


687
GTYHFRLTV
0.900


 


558
QIVLYEWSL
0.810


 


654
GIVFYHWEH
0.810


 


6
GVLSSLLLL
0.810


 


7
VLSSLLLLV
0.600


 


519
LIVNNAVDY
0.600


 


346
TLPDNEVEL
0.600


 


446
TLPLTSALI
0.600


 


394
NLSQLSVGL
0.600


 


347
LPDNEVELK
0.600


 


1062
GASFSYCSK
0.600


 


1045
KMERGNPKV
0.600


 


844
LLNVLDSDI
0.600


 


777
QLTNLVEGV
0.600


 


579
GVQTPYLHL
0.540


 


353
ELKAFVAPA
0.540


 


685
QVGTYHFRL
0.540


 


483
SVDSPVLRL
0.540


 


821
TLQVGVGQL
0.540


 


399
SVGLYVFKV
0.540


 


986
RTKIRKKTK
0.500


 


44
RIMRVSHTF
0.450


 


12
LLLVTIAGC
0.450


 


634
ELIFPVESA
0.405


 


14
LVTIAGCAR
0.400


 


392
TLNLSQLSV
0.400


 


421
NVTVKPARR
0.400


 


805
TVEVQPDPR
0.400


 


209
TQQDPELHY
0.360


 


97
RSYLTFVLR
0.300


 


700
GLSSTSTLT
0.300


 


704
TSTLTVAVK
0.300


 


473
INGPFIEEK
0.270


 


684
LQVGTYHFR
0.270


 


398
LSVGLYVFK
0.225


 


934
HLWMENLIQ
0.200


 


890
HMRLSKEKA
0.200


 


977
CICCCKRQK
0.200


 


905
VLRVDTAGC
0.200


 


625
PVAVAGPDK
0.200


 


914
LLKCSGHGH
0.200


 


279
SLELSSVTV
0.200


 


138
RKDLPFLGK
0.180


 


131
GDSPEDIRK
0.180


 


681
VTGLQVGTY
0.180


 


960
TVLAFTLIV
0.180


 


884
EVARNLHMR
0.180











V2-HLA-A3-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













4
EMSEYADDY
5.400



 


1
GLEEMSEYA
0.900


 


9
ADDYRELEK
0.040


 


5
MSEYADDYR
0.020


 


6
SEYADDYRE
0.001


 


8
YADDYRELE
0.001


 


2
LEEMSEYAD
0.000


 


3
EEMSEYADD
0.000


 


7
EYADDYREL
0.000











V3-HLA-A3-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
RLGWPSPCC
0.300



 


6
WPSPCCARK
0.300


 


5
GWPSPCCAR
0.018


 


4
LGWPSPCCA
0.002


 


2
TRLGWPSPC
0.001


 


8
SPCCARKQC
0.001


 


1
MTRLGWPSP
0.001


 


10
CCARKQCSE
0.000


 


9
PCCARKQCS
0.000


 


7
PSPCCARKQ
0.000











V5-HLA-A3-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













5
RKDLTFLGK
0.120



 


7
DLTFLGKDW
0.030


 


3
DIRKDLTFL
0.027


 


8
LTFLGKDWG
0.005


 


9
TFLGKDWGL
0.004


 


2
EDIRKDLTF
0.002


 


6
KDLTFLGKD
0.000


 


4
IRKDLTFLG
0.000


 


1
PEDIRKDLT
0.000


















TABLE XIII





Start
Subsequence
Score
















V1-HLA-A3-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













934
HLWMENLIQR
60.000



 


346
TLPDNEVELK
60.000


 


847
VLDSDIKVQK
30.000


 


687
GTYHFRLTVK
22.500


 


844
LLNVLDSDIK
20.000


 


397
QLSVGLYVFK
20.000


 


683
GLQVGTYHFR
12.000


 


888
NLHMRLSKEK
10.000


 


973
FTWLCICCCK
7.500


 


655
IVFYHWEHVR
6.000


 


955
SIFYVTVLAF
6.000


 


13
LLVTIAGCAR
6.000


 


825
GVGQLTEQRK
6.000


 


518
ALIVNNAVDY
6.000


 


493
NLDPGNYSFR
6.000


 


865
TVIVFYVQSR
5.400


 


186
GLLPGSEGAF
4.050


 


472
EINGPFIEEK
4.050


 


1039
TIFSREKMER
4.000


 


907
RVDTAGCLLK
4.000


 


997
ILDNMDEQER
4.000


 


394
NLSQLSVGLY
3.600


 


805
TVEVQPDPRK
3.000


 


703
STSTLTVAVK
3.000


 


968
VLTGGFTWLC
2.700


 


1006
RMELRPKYGI
2.700


 


14
LVTIAGCARK
2.000


 


878
KVLKAAEVAR
1.800


 


152
EMSEYSDDYR
1.800


 


112
QLLDYGDMML
1.800


 


777
QLTNLVEGVY
1.800


 


1000
NMDEQERMEL
1.800


 


401
GLYVFKVTVS
1.800


 


895
KEKADFLLFK
1.620


 


128
GIWGDSPEDI
1.350


 


92
KMGPIRSYLT
1.350


 


586
HLSAMQEGDY
1.200


 


1058
SIRNGASFSY
1.200


 


241
TPSSGEVLEK
1.200


 


490
RLSNLDPGNY
1.200


 


700
GLSSTSTLTV
1.200


 


100
LTFVLRPVQR
1.000


 


76
RCYLVSCPHK
1.000


 


836
TLVRQLAVLL
0.900


 


828
QLTEQRKDTL
0.900


 


667
SAVEMENIDK
0.900


 


575
VVMQGVQTPY
0.900


 


843
VLLNVLDSDI
0.900


 


576
VMQGVQTPYL
0.900


 


614
TVIVQPENNR
0.900


 


862
DLSTVIVFYV
0.810


 


781
LVEGVYTFHL
0.810


 


780
NLVEGVYTFH
0.675


 


892
RLSKEKADFL
0.600


 


39
NLETTRIMRV
0.600


 


35
VISPNLETTR
0.600


 


406
KVTVSSENAF
0.600


 


692
RLTVKDQQGL
0.600


 


247
VLEKEKASQL
0.600


 


120
MLNRGSPSGI
0.600


 


45
IMRVSHTFPV
0.600


 


481
KTSVDSPVLR
0.600


 


419
FVNVTVKPAR
0.600


 


416
GEGFVNVTVK
0.540


 


1008
ELRPKYGIKH
0.540


 


988
KIRKKTKYTI
0.540


 


228
KLPERSVLLP
0.540


 


173
KQEPRGSAEY
0.540


 


107
VQRPAQLLDY
0.540


 


680
TVTGLQVGTY
0.540


 


901
LLFKVLRVDT
0.500


 


635
LIFPVESATL
0.450


 


804
ATVEVQPDPR
0.450


 


872
QSRPPFKVLK
0.450


 


1054
SMNGSIRNGA
0.450


 


11
LLLLVTIAGC
0.450


 


396
SQLSVGLYVF
0.405


 


939
NLIQRYIWDG
0.405


 


977
CICCCKRQKR
0.400


 


684
LQVGTYHFRL
0.364


 


7
VLSSLLLLVT
0.300


 


459
STDDTEIVSY
0.300


 


324
SPTTAPRTVK
0.300


 


269
LMPSHSLPPA
0.300


 


217
YLNESASTPA
0.300


 


743
STDDQRIVSY
0.300


 


913
CLLKCSGHGH
0.300


 


223
STPAPKLPER
0.300


 


381
YQGEIKQGHK
0.270


 


729
LVLPNNSITL
0.270


 


960
TVLAFTLIVL
0.270


 


6
GVLSSLLLLV
0.270


 


967
IVLTGGFTWL
0.270


 


149
GLEEMSEYSD
0.270


 


557
HQIVLYEWSL
0.243


 


590
MQEGDYTFQL
0.243


 


564
WSLGPGSEGK
0.225


 


441
QLQELTLPLT
0.225


 


816
GLVELTLQVG
0.203


 


986
RTKIRKKTKY
0.200











V2-HLA-A3-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













5
EMSEYADDYR
1.800



 


9
YADDYRELEK
0.400


 


2
GLEEMSEYAD
0.270


 


4
EEMSEYADDY
0.016


 


7
SEYADDYREL
0.001


 


1
WGLEEMSEYA
0.000


 


6
MSEYADDYRE
0.000


 


3
LEEMSEYADD
0.000


 


10
ADDYRELEKD
0.000


 


8
EYADDYRELE
0.000











V3-HLA-A3-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCCA
0.200



 


5
GWPSPCCARK
0.060


 


4
LGWPSPCCAR
0.045


 


1
MTRLGWPSPC
0.030


 


2
TRLGWPSPCC
0.001


 


8
SPCCARKQCS
0.000


 


10
CCARKQCSEG
0.000


 


7
PSPCCARKQC
0.000


 


6
WPSPCCARKQ
0.000


 


9
PCCARKQCSE
0.000











V5-HLA-A3-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
LTFLGKDWGL
0.450



 


5
IRKDLTFLGK
0.120


 


8
DLTFLGKDWG
0.006


 


4
DIRKDLTFLG
0.002


 


2
PEDIRKDLTF
0.001


 


1
SPEDIRKDLT
0.001


 


7
KDLTFLGKDW
0.000


 


3
EDIRKDLTFL
0.000


 


6
RKDLTFLGKD
0.000


 


10
TFLGKDWGLE
0.000


















TABLE XIV





Start
Subsequence
Score
















V1-HLA-A1101-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













668
AVEMENIDK
4.000



 


983
RQKRTKIRK
3.600


 


870
YVQSRPPFK
2.000


 


15
VTIAGCARK
1.500


 


705
STLTVAVKK
1.500


 


986
RTKIRKKTK
1.500


 


825
GVGQLTEQR
1.200


 


1052
KVSMNGSIR
1.200


 


748
RIVSYLWIR
0.720


 


1062
GASFSYCSK
0.600


 


77
CYLVSCPHK
0.600


 


421
NVTVKPARR
0.400


 


565
SLGPGSEGK
0.400


 


688
TYHFRLTVK
0.400


 


14
LVTIAGCAR
0.400


 


805
TVEVQPDPR
0.400


 


887
RNLHMRLSK
0.360


 


784
GVYTFHLRV
0.240


 


347
LPDNEVELK
0.200


 


625
PVAVAGPDK
0.200


 


6
GVLSSLLLL
0.180


 


684
LQVGTYHFR
0.180


 


258
EQSSNSSGK
0.180


 


39
NLETTRIMR
0.160


 


131
GDSPEDIRK
0.120


 


138
RKDLPFLGK
0.120


 


884
EVARNLHMR
0.120


 


687
GTYHFRLTV
0.120


 


615
VIVQPENNR
0.120


 


281
ELSSVTVEK
0.120


 


1008
ELRPKYGIK
0.120


 


866
VIVFYVQSR
0.120


 


579
GVQTPYLHL
0.120


 


325
PTTAPRTVK
0.100


 


378
PTDTQGEIK
0.100


 


165
KDLLQPSGK
0.090


 


967
IVLTGGFTW
0.090


 


878
KVLKAAEVA
0.090


 


806
VEVQPDPRK
0.090


 


598
QLKVTDSSR
0.080


 


879
VLKAAEVAR
0.080


 


656
VFYHWEHVR
0.080


 


975
WLCICCCKR
0.080


 


1040
IFSREKMER
0.080


 


831
EQRKDTLVR
0.072


 


845
LNVLDSDIK
0.060


 


685
QVGTYHFRL
0.060


 


958
YVTVLAFTL
0.060


 


429
RVNLPPVAV
0.060


 


1010
RPKYGIKHR
0.060


 


907
RVDTAGCLL
0.060


 


960
TVLAFTLIV
0.060


 


47
RVSHTFPVV
0.060


 


101
TFVLRPVQR
0.060


 


399
SVGLYVFKV
0.060


 


846
NVLDSDIKV
0.060


 


406
KVTVSSENA
0.060


 


839
RQLAVLLNV
0.054


 


115
DYGDMMLNR
0.048


 


169
QPSGKQEPR
0.040


 


908
VDTAGCLLK
0.040


 


483
SVDSPVLRL
0.040


 


224
TPAPKLPER
0.040


 


366
TTYNYEWNL
0.040


 


920
HGHCDPLTK
0.040


 


157
SDDYRELEK
0.040


 


655
IVFYHWEHV
0.040


 


977
CICCCKRQK
0.040


 


978
ICCCKRQKR
0.040


 


473
INGPFIEEK
0.040


 


816
GLVELTLQV
0.036


 


654
GIVFYHWEH
0.036


 


974
TWLCICCCK
0.030


 


398
LSVGLYVFK
0.030


 


481
KTSVDSPVL
0.030


 


97
RSYLTFVLR
0.024


 


68
DLAWWFEGR
0.024


 


401
GLYVFKVTV
0.024


 


683
GLQVGTYHF
0.024


 


44
RIMRVSHTF
0.024


 


826
VGQLTEQRK
0.020


 


889
LHMRLSKEK
0.020


 


382
QGEIKQGHK
0.020


 


980
CCKRQKRTK
0.020


 


1064
SFSYCSKDR
0.020


 


848
LDSDIKVQK
0.020


 


773
SVALQLTNL
0.020


 


84
HKENCEPKK
0.020


 


294
TVTPGSTEH
0.020


 


465
IVSYHWEEI
0.020


 


704
TSTLTVAVK
0.020


 


336
TVSAGDNLI
0.020


 


781
LVEGVYTFH
0.020


 


837
LVRQLAVLL
0.020


 


873
SRPPFKVLK
0.020


 


581
QTPYLHLSA
0.020


 


284
SVTVEKSPV
0.020


 


437
VVSPQLQEL
0.020


 


331
TVKELTVSA
0.020


 


351
EVELKAFVA
0.018











V2-HLA-A1101-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
ADDYRELEK
0.040



 


1
GLEEMSEYA
0.012


 


5
MSEYADDYR
0.004


 


4
EMSEYADDY
0.001


 


6
SEYADDYRE
0.000


 


8
YADDYRELE
0.000


 


7
EYADDYREL
0.000


 


2
LEEMSEYAD
0.000


 


3
EEMSEYADD
0.000











V3-HLA-A1101-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













6
WPSPCCARK
0.200



 


5
GWPSPCCAR
0.012


 


3
RLGWPSPCC
0.001


 


1
MTRLGWPSP
0.001


 


4
LGWPSPCCA
0.000


 


10
CCARKQCSE
0.000


 


8
SPCCARKQC
0.000


 


2
TRLGWPSPC
0.000


 


9
PCCARKQCS
0.000


 


7
PSPCCARKQ
0.000











V5-HLA-A1101-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













5
RKDLTFLGK
0.040



 


9
TFLGKDWGL
0.006


 


8
LTFLGKDWG
0.002


 


3
DIRKDLTFL
0.001


 


7
DLTFLGKDW
0.001


 


2
EDIRKDLTF
0.000


 


6
KDLTFLGKD
0.000


 


4
IRKDLTFLG
0.000


 


1
PEDIRKDLT
0.000


















TABLE XV





Start
Subsequence
Score
















V1-HLA-A1101-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













907
RVDTAGCLLK
12.000



 


825
GVGQLTEQRK
6.000


 


687
GTYHFRLTVK
6.000


 


14
LVTIAGCARK
2.000


 


805
TVEVQPDPRK
2.000


 


973
FTWLCICCCK
2.000


 


878
KVLKAAEVAR
1.800


 


76
RCYLVSCPHK
1.200


 


703
STSTLTVAVK
1.000


 


655
IVFYHWEHVR
0.800


 


667
SAVEMENIDK
0.600


 


865
TVIVFYVQSR
0.600


 


614
TVIVQPENNR
0.600


 


869
FYVQSRPPFK
0.600


 


481
KTSVDSPVLR
0.600


 


381
YQGEIKQGHK
0.600


 


100
LTFVLRPVQR
0.400


 


346
TLPDNEVELK
0.400


 


847
VLDSDIKVQK
0.400


 


419
FVNVTVKPAR
0.400


 


844
LNVLDSDIK
0.400


 


241
TPSSGEVLEK
0.400


 


397
QLSVGLYVFK
0.400


 


895
EKADFLLFK
0.360


 


934
HLWMENLIQR
0.320


 


1039
TIFSREKMER
0.320


 


804
ATVEVQPDPR
0.300


 


683
GLQVGTYHFR
0.240


 


888
NLHMRLSKEK
0.200


 


223
STPAPKLPER
0.200


 


82
CPHKENCEPK
0.200


 


324
SPTTAPRTVK
0.200


 


377
HPTDYQGEIK
0.200


 


6
GVLSSLLLLV
0.180


 


597
FQLKVTDSSR
0.180


 


983
RQKRTKIRKK
0.180


 


416
GEGFVNVTVK
0.180


 


1043
REKMERGNPK
0.180


 


919
GHGHCDPLTK
0.120


 


982
KRQKRTKIRK
0.120


 


472
EINGPFIEEK
0.120


 


13
LLVTIAGCAR
0.120


 


168
LQPSGKQEPR
0.120


 


280
LELSSVTVEK
0.090


 


1007
MELRPKYGIK
0.090


 


977
CICCCKRQKR
0.080


 


35
VISPNLETTR
0.080


 


493
NLDPGNYSFR
0.080


 


997
ILDNMDEQER
0.080


 


321
LPISPTTAPR
0.060


 


870
YVQSRPPFKV
0.060


 


257
QEQSSNSSGK
0.060


 


406
KVTVSSENAF
0.060


 


781
LVEGVYTFHL
0.060


 


960
TVLAFTLIVL
0.060


 


429
RVNLPPVAVV
0.060


 


591
QEGDYTFQLK
0.060


 


219
NESASTPAPK
0.060


 


729
LVLPNNSITL
0.060


 


330
RTVKELTVSA
0.045


 


575
VVMQGVQTPY
0.040


 


130
WGDSPEDIRK
0.040


 


20
CARKQCSEGR
0.040


 


137
IRKDLPFLGK
0.040


 


886
ARNLHMRLSK
0.040


 


286
TVEKSPVLTV
0.040


 


717
SPPRARAGGR
0.040


 


156
YSDDYRELEK
0.040


 


336
TVSAGDNLII
0.040


 


386
KQGHKQTLNL
0.036


 


624
PPVAVAGPDK
0.030


 


976
LCICCCKRQK
0.030


 


564
WSLGPGSEGK
0.030


 


985
KRTKIRKKTK
0.030


 


992
KTKYTILDNM
0.030


 


959
VTVLAFTLIV
0.030


 


967
IVLTGGFTWL
0.030


 


986
RTKIRKKTKY
0.030


 


391
QTLNLSQLSV
0.030


 


436
AVVSPQLQEL
0.030


 


539
ITLPQNSITL
0.030


 


727
HVLVLPNNSI
0.030


 


684
LQVGTYHFRL
0.027


 


839
RQLAVLLNVL
0.027


 


1006
RMELRPKYGI
0.024


 


830
TEQRKDTLVR
0.024


 


152
EMSEYSDDYR
0.024


 


988
KIRKKTKYTI
0.024


 


700
GLSSTSTLTV
0.024


 


128
GIWGDSPEDI
0.024


 


979
CCCKRQKRTK
0.020


 


423
TVKPARRVNL
0.020


 


958
YVTVLAFTLI
0.020


 


680
TVTGLQVGTY
0.020


 


366
TTYNYEWNLI
0.020


 


1061
NGASFSYCSK
0.020


 


1019
STEHNSSLMV
0.020


 


284
SVTVEKSPVL
0.020


 


872
QSRPPFKVLK
0.020


 


524
AVDYPPVANA
0.020











V2-HLA-A1101-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
YADDYRELEK
0.400



 


5
EMSEYADDYR
0.024


 


2
GLEEMSEYAD
0.002


 


4
EEMSEYADDY
0.000


 


1
WGLEEMSEYA
0.000


 


8
EYADDYRELE
0.000


 


7
SEYADDYREL
0.000


 


3
LEEMSEYADD
0.000


 


6
MSEYADDYRE
0.000


 


10
ADDYRELEKD
0.000











V3-HLA-A1101-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













5
GWPSPCCARK
0.060



 


3
RLGWPSPCCA
0.012


 


4
LGWPSPCCAR
0.008


 


1
MTRLGWPSPC
0.001


 


10
CCARKQCSEG
0.000


 


8
SPCCARKQCS
0.000


 


2
TRLGWPSPCC
0.000


 


6
WPSPCCARKQ
0.000


 


9
PCCARLQCSE
0.000


 


7
PSPCCARLQC
0.000











V5-HLA-A1101-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
LTFLGKDWGL
0.040



 


5
IRKDLTFLGK
0.040


 


7
KDLTFLGKDW
0.000


 


4
DIRKDLTFLG
0.000


 


10
TFLGKDWGLE
0.000


 


1
SPEDIRKDLT
0.000


 


8
DLTFLGKDWG
0.000


 


2
PEDIRKDLTF
0.000


 


3
EDIRKDLTFL
0.000


 


6
RKDLTFLGKD
0.000


















TABLE XVI





Start
Subsequence
Score
















V1-HLA-A24-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













159
DYRELEKDL
288.000



 


155
EYSDDYREL
264.000


 


869
FYVQSRPPF
150.000


 


367
TYNYEWNLI
90.000


 


636
IFPVESATL
30.000


 


943
RYIWDGESN
15.000


 


228
KLPERSVLL
14.400


 


92
KMGPIRSYL
13.440


 


881
KAAEVARNL
13.440


 


676
KAIATVTGL
12.000


 


105
RPVQRPAQL
12.000


 


814
KSGLVELTL
11.200


 


957
FYVTVLAFT
10.500


 


133
SPEDIRKDL
10.080


 


956
IFYVTVLAF
10.000


 


1012
KYGIKHRST
10.000


 


1018
RSTEHNSSL
9.600


 


441
QLQELTLPL
8.640


 


445
LTLPLTSAL
8.640


 


44
RIMRVSHTF
8.400


 


481
KTSVDSPVL
8.000


 


390
KQTLNLSQL
8.000


 


907
RVDTAGCLL
8.000


 


274
SLPPASLEL
7.920


 


346
TLPDNEVEL
7.920


 


216
HYLNESAST
7.500


 


402
LYVFKVTVS
7.500


 


693
LTVKDQQGL
7.200


 


285
VTVEKSPVL
7.200


 


327
TAPRTVKEL
6.600


 


437
VVSPQLQEL
6.336


 


836
TLVRQLAVL
6.000


 


439
SPQLQELTL
6.000


 


6
GVLSSLLLL
6.000


 


829
LTEQRKDTL
6.000


 


540
TLPQNSITL
6.000


 


821
TLQVGVGQL
6.000


 


730
VLPNNSITL
6.000


 


579
GVQTPYLHL
6.000


 


486
SPVLRLSNL
6.000


 


954
WSIFYVTVL
6.000


 


240
TTPSSGEVL
6.000


 


511
ATNSTTAAL
6.000


 


533
AGPNHTITL
6.000


 


179
SAEYTDWGL
6.000


 


558
QIVLYEWSL
6.000


 


267
EVLMPSHSL
6.000


 


335
LTVSAGDNL
6.000


 


699
QGLSSTSTL
6.000


 


5
TGVLSSLLL
6.000


 


840
QLAVLLNVL
5.760


 


872
QSRPPFKVL
5.760


 


32
SNAVISPNL
5.600


 


469
HWEEINGPF
5.040


 


1032
EFDSDQDTI
5.000


 


594
DYTFQLKVT
5.000


 


498
NYSFRLTVT
5.000


 


785
VYTFHLRVT
5.000


 


885
VARNLHMRL
4.800


 


71
WWFEGRCYL
4.800


 


893
LSKEKADFL
4.800


 


968
VLTGGFTWL
4.800


 


553
SSDDHQIVL
4.800


 


809
QPDPRKSGL
4.800


 


837
LVRQLAVLL
4.800


 


210
QQDPELHYL
4.800


 


339
AGDNLIITL
4.800


 


394
NLSQLSVGL
4.800


 


768
DGSGHSVAL
4.800


 


958
YVTVLAFTL
4.800


 


627
AVAGPDKEL
4.400


 


221
SASTPAPKL
4.400


 


113
LLDYGDMML
4.000


 


685
QVGTYHFRL
4.000


 


261
SNSSGKEVL
4.000


 


773
SVALQLTNL
4.000


 


387
QGHKQTLNL
4.000


 


56
DCTAACCDL
4.000


 


918
SGHGHCDPL
4.000


 


577
MQGVQTPYL
4.000


 


483
SVDSPVLRL
4.000


 


366
TTYNYEWNL
4.000


 


932
CSHLWMENL
4.000


 


961
VLAFTLIVL
4.000


 


61
CCDLSSCDL
4.000


 


495
DPGNYSFRL
4.000


 


136
DIRKDLPFL
4.000


 


892
RLSKEKADF
4.000


 


723
AGGRHVLVL
4.000


 


589
AMQEGDYTF
3.600


 


629
AGPDKELIF
3.600


 


780
NLVEGVYTF
3.600


 


407
VTVSSENAF
3.600


 


965
TLIVLTGGF
3.600


 


1025
SLMVSESEF
3.300


 


142
PFLGKDQGL
3.000


 


1057
GSIRNGASF
3.000


 


683
GLQVGTYHF
3.000


 


187
LLPGSEGAF
3.000


 


349
DNEVELKAF
3.000











V2-HLA-A24-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













7
EYADDYREL
264.000



 


1
GLEEMSEYA
0.180


 


4
EMSEYADDY
0.120


 


5
MSEYADDYR
0.015


 


8
YADDYRELE
0.012


 


3
EEMSEYADD
0.002


 


2
LEEMSEYAD
0.002


 


9
ADDYRELEK
0.001


 


6
SEYADDYRE
0.001











V3-HLA-A24-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
RLGWPSPCC
0.200



 


4
LGWPSPCCA
0.200


 


8
SPCCARKQC
0.120


 


5
GWPSPCCAR
0.015


 


2
TRLGWPSPC
0.015


 


6
WPSPCCARK
0.012


 


9
PCCARKQCS
0.012


 


10
CCARLQCSE
0.010


 


1
MTRLGWPSP
0.010


 


7
PSPCCARKQ
0.0002











V5-HLA-A24-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
TFLGKDWGL
30.000



 


3
DIRKDLTFL
4.000


 


2
EDIRKDLTF
0.300


 


7
DLTFLGKDW
0.120


 


8
LTFLGKDWG
0.010


 


6
KDLTFLGKD
0.003


 


5
RKDLTFLGK
0.002


 


4
IRKDLTFLG
0.001


 


1
PEDIRKDLT
0.001


















TABLE XVII





Start
Subsequence
Score
















V1-HLA-A24-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













957
FYVTVLAFTL
360.000



 


159
DYRELEKDLL
240.000


 


839
RQLAVLLNVL
17.280


 


943
RYIWDGESNC
15.000


 


105
RPVQRPAQLL
14.400


 


897
KADFLLFKVL
11.520


 


402
LYVFKVTVSS
10.500


 


98
SYLTFVLRPV
10.500


 


132
DSPEDIRKDL
10.080


 


868
VFYVQSRPPF
10.000


 


1032
EFDSDQDTIF
10.000


 


692
RLTVKDQQGL
9.600


 


561
LYEWSLGPGS
9.000


 


229
LPERSVLLPL
8.400


 


31
YSNAVISPNL
8.400


 


2
APPTGVLSSL
8.400


 


892
RLSKEKADFL
8.000


 


720
RARAGGRHVL
8.000


 


386
KQGHKQTLNL
8.000


 


722
RAGGRHVLVL
8.000


 


436
AVVSPQLQEL
7.920


 


273
HSLPPASLEL
7.920


 


345
ITLPDNEVEL
7.920


 


367
TYNYEWNLIS
7.500


 


751
SYLWIRDGQS
7.500


 


482
TSVDSPVLRL
7.200


 


539
ITLPQNSITL
7.200


 


209
TQQDPELHYL
7.200


 


967
IVLTGGFTWL
7.200


 


836
TLVRQLAVLL
7.200


 


393
LNLSQLSVGL
7.200


 


729
LNLPNNSITL
7.200


 


808
VQPDPRKSGL
7.200


 


112
QLLDYGDMML
7.200


 


30
TYSNAVISPN
7.000


 


626
VAVAGPDKEL
6.600


 


557
HQIVLYEWSL
6.000


 


684
LQVGTYHFRL
6.000


 


835
DTLVRQLAVL
6.000


 


590
MQEGDYTFQL
6.000


 


438
VSPQLQELTL
6.000


 


247
VLEKEKASQL
6.000


 


820
LTLQVGVGQL
6.000


 


260
SSNSSGKEVL
6.000


 


576
VMQGVQTPYL
6.000


 


179
SAEYTDWGLL
6.000


 


485
DSPVLRLSNL
6.000


 


5
TGVLSSLLLL
6.000


 


960
TVLAFTLIVL
6.000


 


781
LVEGVYTFHL
6.000


 


578
QGVQTPYLHL
6.000


 


772
HSVALQLTNL
6.000


 


338
SAGDNLIITL
5.760


 


769
GSDHSVALQL
5.600


 


926
LTKRCICSHL
5.600


 


326
TTAPRTVKEL
5.280


 


1000
NMDEQERMEL
5.280


 


312
APSESTPSEL
5.280


 


893
LSKEKADFLL
4.800


 


60
ACCDLSSCDL
4.800


 


635
LIFPVESATL
4.800


 


406
KVTVSSENAF
4.800


 


423
TVKPARRVNL
4.800


 


444
ELTLPLTSAL
4.800


 


828
QLTEQRKDTL
4.800


 


884
EVARNLHMRL
4.800


 


384
EIKQGHKQTL
4.800


 


532
NAGPNHTITL
4.800


 


552
QSSDDHQIVL
4.800


 


871
VQSRPPFKVL
4.800


 


178
GSAEYTSWGL
4.800


 


220
ESASTPAPKL
4.400


 


811
DPRKSGLVEL
4.400


 


905
VLRVDTAGCL
4.000


 


141
LPFLGKDWGL
4.000


 


284
SVTVEKSPVL
4.000


 


510
GATNSTTAAL
4.000


 


698
QQGLSSTSTL
4.000


 


334
ELTVSAGDNL
4.000


 


854
VQKIRAHSDL
4.000


 


917
CSGHGHCDPL
4.000


 


931
ICSHLWMENL
4.000


 


365
ETTYNYEWNL
4.000


 


953
EWSIFYVTVL
4.000


 


226
APKLPERSVL
4.000


 


70
AWWFEGRCYL
4.000


 


186
GLLPGSEGAF
3.600


 


964
FTLIVLTGGF
3.600


 


492
SNLDPGNYSF
3.600


 


1024
SSLMVSESEF
3.300


 


1006
RMELRPKYGI
3.000


 


779
TNLVEGVYTF
3.000


 


682
TGLQVGTYHF
3.000


 


588
SAMQEGDYTF
3.000


 


93
MGPIRSYLTF
3.000


 


410
SSENAFGEGF
3.000


 


396
SQLSVGLYVF
3.000


 


648
SSSDDHGIVF
2.400


 


64
LSSCDLAWWF
2.400


 


858
RAHSDLSTVI
2.400











V2-HLA-A24-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













8
EYADDYRELE
0.600



 


7
SEYADDYREL
0.440


 


1
WGLEEMSEYA
0.180


 


2
GLEEMSEYAD
0.018


 


6
MSEYADDYRE
0.015


 


4
EEMSEYADDY
0.015


 


9
YADDYRELEK
0.013


 


5
EMSEYADDYR
0.012


 


3
LEEMSEYADD
0.002


 


10
ADDYRELEKD
0.001











V3-HLA-A24-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCCA
0.200



 


8
SPCCARKQCS
0.120


 


1
MTRLGWPSPC
0.100


 


7
PSPCCARKQC
0.015


 


5
GWPSPCCARK
0.015


 


2
TRLGWPSPCC
0.015


 


6
WPSPCCARKQ
0.013


 


4
LGWPSPCCAR
0.012


 


10
CCARKQCSEG
0.011


 


9
PCCARKQCSE
0.001











V5-HLA-A24-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
LTFLGKDWGL
4.000



 


3
EDIRKDLTFL
0.600


 


1
SPEDIRKDLT
0.180


 


10
TFLGKDWGLE
0.075


 


7
KDLTFLGKDW
0.036


 


2
PEDIRKDLTF
0.020


 


4
DIRKDLTFLG
0.012


 


8
DLTFLGKDWG
0.010


 


6
RKDLTFLGKD
0.002


 


5
IRKDLTFLGK
0.001


















TABLE XVIII





Start
Sequence
Score
















V1-HLA-B7-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













837
LVRQLAVLL
200.00



 


885
VARNLHMRL
120.000


 


627
AVAGPDKEL
90.000


 


105
RPVQRPAQL
80.000


 


486
SPVLRLSNL
80.000


 


495
DPGNYSFRL
80.000


 


439
SPQLQELTL
80.000


 


872
QSRPPFKVL
60.000


 


328
APRTVKELT
60.000


 


136
DIRKDLPFL
40.000


 


133
SPEDIRKDL
36.000


 


267
EVLMPSHSL
30.000


 


579
GVQTPYLHL
30.000


 


809
QPDPRKSGL
24.000


 


437
VVSPQLQEL
20.000


 


685
QVGTYHFRL
20.000


 


773
SVALQLTNL
20.000


 


175
EPRGSAEYT
20.000


 


6
GVLSSLLLL
20.000


 


958
YVTVLAFTL
20.000


 


582
TPYLHLSAM
20.000


 


226
APKLPERSV
18.000


 


221
SASTPAPKL
18.000


 


533
AGPNHTITL
12.000


 


327
TAPRTVKEL
12.000


 


676
KAIATVTGL
12.000


 


881
KAAEVARNL
12.000


 


723
AGGRHVLVL
12.000


 


511
ATNSTTAAL
12.000


 


359
APAPPVETT
9.000


 


483
SVDSPVLRL
9.000


 


3
PPTGVLSSL
8.000


 


296
TPGSTEHSI
8.000


 


37
SPNLETTRI
8.000


 


377
HPTDYQGEI
8.000


 


92
KMGPIRSYL
6.000


 


720
RARAGGRHV
6.000


 


907
RVDTAGCLL
6.000


 


1018
RSTEHNSSL
4.000


 


346
TLPDNEVEL
4.000


 


32
SNAVISPNL
4.000


 


954
WSIFYVTVL
4.000


 


324
SPTTAPRTV
4.000


 


821
TLQVGVGQL
4.000


 


540
TLPQNSITL
4.000


 


918
SGHGHCDPL
4.000


 


927
TKRCICSHL
4.000


 


121
LNRGSPSGI
4.000


 


814
KSGLVELTL
4.000


 


240
TTPSSGEVL
4.000


 


699
QGLSSTSTL
4.000


 


698
VLTGGFTWL
4.000


 


56
DCTAACCDL
4.000


 


445
LTLPLTSAL
4.000


 


932
CSHLWMENL
4.000


 


558
QIVLYEWSL
4.000


 


274
SLPPASLEL
4.000


 


961
VLAFTLIVL
4.000


 


390
KQTLNLSQL
4.000


 


768
DGSDHSVAL
4.000


 


893
LSKEKADFL
4.000


 


577
MQGVQTPYL
4.000


 


730
VLPNNSITL
4.000


 


228
KLPERSVLL
4.000


 


285
VTVEKSPVL
4.000


 


366
TTYNYEWNL
4.000


 


335
LTVSAGDNL
4.000


 


693
LTVKDQQGL
4.000


 


840
QLAVLLNVL
4.000


 


159
DYRELEKDL
4.000


 


567
GPGSEGKHV
4.000


 


836
TLVRQLAVL
4.000


 


5
TGVLSSLLL
4.000


 


387
QGHKQTLNL
4.000


 


261
SNSSGKEVL
4.000


 


481
KTSVDSPVL
4.000


 


441
QLQELTLPL
4.000


 


394
NLSQLSVGL
4.000


 


179
SAEYTDWGL
3.600


 


339
AGDNLIITL
3.600


 


999
DNMDEQERM
3.000


 


106
PVQRPAQLL
3.000


 


304
IPTPPTSAA
3.000


 


924
DPLTKRCIC
3.000


 


111
AQLLDYGDM
3.000


 


34
AVISPNLET
2.250


 


434
PVAVVSPQL
2.000


 


270
MPSHSLPPA
2.000


 


811
DPRKSGLVE
2.000


 


336
TVSAGDNLI
2.000


 


465
IVSYHWEEI
2.000


 


874
RPPFKVLKA
2.000


 


604
SSRQQSTAV
2.000


 


27
EGRTYSNAV
2.000


 


52
FPVVDCTAA
2.000


 


721
ARAGGRHVL
1.800


 


531
ANAGPNHTI
1.800


 


618
QPENNRPPV
1.800


 


517
AALIVNNAV
1.800


 


621
NNRPPVAVA
1.500











V2-HLA-B7-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













7
EYADDYREL
0.400



 


1
GLEEMSEYA
0.030


 


4
EMSEYADDY
0.020


 


8
YADDYRELE
0.013


 


3
EEMSEYADD
0.003


 


5
MSEYADDYR
0.003


 


6
SEYADDYRE
0.001


 


9
ADDYRELEK
0.001


 


2
LEEMSEYAD
0.000











V3-HLA-B7-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













8
SPCCARKQC
3.000



 


6
WPSPCCARK
0.200


 


3
RLGWPSPCC
0.150


 


1
MTRLGWPSP
0.100


 


4
LGWPSPCCA
0.100


 


10
CCARKQCSE
0.010


 


2
TRLGWPSPC
0.010


 


9
PCCARKQCS
0.002


 


5
GWPSPCCAR
0.002


 


7
PSPCCARKQ
0.001











V5-HLA-B7-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
DIRKDLTFL
40.00



 


9
TFLGKDWGL
0.400


 


7
DLTFLGKDW
0.020


 


8
LTFLGKDWG
0.010


 


2
EDIRKDLTF
0.002


 


4
IRKDLTFLG
0.001


 


6
KDLTFLGKD
0.001


 


1
PEDIRKDLT
0.000


 


5
RKDLTFLGK
0.000


















TABLE XIX





Start
Subsequence
Score
















V1-HLA-B7-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













811
DPRKSGLVEL
800.00



 


226
APKLPERSVL
360.000


 


312
APSESTPSEL
240.000


 


2
APPTGVLSSL
240.000


 


720
RARAGGRHVL
180.000


 


105
RPVQRPAQLL
120.000


 


328
APRTVKELTV
120.000


 


141
LPFLGKDWGL
80.000


 


436
AVVSPQLQEL
60.000


 


662
HVRGPSAVEM
50.000


 


905
VLRVDTAGCL
40.000


 


423
TVKPARRVNL
30.000


 


229
LPERSVLLPL
24.000


 


37
SPNLETTRIM
20.000


 


284
SVTVEKSPVL
20.000


 


967
IVLTGGFTWL
20.000


 


960
TVLAFTLIVL
20.000


 


729
LVLPNNSITL
20.000


 


884
EVARNLHMRL
20.000


 


626
VAVAGPDKEL
18.000


 


338
SAGDNLIITL
12.000


 


722
RAGGRHVLVL
12.000


 


60
ACCDLSSCDL
12.000


 


510
GATNSTTAAL
12.000


 


532
NAGPNHTITL
12.000


 


665
GPSAVEMENI
8.000


 


1050
NPKVSMNGSI
8.000


 


3
PPTGVLSSLL
8.000


 


433
PPVAVVSPQL
8.000


 


781
LVEGVYTFHL
6.000


 


871
VQSRPPFKVL
6.000


 


578
QGVQTPYLHL
6.000


 


627
AVAGPDKELI
6.000


 


220
ESASTPAPKL
6.000


 


482
TSVDSPVLRL
6.000


 


132
DSPEDIRKDL
6.000


 


892
RLSKEKADFL
4.000


 


260
SSNSSGKEVL
4.000


 


828
QLTEQRKDTL
4.000


 


384
EIKQGHKQTL
4.000


 


159
DYRELEKDLL
4.000


 


917
CSGHGHCDPL
4.000


 


438
VSPQLQELTL
4.000


 


485
DSPVLRLSNL
4.000


 


893
LSKEKADFLL
4.000


 


27
EGRTYSNAVI
4.000


 


326
TTAPRTVKEL
4.000


 


698
QQGLSSTSTL
4.000


 


393
LNLSQLSVGL
4.000


 


365
ETTYNYEWNL
4.000


 


238
LPTTPSSGEV
4.000


 


386
KQGHKQTLNL
4.000


 


95
PIRSYLTFVL
4.000


 


835
DTLVRQLAVL
4.000


 


820
LTLQVGVGQL
4.000


 


31
YSNAVISPNL
4.000


 


926
LTKRCICSHL
4.000


 


539
ITLPQNSITL
4.000


 


692
RLTVKDQQGL
4.000


 


5
TGVLSSLLLL
4.000


 


635
LIFPVESATL
4.000


 


557
HQIVLYEWSL
4.000


 


854
VQKIRAHSDL
4.000


 


836
TLVRQLAVLL
4.000


 


552
QSSDDHQIVL
4.000


 


740
GSRSTDDQRI
4.000


 


475
GPFIEEKTSV
4.000


 


112
QLLDYGDMML
4.000


 


345
ITLPDNEVEL
4.000


 


334
ELTVSAGDNL
4.000


 


273
HSLPPASLEL
4.000


 


988
KIRKKTKYTI
4.000


 


746
DQRIVSYLWI
4.000


 


444
ELTLPLTSAL
4.000


 


576
VMQGVQTPYL
4.000


 


684
LQVGTYHFRL
4.000


 


772
HSVALQLTNL
4.000


 


839
RQLAVLLNVL
4.000


 


567
GPGSEGKHVV
4.000


 


931
ICSHLWMENL
4.000


 


209
TQQDPELHYL
4.000


 


178
GSAEYTDWGL
4.000


 


808
VQPDPRKSGL
4.000


 


94
GPIRSYLTFV
4.000


 


897
KADFLLFKVL
3.600


 


179
SAEYTDWGLL
3.600


 


111
AQLLDYGDMM
3.000


 


317
TPSELPISPT
3.000


 


727
HVLVLPNNSI
3.000


 


882
AAEVARNLHM
2.700


 


175
EPRGSAEYTD
2.000


 


52
FPVVDCTAAC
2.000


 


336
TVSAGDNLII
2.000


 


45
IMRVSHTFPV
2.000


 


495
DPGNYSFRLT
2.0


 


874
RPPFKVLKAA
2.000


 


958
YVTVLAFTLI
2.000


 


604
SSRQQSTAVV
2.000


 


70
AWWFEGRCYL
1.800


 


91
KKMGPIRSYL
1.800











V2-HLA-B7-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













7
SEYADDYREL
0.400



 


1
WGLEEMSEYA
0.100


 


5
EMSEYADDYR
0.100


 


9
YADDYRELEK
0.009


 


4
EEMSEYADDY
0.006


 


2
GLEEMSEYAD
0.003


 


6
MSEYADDYRE
0.003


 


8
EYADDYRELE
0.002


 


10
ADDYRELEKD
0.001


 


3
LEEMSEYADD
0.000











V3-HLA-B7-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
MTRLGWPSPC
1.000



 


8
SPCCARKQCS
0.400


 


6
WPSPCCARKQ
0.200


 


3
RLGWPSPCCA
0.100


 


7
PSPCCARKQC
0.015


 


2
TRLGWPSPCC
0.015


 


4
LGWPSPCCAR
0.015


 


10
CCARKQCSEG
0.010


 


9
PCCARKQCSE
0.001


 


5
GWPSPCCARK
0.001











V5-HLA-B7-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
LTFLGKDWGL
4.000



 


1
SPEDIRKDLT
0.600


 


3
EDIRKDLTFL
0.400


 


4
DIRKDLTFLG
0.100


 


8
DLTFLGKDWG
0.01


 


7
KDLTFLGKDW
0.002


 


10
TFLGKDWGLE
0.001


 


5
IRDKLTFLGK
0.001


 


6
RKDLTFLGKD
0.000


 


2
PEDIRKDLTF
0.000


















TABLE XX





Start
Sequence
Score
















V1-HLA-B3501-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













105
RPVQRPAQL
40.000



 


582
TPYLHLSAM
40.000


 


893
LSKEKADFL
30.000


 


1018
RSTEHNSSL
20.000


 


94
GPIRSYLTF
20.000


 


495
DPGNYSFRL
20.000


 


439
SPQLQELTL
20.000


 


486
SPVLRLSNL
20.000


 


377
HPTDYQGEI
16.000


 


491
LSNLDPGNY
15.000


 


872
QSRPPFKVL
15.0


 


133
SPEDIRKDL
12.000


 


226
APKLPERSV
12.000


 


881
KAAEVARNL
12.000


 


37
SPNLETTRI
12.000


 


587
LSAMQEGDY
10.000


 


814
KSGLVELTL
10.000


 


262
NSSGKEVLM
10.000


 


65
SSCDLAWWF
10.000


 


395
LSQLSVGLY
10.000


 


885
VARNLHMRL
9.000


 


296
TPGSTEHSI
8.000


 


362
PPVETTYNY
8.000


 


949
ESNCEWSIF
7.500


 


742
RSTDDQRIV
6.000


 


999
DNMDEQERM
6.000


 


148
WGLEEMSEY
6.000


 


676
KAIATVTGL
6.000


 


23
KQCSEGRTY
6.000


 


567
GPGSEGKHV
6.000


 


175
EPRGSAEYT
6.000


 


809
QPDPRKSGL
6.000


 


1050
NPKVSMNGS
6.000


 


328
APRTVKELT
6.000


 


932
CSHLWMENL
5.000


 


1057
GSIRNGASF
5.000


 


954
WSIFYVTVL
5.000


 


136
DIRKDLPFL
4.500


 


228
KLPERSVLL
4.000


 


929
RCICSHLWM
4.000


 


874
RPPFKVLKA
4.000


 


112
QLLDYGDMM
4.000


 


950
SNCEWSIFY
4.000


 


209
TQQDPELHY
4.000


 


152
EMSEYSDDY
4.000


 


324
SPTTAPRTV
4.000


 


64
LSSCDLAWW
3.750


 


720
RARAGGRHV
3.600


 


327
TAPRTVKEL
3.000


 


649
SSDDHGIVF
3.000


 


552
QSSDDHQIV
3.000


 


221
SASTPAPKL
3.000


 


52
FPVVDCTAA
3.000


 


337
VSAGDNLII
3.000


 


604
SSRQQSTAV
3.000


 


647
SSSSDDHGI
3.000


 


475
GPFIEEKTS
3.000


 


569
GSEGKHVVM
3.000


 


361
APPVETTYN
3.000


 


188
LPGSEGAFN
3.000


 


553
SSDDHQIVL
3.000


 


648
SSSDDHGIV
3.000


 


892
RLSKEKADF
3.000


 


111
AQLLDYGDM
3.000


 


481
KTSVDSPVL
3.000


 


837
LVRQLAVLL
3.000


 


458
QSTDDTEIV
3.000


 


759
QSPAAGDVI
2.000


 


780
NLVEGVYTF
2.000


 


346
TLPDNEVEL
2.000


 


681
VTGLQVGTY
2.000


 


304
IPTPPTSAA
2.000


 


541
LPQNSITLN
2.000


 


125
SPSGIWGDS
2.000


 


275
LPPASLELS
2.000


 


862
DLSTVIVFY
2.000


 


236
LPLPTTPSS
2.000


 


373
NLISHPTDY
2.000


 


665
GPSAVEMEN
2.000


 


9
SSLLLLVTI
2.000


 


270
MPSHSLPPA
2.000


 


441
QLQELTLPL
2.000


 


589
AMQEGDYTF
2.000


 


576
VMQGVQTPY
2.000


 


519
LIVNNAVDY
2.000


 


924
DPLTKRCIC
2.000


 


629
AGPDKELIF
2.000


 


359
APAPPVETT
2.000


 


778
LTNLVEGVY
2.000


 


3
PPTGVLSSL
2.000


 


608
QSTAVVTVI
2.000


 


306
TPPTSAAPS
2.000


 


285
VTVEKSPVL
2.000


 


315
ESTPSELPI
2.000


 


44
RIMRVSHTF
2.000


 


2
APPTGVLSS
2.000


 


390
KQTLNLSQL
2.000


 


1038
DTIFSREKM
2.000


 


92
KMGPIRSYL
2.000


 


768
DGSDHSVAL
2.000











V2-HLA-B3501-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













4
EMSEYADDY
4.000



 


7
EYADDYREL
0.300


 


1
GLEEMSEYA
0.060


 


8
YADDYRELE
0.018


 


5
MSEYADDYR
0.015


 


6
SEYADDYRE
0.002


 


3
EEMSEYADD
0.002


 


2
LEEMSETAD
0.000


 


9
ADDYRELEK
0.000











V3-HLA-B3501-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













8
SPCCARKQC
2.000



 


3
RLGWPSPCC
0.200


 


6
WPSPCCARK
0.200


 


4
LGWPSPCCA
0.100


 


1
MTRLGWPSP
0.030


 


10
CCARKQCSE
0.010


 


9
PCCARKQCS
0.010


 


2
TRLGWPSPC
0.010


 


7
PSPCCARKQ
0.010


 


5
GWPSPCCAR
0.001











V5-HLA-B3501-



9mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
DIRKDKTFL
4.500



 


7
DLTFLGKDW
0.500


 


9
TFLGKDWGL
0.100


 


2
EDIRKDLTF
0.100


 


8
LTFLGKDWG
0.010


 


4
IRKDLTFLG
0.006


 


6
KDLTFLGKD
0.002


 


5
RKDLTFLGK
0.001


 


1
PEDIRKDLT
0.0000


















TABLE XXI





Start
Subsequence
Score
















V1-HLA-B3501-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













226
APKLPERSVL
90.000



 


811
DPRKSGLVEL
60.000


 


361
APPVETTYNY
40.000


 


312
APSESTPSEL
40.000


 


1018
RSTEHNSSLM
40.000


 


359
APAPPVETTY
40.000


 


37
SPNLETTRIM
40.000


 


105
RPVQRPAQLL
40.000


 


893
LSKEKADFLL
30.000


 


1050
NPKVSMNGSI
24.000


 


141
LPFLGKDWGL
20.000


 


2
APPTGVLSSL
20.000


 


720
RARAGGRHVI
18.00


 


986
RTKIRKKTKY
12.000


 


1010
RPKYGIKHRS
12.000


 


992
KTKYTILDNM
12.000


 


144
LGKDWGLEEM
12.000


 


665
GPSAVEMENI
12.000


 


328
APRTVKELTV
12.000


 


552
QSSDDHQIVL
10.000


 


648
SSSDDHGIVF
10.000


 


132
DSPEDIRKDL
10.000


 


178
GSAEYTDWGL
10.000


 


482
TSVDSPVLRL
10.000


 


949
ESNCEWSIFY
10.000


 


69
LAWWFEGRCY
9.000


 


740
GSRSTDDQRI
9.000


 


553
SSDDHQIVLY
6.000


 


649
SSDDHGIVFY
6.000


 


475
GPFIEEKTSV
6.000


 


89
EPKKMGPIRS
6.000


 


490
RLSNLDPGNY
6.000


 


722
RAGGRHVLVL
6.000


 


1058
SIRNGASFSY
6.000


 


107
VQRPAQLLDY
6.000


 


338
SAGDNLIITL
6.000


 


229
LPERSVLLPL
6.000


 


662
HVRGPSAVEM
6.000


 


485
DSPVLRLSNL
5.000


 


260
SSNSSGKEVL
5.000


 


31
YSNAVISPNL
5.000


 


1024
SSLMVSESEF
5.000


 


64
LSSCDLAWWF
5.000


 


917
CSGHGHCDPL
5.000


 


220
ESASTPAPKL
5.000


 


772
HSVALQLTNL
5.000


 


273
HSLPPASLEL
5.000


 


438
VSPQLQELTL
5.000


 


567
GPGSEGKHVV
4.000


 


94
GPIRSYLTFV
4.000


 


238
LPTTPSSGEV
4.000


 


317
TPSELPISPT
4.000


 


874
RPPFKVLKAA
4.000


 


646
GSSSSDDHGI
3.000


 


628
VAGPDKELIF
3.000


 


510
GATNSTTAAL
3.000


 


209
TQQDPELHYL
3.000


 


905
VLRVDTAGCL
3.000


 


456
GSQSTDDTEI
3.000


 


692
RLTVKDQQGL
3.000


 


854
VQKIRAHSDL
3.000


 


36
ISPNLETTRI
3.000


 


588
SAMQEGDYTF
3.000


 


926
LTKRCICSHL
3.000


 


423
TVKPARRVNL
3.000


 


1041
FSREKMERGN
3.000


 


604
SSRQQSTAVV
3.000


 


532
NAGPNHTITL
3.000


 


384
EIKQGHKQTL
3.000


 


626
VAVAGPDKEL
3.000


 


858
RAHSDLSTVI
2.400


 


988
KIRKKTKTYI
2.400


 


892
RLSKEKADFL
2.000


 


208
ETQQDPELHY
2.000


 


495
DPGNYSFRLT
2.000


 


188
LPGSEGAFNS
2.000


 


278
ASLELSSVTV
2.000


 


270
MPSHSLPPAS
2.000


 


372
WNLISHPTDY
2.000


 


777
QLTNLVEGVY
2.000


 


581
QTPYLHLSAM
2.000


 


828
QLTEQRKDTL
2.000


 


808
VQPDPRKSGL
2.000


 


275
LPPASLELSS
2.000


 


3
PPTGVLSSLL
2.000


 


924
DPLTKRCICS
2.000


 


680
TVTGLQVGTY
2.000


 


575
VVMQGVQTPY
2.000


 


492
SNLDPGNYSF
2.000


 


386
KQGHKQTLNL
2.000


 


52
FPVVDCTAAC
2.000


 


112
QLLDYGDMML
2.000


 


111
AQLLDYGDMM
2.000


 


433
PPVAVVSPQL
2.000


 


290
SPVLTVTPGS
2.000


 


527
YPPVANAGPN
2.000


 


586
HLSAMQEGDY
2.000


 


742
RSTDDQRIVS
2.000


 


224
TPAPKLPERS
2.000


 


8
LSSLLLLVTI
2.000











V2-HLA-B3501-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
WGLEEMSEYA
0.200



 


4
EEMSEYADDY
0.200


 


7
SEYADDYREL
0.150


 


6
MSEYADDYRE
0.023


 


5
EMSEYADDYR
0.020


 


9
TADDYRELEK
0.018


 


2
GLEEMSEYAD
0.006


 


8
EYADDYRELE
0.002


 


10
ADDYRELEKD
0.000


 


3
LEEMSEYADD
0.000











V3-HLA-B3501-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













8
SPCCARKQCS
2.000



 


1
MTRLGWPSPC
0.300


 


6
WPSPCCARKQ
0.200


 


3
RLGWPSPCCA
0.200


 


7
PSPCCARKQC
0.050


 


10
CCARKQCSEG
0.010


 


4
LGWPSPCCAR
0.010


 


2
TRLGWPSPCC
0.010


 


9
PCCARKQCSE
0.001


 


5
GWPSPCCARK
0.001











V5-HLA-B3501-



10mers-254P1D68


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
SPEDIRKDLT
1.200



 


9
LTFLGKDWGL
1.000


 


3
EDIRKDLTFL
0.150


 


7
KDLTFLGKDW
0.100


 


4
DIRKDLTFLG
0.030


 


8
DLTFLGKDWG
0.010


 


5
IRKDLTFLGK
0.006


 


2
PEDIRKDLTF
0.003


 


10
TFLGKDWGLE
0.002


 


6
RKDLTFLGKD
0.001


















TABLE XXII





Pos
123456789
score
















V1-HLA-A1-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













554
SDDHQIVLY
31



 


650
SDDHGIVFY
29


 


182
YTDWGLLPG
26


 


743
STDDQRIVS
26


 


460
TDDTEIVSY
25


 


681
VTGLQVGTY
25


 


744
TDDQRIVSY
25


 


936
WMENLIQRY
25


 


778
LTNLVEGVY
24


 


108
QRPAQLLDY
23


 


459
STDDTEIVS
23


 


209
TQQDPELHY
22


 


395
LSQLSVGLY
22


 


649
SSDDHGIVF
22


 


360
PAPPVETTY
21


 


553
SSDDHQIVL
21


 


587
LSAMQEGDY
21


 


950
SNCEWCIFY
21


 


138
RKDLPFLGK
20


 


156
YSDDYRELE
20


 


483
SVDSPVLRL
20


 


695
VKDQQGLSS
20


 


792
VTDSQGASD
20


 


1019
STEHNSSLM
20


 


229
LPERSVLLP
19


 


378
PTDYQGEIK
19


 


410
SSENAFGEG
19


 


491
LSNLDPGNY
19


 


576
VMQGVQTPY
19


 


157
SDDYRELEK
18


 


190
GSEGAFNSS
18


 


299
STEFSIPTP
18


 


462
DTEIVSYHW
18


 


493
NLDPGNYSF
18


 


505
VTDSDGATN
18


 


601
VTDSSRQQS
18


 


862
DLSTVIVFY
18


 


1005
ERMELRPKY
18


 


1028
VSESEFDSD
18


 


1034
DSDQDTIFS
18


 


39
NLETTRIMR
17


 


70
AWWFEGRCY
17


 


91
KKMGPIRSY
17


 


162
ELEKDLLQP
17


 


174
QEPRGSAEY
17


 


769
GSDHSVALQ
17


 


849
DSDIKVQKI
17


 


987
TKIRKKTKY
17


 


23
KQCSEGRTY
16


 


152
EMSEYSDDY
16


 


212
DPELHYLNE
16


 


373
NLISHPTDY
16


 


569
GSEGKHVVM
16


 


638
PVESATLDG
16


 


668
AVEMENIDK
16


 


800
DTDTATVEV
16


 


829
LTEQRKDTL
16


 


1003
EQERMELRP
16


 


1059
IRNGASFSY
16


 


25
CSEGRTYSN
15


 


148
WGLEEMSEY
15


 


173
KQEPRGSAE
15


 


223
STPAPKLPE
15


 


318
PSELPISPT
15


 


339
AGDNLIITL
15


 


362
PPVETTYNY
15


 


507
DSDGATNST
15


 


519
LIVNNAVDY
15


 


592
EGDYTFQLK
15


 


798
ASDTDTATV
15


 


909
DTAGCLLKC
15


 


1045
KMERGNPKV
15











V2-HLA-A1-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
ADDYRELEK
17



 


4
EMSEYADDY
16


 


8
YADDYRELE
16


 


5
MSEYADDYR
14


 


1
GLEEMSEYA
11


 


2
LEEMSEYAD
10











V3-HLA-A1-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













1
MTRLGWPSP
8



 


7
PSPCCARKQ
6


 


4
LGWPSPCCA
4


 


6
WPSPCCARK
4


 


8
SPCCARKQC
3











V5-HLA-A1-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













5
RKDLTFLGK
19



 


1
PEDIRKDLT
12


















TABLE XXIII





Pos
123456789
score
















V1-HLA-A0201-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













840
QLAVLLNVL
28



 


900
FLLFKVLRV
28


 


7
VLSSLLLLV
27


 


274
SLPPASLEL
27


 


401
GLYVFKVTV
27


 


816
GLVELTLQV
27


 


441
QLQELTLPL
26


 


673
NIDKAIATV
26


 


821
TLQVGVGQL
26


 


836
TLVRQLAVL
26


 


961
VLAFTLIVL
26


 


228
KLPERSVLL
25


 


279
SLELSSVTV
25


 


346
TLPDNEVEL
25


 


777
QLTNLVEGV
25


 


99
YLTFVLRPV
24


 


392
TLNLSQLSV
24


 


394
NLSQLSVGL
24


 


445
LTLPLTSAL
24


 


766
VIDGSDHSV
24


 


968
VLTGGFTWL
24


 


10
SLLLLVTIA
23


 


113
LLDYGDMML
23


 


344
IITLPDNEV
23


 


399
SVGLYVFKV
23


 


437
VVSPQLQEL
23


 


452
ALIDGSQST
23


 


728
VLVLPNNSI
23


 


730
VLPNNSITL
23


 


1045
KMERGNPKV
23


 


6
GVLSSLLLL
22


 


136
DIRKDLPFL
22


 


186
GLLPGSEGA
22


 


430
VNLPPVAVV
22


 


483
SVDSPVLRL
22


 


511
ATNSTTAAL
22


 


540
TLPQNSITL
22


 


609
STAVVTVIV
22


 


627
AVAGPDKEL
22


 


676
KAIATVTGL
22


 


703
STSTLTVAV
22


 


773
SVALQLTNL
22


 


844
LLNVLDSDI
22


 


9
SSLLLLVTI
21


 


12
LLLVTIAGC
21


 


35
VISPNLETT
21


 


92
KMGPIRSYL
21


 


558
QIVLYEWSL
21


 


774
VALQLTNLV
21


 


780
NLVEGVYTF
21


 


897
KADFLLFKV
21


 


95
PIRSYLTFV
20


 


221
SASTPAPKL
20


 


233
SVLLPLPTT
20


 


446
TLPLTSALI
20


 


517
AALIVNNAV
20


 


687
GTYHFRLTV
20


 


858
RAHSDLSTV
20


 


960
TVLAFTLIV
20


 


285
VTVEKSPVL
19


 


327
TAPRTVKEL
19


 


339
AGDNLIITL
19


 


429
RVNLPPVAV
19


 


538
TITLPQNSI
19


 


634
ELIFPVESA
19


 


721
ARAGGRHVL
19


 


800
DTDTATVEV
19


 


837
LVRQLAVLL
19


 


843
VLLNVLDSD
19


 


846
NVLDSDIKV
19


 


881
KAAEVARNL
19


 


112
QLLDYGDMM
18


 


234
VLLPLPTTP
18


 


287
VEKSPVLTV
18


 


414
AFGEGFVNV
18


 


531
ANAGPNHTI
18


 


607
QQSTAVVTV
18


 


635
LIFPVESAT
18


 


722
RAGGRHVLV
18


 


784
GVYTFHLRV
18


 


798
ASDTDTATV
18


 


955
SIFYVTVLA
18


 


958
YVTVLAFTL
18


 


962
LAFTLIVLT
18


 


11
LLLLVTIAG
17


 


103
VLRPVQRPA
17


 


210
QQDPELHYL
17


 


217
YLNESASTP
17


 


267
EVLMPSHSL
17


 


272
SHSLPPASL
17


 


277
PASLELSSV
17


 


303
SIPTPPTSA
17


 


342
NLIITLPDN
17


 


353
ELKAFVAPA
17


 


359
APAPPVETT
17


 


397
QLSVGLYVF
17


 


427
ARRVNLPPV
17


 


444
ELTLPLTSA
17


 


493
NLDPGNYSF
17


 


565
SLGPGSEGK
17


 


579
GVQTPYLHL
17


 


589
AMQEGDYTF
17


 


693
LTVKDQQGL
17


 


701
LSSTSTLTV
17


 


723
AGGRHVLVL
17


 


736
ITLDGSRST
17


 


818
VELTLQVGV
17


 


829
LTEQRKDTL
17


 


835
DTLVRQLAV
17


 


839
RQLAVLLNV
17


 


901
LLFKVLRVD
17


 


1054
SMNGSIRNG
17


 


13
LLVTIAGCA
16


 


34
AVISPNLET
16


 


120
MLNRGSPSG
16


 


197
SSVGDSPAV
16


 


292
VLTVTPGST
16


 


331
TVKELTVSA
16


 


335
LTVSAGDNL
16


 


366
TTYNYEWNL
16


 


385
IKQGHKQTL
16


 


422
VTVKPARRV
16


 


481
KTSVDSPVL
16


 


486
SPVLRLSNL
16


 


497
GNYSFRLTV
16


 


518
ALIVNNAVD
16


 


533
AGPNHTITL
16


 


560
VLYEWSLGP
16


 


593
GDYTFQLKV
16


 


605
SRQQSTAVV
16


 


636
IFPVESATL
16


 


655
IVFYHWEHV
16


 


678
IATVTGLQV
16


 


683
GLQVGTYHF
16


 


699
QGLSSTSTL
16


 


720
RARAGGRHV
16


 


812
PRKSGLVEL
16


 


877
FKVLKAAEV
16


 


885
VARNLHMRL
16


 


888
NLHMRLSKE
16


 


905
VLRVDTAGC
16


 


954
WSIFYVTVL
16


 


965
TLIVLTGGF
16


 


32
SNAVISPNL
15


 


40
LETTRIMRV
15


 


47
RVSHTFPVV
15


 


50
HTFPVVDCT
15


 


71
WWFEGRCYL
15


 


78
YLVSCPHKE
15


 


128
GIWGDSPED
15


 


179
SAEYTDWGL
15


 


187
LLPGSEGAF
15


 


191
SEGAFNSSV
15


 


235
LLPLPTTPS
15


 


284
SVTVEKSPV
15


 


336
TVSAGDNLI
15


 


338
SAGDNLIIT
15


 


350
NEVELKAFV
15


 


396
SQLSVGLYV
15


 


439
SPQLQELTL
15


 


465
IVSYHWEEI
15


 


516
TAALIVNNA
15


 


525
VDYPPVANA
15


 


547
TLNGNQSSD
15


 


628
VAGPDKELI
15


 


685
QVGTYHFRL
15


 


700
GLSSTSTLT
15


 


754
WIRDGQSPA
15


 


833
RKDTLVRQL
15


 


862
DLSTVIVFY
15


 


863
LSTVIVFYV
15


 


866
VIVFYVQSR
15


 


940
LIQRYIWDG
15


 


988
KIRKKTKYT
15


 


1025
SLMVSESEF
15


 


3
PPTGVLSSL
14


 


16
TIAGCARKQ
14


 


96
IRSYLTFVL
14


 


166
DLLQPSGKQ
14


 


207
AETQQDPEL
14


 


226
APKLPERSV
14


 


239
PTTPSSGEV
14


 


240
TTPSSGEVL
14


 


247
VLEKEKASQ
14


 


248
LEKEKASQL
14


 


260
SSNSSGKEV
14


 


261
SNSSGKEVL
14


 


268
VLMPSHSLP
14


 


326
TTAPRTVKE
14


 


337
VSAGDNLII
14


 


356
AFVAPAPPV
14


 


358
VAPAPPVET
14


 


390
KQTLNLSQL
14


 


416
GEGFVNVTV
14


 


431
NLPPVAVVS
14


 


434
PVAVVSPQL
14


 


453
LIDGSQSTD
14


 


539
ITLPQNSIT
14


 


575
VVMQGVQTP
14


 


591
QEGDYTFQL
14


 


643
TLDGSSSSD
14


 


669
VEMENIDKA
14


 


677
AIATVTGLQ
14


 


706
TLTVAVKKE
14


 


729
LVLPNNSIT
14


 


737
TLDGSRSTD
14


 


782
VEGVYTFHL
14


 


814
KSGLVELTL
14


 


828
QLTEQRKDT
14


 


847
VLDSDIKVQ
14


 


849
DSDIKVQKI
14


 


860
HSDLSTVIV
14


 


871
VQSRPPFKV
14


 


890
HMRLSKEKA
14


 


893
LSKEKADFL
14


 


907
RVDTAGCLL
14


 


909
DTAGCLLKC
14


 


918
SGHGHCDPL
14


 


944
YIWDGESNC
14


 


966
LIVLTGGFT
14


 


37
SPNLETTRI
13


 


121
LNRGSPSGI
13


 


142
PFLGKDWGL
13


 


145
GKDWDLEEM
13


 


167
LLQPSGKQE
13


 


180
AEYTDWGLL
13


 


182
YTDWGLLPG
13


 


214
ELHYLNESA
13


 


281
ELSSVTVEK
13


 


319
SELPISPTT
13


 


320
ELPISPTTA
13


 


324
SPTTAPRTV
13


 


343
LIITLPDNE
13


 


374
LISHPTDYQ
13


 


387
QGHKQTLNL
13


 


403
YVFKVTVSS
13


 


419
FVNVTVKPA
13


 


424
VKPARRVNL
13


 


476
PFIEEKTSV
13


 


477
FIEEKTSVD
13


 


490
RLSNLDPGN
13


 


515
TTAALIVNN
13


 


522
NNAVDYPPV
13


 


530
VANAGPNHT
13


 


553
SSDDHQIVL
13


 


577
MQGVQTPYL
13


 


604
SSRQQSTAV
13


 


621
NNRPPVAVA
13


 


631
PDKELIFPV
13


 


642
ATLDGSSSS
13


 


648
SSSDDHGIV
13


 


680
TVTGLQVGT
13


 


696
KDQQGLSST
13


 


745
DDQRIVSYL
13


 


748
RIVSYLWIR
13


 


752
YLWIRDGQS
13


 


758
GQSPAAGDV
13


 


768
DGSDHSVAL
13


 


770
SDHSVALQL
13


 


775
ALQLTNLVE
13


 


809
QPDPRKSGL
13


 


842
AVLLNVLDS
13


 


879
VLKAAEVAR
13


 


898
ADFLLFKVL
13


 


906
LRVDTAGCL
13


 


914
LLKCSGHGH
13


 


933
SHLWMENLI
13


 


951
NCEWSIFYV
13


 


959
VTVLAFTLI
13


 


996
TILDNMDEQ
13


 


1007
MELRPKYGI
13


 


1018
RSTEHNSSL
13











V2-HLA-A0201-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













1
GLEEMSEYA
16



 


7
EYADDYREL
12


 


4
EMSEYADDY
8


 


8
YADDYRELE
8











V3-HLA-A0201-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
RLGWPSPCC
12



 


1
MTRLGWPSP
9


 


4
LGWPSPCCA
9


 


10
CCARLQCSE
5











254P1D6B v5-



HLA-0201-p-mers


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
DIRKDLTFL
21



 


9
TFLGKDWGL
16


 


6
KDLTFLGKD
10

















TABLE XXIV







V1-



HLA-A0203-


9mers-


254P1D6B





NoResultsFound.





V2-


HLA-A0203-


9mers-


254P1D6B





NoResultsFound.





V3-


HLA-A0203-


9mers-


254P1D6B





NoResultsFound.





V5-


HLA-A0203-


9mers-


254P1D6B





NoResultsFound.

















TABLE XXV







V1-



HLA-A0203-


9mers-


254P1D6B





NoResultsFound.





V2-


HLA-A0203-


9mers-


254P1D6B





NoResultsFound.





V3-HLA-A3-


9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 7; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.












Pos
123456789
score





6
WPSPCCARK
16


 


3
RLGWPSPCC
14


 


1
MTRLGWPSP
8


 


2
TRLGWPSPC
8


 


10
CCARKQCSE
7











V5-HLA-A3-



9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 11; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.












Pos
123456789
score





2
EDIRKDLTF
18


 


5
RKDLTFLGK
18


 


7
DLTFLGKDW
13


 


3
DIRKDLTFL
12


















TABLE XXVI





Pos
123456789
score
















V1-HLA-A26-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













267
EVLMPSHSL
29



 


884
EVARNLHMR
26


 


483
SVDSPVLRL
25


 


6
GVLSSLLLL
24


 


135
EDIRKDLPF
24


 


136
DIRKDLPFL
24


 


246
EVLEKEKAS
24


 


681
VTGLQVGTY
24


 


1005
ERMELRPKY
24


 


285
VTVEKSPVL
23


 


437
VVSPQLQEL
23


 


745
DDQRIVSYL
23


 


765
DVIDGSDHS
23


 


773
SVALQLTNL
23


 


152
EMSEYSDDY
22


 


335
LTVSAGDNL
22


 


407
VTVSSENAF
22


 


807
EVQPDPRKS
22


 


862
DLSTVIVFY
22


 


909
DTAGCLLKC
22


 


41
ETTRIMRVS
21


 


349
DNEVELKAF
21


 


351
EVELKAFVA
21


 


958
YVTVLAFTL
21


 


1038
DTIFSREKM
21


 


365
ETTYNYEWN
20


 


445
LTLPLTSAL
20


 


693
LTVKDQQGL
20


 


155
EYSDDYREL
20


 


159
DYRELEKDL
19


 


240
TTPSSGEVL
19


 


417
EGFVNVTVK
19


 


434
PVAVVSPQL
19


 


464
EIVSYHWEE
19


 


519
LIVNNAVDY
19


 


579
GVQTPYLHL
19


 


611
AVVTVIVQP
19


 


634
ELIFPVESA
19


 


778
LTNLVEGVY
19


 


780
NLVEGVYTF
19


 


837
LVRQLAVLL
19


 


907
RVDTAGCLL
19


 


949
ESNCEWSIF
19


 


4
PTGVLSSLL
18


 


106
PVQRPAQLL
18


 


208
ETQQDPELH
18


 


461
DDTEIVSYH
18


 


486
SPVLRLSNL
18


 


511
ATNSTTAAL
18


 


627
AVAGPDKEL
18


 


672
ENIDKAIAT
18


 


685
QVGTYHFRL
18


 


768
DGSDHSVAL
18


 


835
DTLVRQLAV
18


 


50
HTFPVVDCT
17


 


56
DCTAACCDL
17


 


366
TTYNYEWNL
17


 


436
AVVSPQLQE
17


 


558
QIVLYEWSL
17


 


612
VVTVIVQPE
17


 


802
DTATVEVQP
17


 


829
LTEQRKDTL
17


 


836
TLVRQLAVL
17


 


987
TKIRKKTKY
17


 


34
AVISPNLET
16


 


53
PVVDCTAAC
16


 


162
ELEKDLLQP
16


 


233
SVLLPLPTT
16


 


330
RTVKELTVS
16


 


362
PPVETTYNY
16


 


390
KQTLNLSQL
16


 


399
SVGLYVFKV
16


 


444
ELTLPLTSA
16


 


460
TDDTEIVSY
16


 


462
DTEIVSYHW
16


 


481
KTSVDSPVL
16


 


495
DPGNYSFRL
16


 


574
HVVMQGVQT
16


 


661
EHVRGPSAV
16


 


676
KAIATVTGL
16


 


679
ATVTGLQVG
16


 


744
TDDQRIVSY
16


 


800
DTDTATVEV
16


 


819
ELTLQVGVG
16


 


842
AVLLNVLDS
16


 


865
TVIVFYVQS
16


 


896
EKADFLLFK
16


 


954
WSIFYVTVL
16


 


3
PPTGVLSSL
15


 


74
EGRCYLVSC
15


 


91
KKMGPIRSY
15


 


108
QRPAQLLDY
15


 


132
DSPEDIRKD
15


 


231
ERSVLLPLP
15


 


251
EKASQLQEQ
15


 


288
EKSPVLTVT
15


 


293
LTVTPGSTE
15


 


331
TVKELTVSA
15


 


339
AGDNLIITL
15


 


373
NLISHPTDY
15


 


384
EIKQGHKQT
15


 


395
LSQLSVGLY
15


 


403
YVFKVTVSS
15


 


472
EINGPFIEE
15


 


479
EEKTSVDSP
15


 


504
TVTDSDGAT
15


 


514
STTAALIVN
15


 


554
SDDHQIVLY
15


 


555
DDHQIVLYE
15


 


571
EGKHVVMQG
15


 


575
VVMQGVQTP
15


 


614
TVIVQPENN
15


 


650
SDDHGIVFY
15


 


821
TLQVGVGQL
15


 


861
SDLSTVIVF
15


 


867
IVFYVQSRP
15


 


936
WMENLIQRY
15


 


965
TLIVLTGGF
15


 


1021
EHNSSLMVS
15


 


1057
GSIRNGASF
15


 


5
TGVLSSLLL
14


 


71
WWFEGRCYL
14


 


94
GPIRSYLTF
14


 


102
FVLRPVQRP
14


 


181
EYTDWGLLP
14


 


230
PERSVLLPL
14


 


299
STEHSIPTP
14


 


316
STPSELPIS
14


 


353
ELKAFVAPA
14


 


419
FVNVTVKPA
14


 


471
EEINGPFIE
14


 


515
TTAALIVNN
14


 


520
IVNNAVDYP
14


 


595
YTFQLKVTD
14


 


651
DDHGIVFYH
14


 


655
IVFYHWEHV
14


 


783
EGVYTFHLR
14


 


786
YTFHLRVTD
14


 


791
RVTDSQGAS
14


 


804
ATVEVQPDP
14


 


817
LVELTLQVG
14


 


833
RKDTLVRQL
14


 


849
DSDIKVQKI
14


 


906
LRVDTAGCL
14


 


950
SNCEWSIFY
14


 


956
IFYVTVLAF
14











V2A26-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













4
EMSEYADDY
22



 


7
EYADDYREL
19


 


3
EEMSEYADD
11











V3-A26-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.












1
MTRLGWPSP
9











V5-A26-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













2
EDIRKDLTF
25



 


3
DIRKDLTFL
24


 


8
LTFLGKDWG
12


















TABLE XXVII





Pos
123456789
score
















V1-HLA-B0702-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













359
APAPPVETT
24



 


304
IPTPPTSAA
23


 


3
PPTGVLSSL
22


 


105
RPVQRPAQL
22


 


439
SPQLQELTL
22


 


809
QPDPRKSGL
22


 


133
SPEDIRKDL
21


 


175
EPRGSAEYT
21


 


226
APKLPERSV
21


 


495
DPGNYSFRL
21


 


270
MPSHSLPPA
20


 


328
APRTVKELT
20


 


486
SPVLRLSNL
20


 


874
RPPFKVLKA
20


 


618
QPENNRPPV
19


 


37
SPNLETTRI
18


 


52
FPVVDCTAA
18


 


94
GPIRSYLTF
18


 


567
GPGSEGKHV
18


 


627
AVAGPDKEL
18


 


872
QSRPPFKVL
18


 


875
PPFKVLKAA
18


 


296
TPGSTEHSI
17


 


483
SVDSPVLRL
17


 


582
TPYLHLSAM
17


 


721
ARAGGRHVL
17


 


723
AGGRHVLVL
17


 


811
DPRKSGLVE
17


 


221
SASTPAPKL
16


 


272
SHSLPPASL
16


 


312
APSESTPSE
16


 


321
LPISPTTAP
16


 


324
SPTTARPTV
16


 


377
HPTDYQGEI
16


 


2
APPTGVLSS
15


 


96
IRSYLTFVL
15


 


136
DIRKDLPFL
15


 


169
QPSGKQEPR
15


 


230
PERSVLLPL
15


 


301
EHSIPTPPT
15


 


481
KTSVDSPVL
15


 


511
ATNSTTAAL
15


 


579
GVQTPYLHL
15


 


621
NNRPPVAVA
15


 


768
DGSDHSVAL
15


 


89
EPKKMGPIR
14


 


92
KMGPIRSYL
14


 


125
SPSGIWGDS
14


 


188
LPGSEGAFN
14


 


202
SPAVPAETQ
14


 


241
TPSSGEVLE
14


 


267
EVLMPSHSL
14


 


356
AFVAPAPPV
14


 


361
APPVETTYN
14


 


387
QGHKQTLNL
14


 


394
NLSQLSVGL
14


 


424
VKPARRVNL
14


 


441
QLQELTLPL
14


 


431
ANAGPNHTI
14


 


676
KAIATVTGL
14


 


715
NNSPPRARA
14


 


760
SPAAGDVID
14


 


814
KSGLVELTL
14


 


837
LVRQLAVLL
14


 


898
ADFLLFKVL
14


 


968
VLTGGFTWL
14


 


34
AVISPNLET
13


 


106
PVQRPAQLL
13


 


155
EYSDDYREL
13


 


199
VGDSPAVPA
13


 


207
AETQQDPEL
13


 


224
TPAPKLPER
13


 


227
PKLPERSVL
13


 


228
KLPERSVLL
13


 


229
LPERSVLLP
13


 


236
LPLPTTPSS
13


 


238
LPTTPSSGE
13


 


261
SNSSGKEVL
13


 


274
SLPPASLEL
13


 


276
PPASLELSS
13


 


290
SPVLTVTPG
13


 


339
AGDNLIITL
13


 


385
IKQGHKQTL
13


 


425
KPARRVNLP
13


 


429
RVNLPPVAV
13


 


430
VNLPPVAVV
13


 


432
LPPVAVVSP
13


 


433
PPVAVVSPQ
13


 


437
VVSPQLQEL
13


 


445
LTLPLTSAL
13


 


533
AGPNHTITL
13


 


577
MQGVQTPYL
13


 


620
ENNRPPVAV
13


 


623
RPPVAVAGP
13


 


630
GPDKELIFP
13


 


665
GPSAVEMEN
13


 


718
PPRARAGGR
13


 


833
RKDTLVRQL
13


 


907
RVDTAGCLL
13


 


918
SGHGHCDPL
13


 


954
WSIFYVTVL
13


 


1047
ERGNPKVSM
13


 


5
TGVLSSLLL
13


 


6
GVLSSLLLL
12


 


32
SNAVISPNL
12


 


47
RVSHTFPVV
12


 


109
RPAQLLDYG
12


 


142
PFLGKDWGL
12


 


159
DYRELEKDL
12


 


180
AEYTDWGLL
12


 


210
QQDPELHYL
12


 


212
DPELHYLNE
12


 


240
TTPSSGEVL
12


 


262
NSSGKEVLM
12


 


285
VTVEKSPVL
12


 


287
VEKSPVLTV
12


 


288
EKSPVLTVT
12


 


306
TPPTSAAPS
12


 


317
TPSELPISP
12


 


346
TLPDNEVEL
12


 


347
LPDNEVELK
12


 


358
VAPAPPVET
12


 


414
AFGEGFVNV
12


 


427
ARRVNLPPV
12


 


434
PVAVVSPQL
12


 


447
LPLTSALID
12


 


525
VDYPPVANA
12


 


528
PPVANAGPN
12


 


553
SSDDHQIVL
12


 


591
QEGDYTFQL
12


 


624
PPVAVAGPD
12


 


636
IFPVESATL
12


 


703
STSTLTVAV
12


 


717
SPPRARAGG
12


 


722
RAGGRHVLV
12


 


755
IRDGQSPAA
12


 


770
SDHSVALQL
12


 


773
SVALQLTNL
12


 


782
VEGVYTFHL
12


 


812
PRKSGLVEL
12


 


813
RKSGLVELT
12


 


836
TLVRQLAVL
12


 


840
QLAVLLNVL
12


 


859
AHSDLSTVI
12


 


881
KAAEVARNL
12


 


885
VARNLHMRL
12


 


927
TKRCICSHL
12


 


961
VLAFTLIVL
12


 


990
RKKTKYTIL
12


 


4
PTGVLSSLL
11


 


8
LSSLLLLVT
11


 


56
DCTAACCDL
11


 


61
CCDLSSCDL
11


 


71
WWFEGRCYL
11


 


82
CPHKENCEP
11


 


113
LLDYGDMML
11


 


205
VPAETQQDP
11


 


275
LPPASLELS
11


 


307
PPTSAAPSE
11


 


309
TSAAPSEST
11


 


315
ESTPSELPI
11


 


327
TAPRTVKEL
11


 


335
LTVSAGDNL
11


 


337
VSAGDNLII
11


 


353
ELKAFVAPA
11


 


362
PPVETTYNY
11


 


390
KQTLNLSQL
11


 


444
ELTLPLTSA
11


 


527
YPPVANAGP
11


 


534
GPNHTITLP
11


 


541
LPQNSITLN
11


 


569
GSEGKHVVM
11


 


607
QQSTAVVTV
11


 


634
ELIFPVESA
11


 


637
FPVESATLD
11


 


685
QVGTYHFRL
11


 


693
LTVKDQQGL
11


 


699
QGLSSTSTL
11


 


701
LSSTSTLTV
11


 


720
RARAGGRHV
11


 


731
LPNNSITLD
11


 


745
DDQRIVSYL
11


 


798
ASDTDTATV
11


 


821
TLQVGVGQL
11


 


871
VQSRPPFKV
11


 


883
AEVARNLHM
11


 


892
RLSKEKADF
11


 


893
LSKEKADFL
11


 


894
SKEKADFLL
11


 


895
KEKADFLLF
11


 


924
DPLTKRCIC
11


 


953
EWSIFYVTV
11


 


956
IFYVTVLAF
11


 


988
KIRKKTKYT
11


 


1001
MDEQERMEL
11


 


1010
RPKYGIKHR
11


 


1018
RSTEHNSSL
11











V2-HLA-B0702-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













7
EYADDYREL
12



 


1
GLEEMSEYA
6


 


9
ADDYRELEK
5











V3-HLA-B0702-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













6
WPSPCCARK
14



 


8
SPCCARKQC
11


 


4
LGWPSPCCA
7


 


3
RLGWPSPCC
6











V5-HLA-B0702-



9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
DIRKDLTFL
15



 


9
TFLGKDWGL
12


 


2
EDIRKDLTF
9


 


1
PEDIRKDLT
7


















TABLE XXVIII





Pos
123456789
score
















V1-HLA-B08-



9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 3; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide is


the start position plus


eight.













248
LEKEKASQL
32



 


893
LSKEKADFL
32


 


990
RKKTKYTIL
30


 


228
KLPERSVLL
27


 


486
SPVLRLSNL
27


 


105
RPVQRPAQL
24


 


809
QPDPRKSGL
24


 


1008
ELRPKYGIK
24


 


1014
GIKHRSTEH
24


 


285
VTVEKSPVL
23


 


812
PRKSGLVEL
22


 


981
CKRQKRTKI
22


 


885
VARNLHMRL
21


 


988
KIRKKTKYT
21


 


136
DIRKDLPFL
20


 


142
PFLGKDWGL
20


 


424
VKPARRVNL
20


 


718
PPRARAGGR
20


 


133
SPEDIRKDL
19


 


159
DYRELEKDL
19


 


274
SLPPASLEL
19


 


353
ELKAFVAPA
19


 


439
SPQLQELTL
19


 


854
VQKIRAHSD
19


 


879
VLKAAEVAR
19


 


986
RTKIRKKTK
19


 


1010
RPKYGIKHR
19


 


1041
FSREKMERG
19


 


89
EPKKMGPIR
18


 


135
EDIRKDLPF
18


 


346
TLPDNEVEL
18


 


441
QLQELTLPL
18


 


821
TLQVGVGQL
18


 


829
LTEQRKDTL
18


 


900
FLLFKVLRV
18


 


113
LLDYGDMML
17


 


179
SAEYTDWGL
17


 


224
TPAPKLPER
17


 


226
APKLPERSV
17


 


327
TAPRTVKEL
17


 


384
EIKQGHKQT
17


 


394
NLSQLSVGL
17


 


477
FIEEKTSVD
17


 


598
QLKVTDSSR
17


 


692
RLTVKDQQG
17


 


730
VLPNNSITL
17


 


837
LVRQLAVLL
17


 


840
QLAVLLNVL
17


 


849
DSDIKVQKI
17


 


872
QSRPPFKVL
17


 


874
RPPFKVLKA
17


 


961
VLAFTLIVL
17


 


968
VLTGGFTWL
17


 


984
QKRTKIRKK
17


 


989
IRKKTKYTI
17


 


1050
NPKVSMNGS
17


 


3
PPTGVLSSL
16


 


88
CEPKKMGPI
16


 


169
QPSGKQEPR
16


 


221
SASTPAPKL
16


 


230
PERSVLLPL
16


 


246
EVLEKEKAS
16


 


495
DPGNYSFRL
16


 


540
TLPQNSITL
16


 


629
AGPDKELIF
16


 


836
TLVRQLAVL
16


 


881
KAAEVARNL
16


 


895
KEKADFLLF
16


 


914
LLKCSGHGH
16


 


924
DPLTKRCIC
16


 


927
TKRCICSHL
16


 


1025
SLMVSESEF
16


 


37
SPNLETTRI
15


 


425
KPARRVNLP
15


 


488
VLRLSNLDP
15


 


558
QIVLYEWSL
15


 


676
KAIATVTGL
15


 


709
VAVKKENNS
15


 


728
VLVLPNNSI
15


 


780
NLVEGVYTF
15


 


851
DIKVQKIRA
15











V2-HLA-B08-



9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 5; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide is


the start position plus


eight.













7
EYADDYREL
13



 


9
ADDYRELEK
10


 


1
GLEEMSEYA
9











V3-HLA-B08-



9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 7; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide is


the start position plus


eight.













10
CCARKQCSE
10



 


8
SPCCARKQC
9


 


9
PCCARKQCS
8


 


1
MTRLGWPSP
7


 


3
RLGWPSPCC
6


 


6
WPSPCCARK
6











V5-HLA-B08-



9mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 11; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide is


the start position plus


eight.













3
DIRKDLTFL
20



 


9
TFLGKDWGL
20


 


2
EDIRKDLTF
18


 


4
IRKDLTFLG
11


















TABLE XXIX





Pos
123456789
score
















V1-HLA-B1510-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













272
SHSLPPASL
23



 


155
EYSDDYREL
16


 


346
TLPDNEVEL
16


 


721
ARAGGRHVL
16


 


96
IRSYLTFVL
15


 


227
PKLPERSVL
15


 


261
SNSSGKEVL
15


 


385
IKQGHKQTL
15


 


481
KTSVDSPVL
15


 


658
YHWEHVRGP
15


 


768
DGSDHSVAL
15


 


872
QSRPPFKVL
15


 


49
SHTFPVVDC
14


 


285
VTVEKSPVL
14


 


301
EHSIPTPPT
14


 


394
NLSQLSVGL
14


 


437
VVSPQLQEL
14


 


483
SVDSPVLRL
14


 


540
TLPQNSITL
14


 


627
AVAGPDKEL
14


 


636
IFPVESATL
14


 


661
EHVRGPSAV
14


 


812
PRKSGLVEL
14


 


821
TLQVGVGQL
14


 


829
LTEQRKDTL
14


 


840
QLAVLLNVL
14


 


859
AHSDLSTVI
14


 


881
KAAEVARNL
14


 


1001
MDEQERMEL
14


 


32
SNAVISPNL
13


 


71
WWFEGRCYL
13


 


92
KMGPIRSYL
13


 


133
SPEDIRKDL
13


 


160
YRELEKDLL
13


 


207
AETQQDPEL
13


 


221
SASTPAPKL
13


 


228
KLPERSVLL
13


 


240
TTPSSGEVL
13


 


274
SLPPASLEL
13


 


313
PSESTPSEL
13


 


327
TAPRTVKEL
13


 


424
VKPARRVNL
13


 


434
PVAVVSPQL
13


 


445
LTLPLTSAL
13


 


468
YHWEEINGP
13


 


553
SSDDHQIVL
13


 


569
GSEGKHVVM
13


 


573
KHVVMQGVQ
13


 


689
YHFRLTVKD
13


 


723
AGGRHVLVL
13


 


809
QPDPRKSGL
13


 


833
RKDTLVRQL
13


 


836
TLVRQLAVL
13


 


837
LVRQLAVLL
13


 


921
GHCDPLTKR
13


 


954
WSIFYVTVL
13


 


958
YVTVLAFTL
13


 


961
VLAFTLIVL
13


 


1021
EHNSSLMVS
13


 


83
PHKENCEPK
12


 


105
RPVQRPAQL
12


 


136
DIRKDLPFL
12


 


210
QQDPELHYL
12


 


215
LHYLNESAS
12


 


267
EVLMPSHSL
12


 


339
AGDNLIITL
12


 


388
GHKQTLNLS
12


 


495
DPGNYSFRL
12


 


577
MQGVQTPYL
12


 


579
GVQTPYLHL
12


 


585
LHLSAMQEG
12


 


685
QVGTYHFRL
12


 


730
VLPNNSITL
12


 


771
DHSVALQLT
12


 


885
VARNLHMRL
12


 


894
SKEKADFLL
12


 


898
ADFLLFKVL
12


 


919
GHGHCDPLT
12


 


968
VLTGGFTWL
12


 


3
PPTGVLSSL
11


 


4
PTGVLSSLL
11


 


5
TGVLSSLLL
11


 


6
GVLSSLLLL
11


 


106
PVQRPAQLL
11


 


113
LLDYGDMML
11


 


142
PFLGKDWGL
11


 


159
DYRELEKDL
11


 


179
SAEYTDWGL
11


 


248
LEKEKASQL
11


 


366
TTYNYEWNL
11


 


387
QGHKQTLNL
11


 


390
KQTLNLSQL
11


 


397
QLSVGLYVF
11


 


439
SPQLQELTL
11


 


441
QLQELTLPL
11


 


511
ATNSTTAAL
11


 


533
AGPNHTITL
11


 


536
NHTITLPQN
11


 


556
DHQIVLYEW
11


 


591
QEGDYTFQL
11


 


663
VRGPSAVEM
11


 


676
KAIATVTGL
11


 


693
LTVKDQQGL
11


 


699
QGLSSTSTL
11


 


726
RHVLVLPNN
11


 


745
DDQRIVSYL
11


 


773
SVALQLTNL
11


 


782
VEGVYTFHL
11


 


814
KSGLVELTL
11


 


889
LHMRLSKEK
11


 


893
LSKEKADFL
11


 


906
LRVDTAGCL
11


 


918
SGHGHCDPL
11


 


927
TKRCICSHL
11


 


932
CSHLWMENL
11


 


956
IFYVTVLAF
11


 


1018
RSTEHNSSL
11


 


1047
ERGNPKVSM
11











V2-HLA-B1510-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.












7
YADDYREL
16











V3-HLA-B1510-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













6
WPSPCCARK
58



 


2
TRLGWPSPC
3


 


4
LGWPSPCCA
3


 


5
GWPSPCCAR
3


 


1
MTRLGWPSP
2


 


3
RLGWPSPCC
2


 


7
PSPCCARKQ
2











V5-HLA-B1510-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
TFLGKDWGL
12



 


3
DIRKDLTFL
11


 


2
EDIRKDLTF
8


















TABLE XXX





Pos
123456789
score















V1-HLA-B2705-9mers-254P1D6B





NoResultsFound.











V2-B2705-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
ADDYRELEK
13



 


5
MSEYADDYR
11


 


7
EYADDYREL
11


 


4
EMSEYADDY
10


 


6
SEYADDYRE
6











V3-B2705-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













2
TRLGWPSPC
15



 


5
GWPSPCCAR
14


 


6
WPSPCCARK
14


 


3
RLGWPSPCC
7











V5-B2705-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
TFLGKDWGL
17



 


2
EDIRKDLTF
16


 


5
RKDLTFLGK
16


 


3
DIRKDLTFL
15


 


4
IRKDLTFLG
12
















TABLE XXXI







V1-HLA-B2709-9mers-254P1D6B





NoResultsFound.





V2-HLA-B2709-9mers-254P1D6B





NoResultsFound.





V3-HLA-B2709-9mers-254P1D6B





NoResultsFound.





V5-HLA-B2709-9mers-254P1D6B





NoResultsFound.


















TABLE XXXII





Pos
123456789
score















V1-HLA-


B2709-9mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













28
GRTYSNAVI
22



 


812
PRKSGLVEL
22


 


906
LRVDTAGCL
22


 


96
IRSYLTFVL
21


 


663
VRGPSAVEM
21


 


721
ARAGGRHVL
21


 


46
MRVSHTFPV
20


 


160
YRELEKDLL
20


 


329
PRTVKELTV
20


 


427
ARRVNLPPV
20


 


741
SRSTDDQRI
20


 


747
QRIVSYLWI
20


 


989
IRKKTKYTI
20


 


1047
ERGNPKVSM
19


 


605
SRQQSTAVV
18


 


6
GVLSSLLLL
17


 


105
RPVQRPAQL
16


 


428
RRVNLPPVA
16


 


833
RKDTLVRQL
16


 


839
RQLAVLLNV
16


 


497
GNYSFRLTV
15


 


725
GRHVLVLPN
15


 


784
GVYTFHLRV
15


 


1018
RSTEHNSSL
15


 


75
GRCYLVSCP
14


 


92
KMGPIRSYL
14


 


180
AEYTDWGLL
14


 


390
KQTLNLSQL
14


 


401
GLYVFKVTV
14


 


481
KTSVDSPVL
14


 


483
SVDSPVLRL
14


 


579
GVQTPYLHL
14


 


593
GDYTFQLKV
14


 


676
KAIATVTGL
14


 


687
GTYHFRLTV
14


 


742
RSTDDQRIV
14


 


770
SDHSVALQL
14


 


816
GLVELTLQV
14


 


858
RAHSDLSTV
14


 


881
KAAEVARNL
14


 


907
RVDTAGCLL
14


 


929
RCICSHLWM
14


 


985
KRTKIRKKT
14


 


990
RKKTKYTIL
14


 


32
SNAVISPNL
13


 


47
RVSHTFPW
13


 


94
GPIRSYLTF
13


 


207
AETQQDPEL
13


 


227
PKLPERSVL
13


 


228
KLPERSVLL
13


 


335
LTVSAGDNL
13


 


366
TTYNYEWNL
13


 


429
RVNLPPVAV
13


 


445
LTLPLTSAL
13


 


489
LRLSNLDPG
13


 


622
NRPPVAVAG
13


 


691
FRLTVKDQQ
13


 


699
QGLSSTSTL
13


 


722
RAGGRHVLV
13


 


723
AGGRHVLVL
13


 


758
GQSPAAGDV
13


 


814
KSGLVELTL
13


 


891
MRLSKEKAD
13


 


898
ADFLLFKVL
13


 


900
FLLFKVLRV
13


 


956
IFYVTVLAF
13


 


5
TGVLSSLLL
12


 


43
TRIMRVSHT
12


 


44
RIMRVSHTF
12


 


71
WWFEGRCYL
12


 


111
AQLLDYGDM
12


 


136
DIRKDLPFL
12


 


142
PFLGKDWGL
12


 


176
PRGSAEYTD
12


 


221
SASTPAPKL
12


 


230
PERSVLLPL
12


 


248
LEKEKASQL
12


 


267
EVLMPSHSL
12


 


274
SLPPASLEL
12


 


285
VTVEKSPVLI
12


 


356
AFVAPAPPV
12


 


387
QGHKQTLNL
12


 


396
SQLSVGLYV
12


 


416
GEGFVNVTV
12


 


424
VKPARRVNL
12


 


430
VNLPPVAVV
12


 


434
PVAVVSPQL
12


 


486
SPVLRLSNL
12


 


501
FRLTVTDSD
12


 


511
ATNSTTAAL
12


 


567
GPGSEGKHV
12


 


569
GSEGKHVVM
12


 


678
IATVTGLQV
12


 


683
GLQVGTYHF
12


 


693
LTVKDQQGL
12


 


720
RARAGGRHV
12


 


745
DDQRIVSYL
12


 


755
IRDGQSPAA
12


 


790
LRVTDSQGA
12


 


821
TLQVGVGQL
12


 


832
QRKDTLVRQ
12


 


837
LVRQLAVLL
12


 


838
VRQLAVLLN
12


 


857
IRAHSDLST
12


 


861
SDLSTVIVF
12


 


873
SRPPFKVLK
12


 


892
RLSKEKADF
12


 


895
KEKADFLLF
12


 


928
KRCICSHLW
12


 


942
QRYIWDGES
12


 


954
WSIFYVTVL
12


 


958
YVTVLAFTL
12


 


982
KRQKRTKIR
12


 


993
TKYTILDNM
12


 


1057
GSIRNGASF
12


 


3
PPTGVLSSL
11


 


9
SSLLLLVTI
11


 


21
ARKQCSEGR
11


 


56
DCTAACCDL
11


 


104
LRPVQRPAQ
11


 


106
PVQRPAQLL
11


 


108
QRPAQLLDY
11


 


122
NRGSPSGIW
11


 


133
SPEDIRKDL
11


 


137
IRKDLPFLG
11


 


145
GKDWGLEEM
11


 


155
EYSDDYREL
11


 


197
SSVGDSPAV
11


 


210
QQDPELHYL
11


 


231
ERSVLLPLP
11


 


240
TTPSSGEVL
11


 


261
SNSSGKEVL
11


 


287
VEKSPVLTV
11


 


313
PSESTPSEL
11


 


315
ESTPSELPI
11


 


327
TAPRTVKEL
11


 


339
AGDNLIITL
11


 


346
TLPDNEVEL
11


 


385
IKQGHKQTL
11


 


394
NLSQLSVGL
11


 


414
AFGEGFVNV
11


 


422
VTVKPARRV
11


 


437
VVSPQLQEL
11


 


439
SPQLQELTL
11


 


441
QLQELTLPL
11


 


495
DPGNYSFRL
11


 


513
NSTTAALIV
11


 


517
AALIVNNAV
11


 


533
AGPNHTITL
11


 


551
NQSSDDHQI
11


 


558
QIVLYEWSL
11


 


572
GKHVVMQGV
11


 


577
MQGVQTPYL
11


 


591
QEGDYTFQL
11


 


627
AVAGPDKEL
11


 


636
IFPVESATL
11


 


655
IVFYHWEHV
11


 


685
QVGTYHFRL
11


 


719
PRARAGGRH
11


 


768
DGSDHSVAL
11


 


773
SVALQLTNL
11


 


780
NLVEGVYTF
11


 


809
QPDPRKSGL
11


 


818
VELTLQVGV
11


 


835
DTLVRQLAV
11


 


836
TLVRQLAVL
11


 


855
QKIRAHSDL
11


 


863
LSTVIVFYV
11


 


872
QSRPPFKVL
11


 


883
AEVARNLHM
11


 


885
VARNLHMRL
11


 


886
ARNLHMRLS
11


 


893
LSKEKADFL
11


 


927
TKRCICSHL
11


 


932
CSHLWMENL
11


 


948
GESNCEWSI
11


 


960
TVLAFTLIV
11


 


968
VLTGGFTWL
11


 


1005
ERMELRPKY
11


 


1007
MELRPKYGI
11


 


1045
KMERGNPKV
11


 


1059
IRNGASFSY
11


 


4
PTGVLSSLL
10


 


7
VLSSLLLLV
10


 


38
PNLETTRIM
10


 


40
LETTRIMRV
10


 


61
CCDLSSCDL
10


 


85
KENCEPKKM
10


 


112
QLLDYGDMM
10


 


113
LLDYGDMML
10


 


135
EDIRKDLPF
10


 


159
DYRELEKDL
10


 


179
SAEYTDWGL
10


 


239
PTTPSSGEV
10


 


272
SHSLPPASL
10


 


337
VSAGDNLII
10


 


344
IITLPDNEV
10


 


407
VTVSSENAF
10


 


458
QSTDDTEIV
10


 


480
EKTSVDSPV
10


 


493
NLDPGNYSF
10


 


522
NNAVDYPPV
10


 


540
TLPQNSITL
10


 


553
SSDDHQIVL
10


 


582
TPYLHLSAM
10


 


589
AMQEGDYTF
10


 


607
QQSTAVVTV
10


 


608
QSTAVVTVI
10


 


628
VAGPDKELI
10


 


629
AGPDKELIF
10


 


647
SSSSDDHGI
10


 


730
VLPNNSITL
10


 


774
VALQLTNLV
10


 


777
QLTNLVEGV
10


 


782
VEGVYTFHL
10


 


798
ASDTDTATV
10


 


829
LTEQRKDTL
10


 


840
QLAVLLNVL
10


 


846
NVLDSDIKV
10


 


869
FYVQSRPPF
10


 


877
FKVLKAAEV
10


 


894
SKEKADFLL
10


 


897
KADFLLFKV
10


 


918
SGHGHCDPL
10


 


933
SHLWMENLI
10


 


961
VLAFTLIVL
10


 


1001
MDEQERMEL
10


 


1009
LRPKYGIKH
10


 


1017
HRSTEHNSS
10


 


1032
EFDSDQDTI
10


 


1042
SREKMERGN
10


 


1051
PKVSMNGSI
10


















TABLE XXXI





Pos
123456789
score
















V2-HLA-B2709-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













7
EYADDYREL
11



 


6
SEYADDYRE
5











V3-HLA-B2709-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













2
TRLGWPSPC
12



 


3
RLGWPSPCC
5











V5-HLA-B2709-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













9
TFLGKDWGL
12



 


3
DIRKDLTFL
11


 


4
IRKDLTFLG
11


 


2
EDIRKDLTF
10


 


5
RKDLTFLGK
5


















TABLE XXXII





Pos
123456789
score
















V1-HLA-B4402-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













180
AEYTDWGLL
25



 


895
KEKADFLLF
24


 


207
AETQQDPEL
23


 


591
QEGDYTFQL
23


 


174
QEPRGSAEY
22


 


230
PERSVLLPL
22


 


248
LEKEKASQL
22


 


364
VETTYNYEW
21


 


411
SENAFGEGF
21


 


782
VEGVYTFHL
21


 


937
MENLIQRYI
21


 


339
AGDNLIITL
20


 


898
ADFLLFKVL
20


 


948
GESNCEWSI
20


 


1007
MELRPKYGI
20


 


88
CEPKKMGPI
19


 


470
WEEINGPFI
19


 


533
AGPNHTITL
18


 


91
KKMGPIRSY
17


 


135
EDIRKDLPF
17


 


319
SELPISPTT
17


 


445
LTLPLTSAL
17


 


471
EEINGPFIE
17


 


554
SDDHQIVLY
17


 


721
ARAGGRHVL
17


 


723
AGGRHVLVL
17


 


872
QSRPPFKVL
17


 


92
KMGPIRSYL
16


 


94
GPIRSYLTF
16


 


133
SPEDIRKDL
16


 


210
QQDPELHYL
16


 


227
PKLPERSVL
16


 


274
SLPPASLEL
16


 


460
TDDTEIVSY
16


 


511
ATNSTTAAL
16


 


627
AVAGPDKEL
16


 


629
AGPDKELIF
16


 


650
SDDHGIVFY
16


 


669
VEMENIDKA
16


 


670
EMENIDKAI
16


 


744
TDDQRIVSY
16


 


862
DLSTVIVFY
16


 


1046
MERGNPKVS
16


 


40
LETTRIMRV
15


 


85
KENCEPKKM
15


 


155
EYSDDYREL
15


 


219
NESASTPAP
15


 


221
SASTPAPKL
15


 


228
KLPERSVLL
15


 


327
TAPRTVKEL
15


 


349
DNEVELKAF
15


 


352
VELKAFVAP
15


 


360
PAPPVETTY
15


 


373
NLISHPTDY
15


 


383
GEIKQGHKQ
15


 


390
KQTLNLSQL
15


 


437
WSPQLQEL
15


 


443
QELTLPLTS
15


 


493
NLDPGNYSF
15


 


553
SSDDHQIVL
15


 


649
SSDDHGIVF
15


 


676
KAIATVTGL
15


 


730
VLPNNSITL
15


 


768
DGSDHSVAL
15


 


809
QPDPRKSGL
15


 


833
RKDTLVRQL
15


 


861
SDLSTVIVF
15


 


883
AEVARNLHM
15


 


954
WSIFYVTVL
15


 


987
TKIRKKTKY
15


 


1005
ERMELRPKY
15


 


1057
GSIRNGASF
15


 


6
GVLSSLLLL
14


 


9
SSLLLLVTI
14


 


44
RIMRVSHTF
14


 


63
DLSSCDLAW
14


 


70
AWWFEGRCY
14


 


187
LLPGSEGAF
14


 


267
EVLMPSHSL
14


 


272
SHSLPPASL
14


 


314
SESTPSELP
14


 


315
ESTPSELPI
14


 


346
TLPDNEVEL
14


 


370
YEWNLISHP
14


 


424
VKPARRVNL
14


 


439
SPQLQELTL
14


 


463
TEIVSYHWE
14


 


479
EEKTSVDSP
14


 


483
SVDSPVLRL
14


 


486
SPVLRLSNL
14


 


519
LIVNNAVDY
14


 


531
ANAGPNHTI
14


 


540
TLPQNSITL
14


 


589
AMQEGDYTF
14


 


619
PENNRPPVA
14


 


633
KELIFPVES
14


 


770
SDHSVALQL
14


 


814
KSGLVELTL
14


 


855
QKIRAHSDL
14


 


859
AHSDLSTVI
14


 


936
WMENLIQRY
14


 


938
ENLIORYIW
14


 


956
IFYVTVLAF
14


 


965
TLIVLTGGF
14


 


1031
SEFDSDQDT
14


 


5
TGVLSSLLL
13


 


23
KQCSEGRTY
13


 


65
SSCDLAWWF
13


 


71
WWFEGRCYL
13


 


73
FEGRCYLVS
13


 


96
IRSYLTFVL
13


 


105
RPVQRPAQL
13


 


106
PVQRPAQLL
13


 


108
QRPAQLLDY
13


 


134
PEDIRKDLP
13


 


140
DLPFLGKDW
13


 


151
EEMSEYSDD
13


 


152
EMSEYSDDY
13


 


161
RELEKDLLQ
13


 


213
PELHYLNES
13


 


250
KEKASQLQE
13


 


261
SNSSGKEVL
13


 


266
KEVLMPSHS
13


 


280
LELSSVTVE
13


 


287
VEKSPVLTV
13


 


333
KELTVSAGD
13


 


394
NLSQLSVGL
13


 


395
LSQLSVGLY
13


 


397
QLSVGLYVF
13


 


407
VTVSSENAF
13


 


481
KTSVDSPVL
13


 


570
SEGKHVVMQ
13


 


681
VTGLQVGTY
13


 


699
QGLSSTSTL
13


 


713
KENNSPPRA
13


 


745
DDQRIVSYL
13


 


773
SVALQLTNL
13


 


780
NLVEGVYTF
13


 


818
VELTLQVGV
13


 


836
TLVRQLAVL
13


 


837
LVRQLAVLL
13


 


840
QLAVLLNVL
13


 


881
KAAEVARNL
13


 


907
RVDTAGCLL
13


 


928
KRCICSHLW
13


 


952
CEWSIFYVT
13


 


961
VLAFTLIVL
13


 


967
IVLTGGFTW
13


 


3
PPTGVLSSL
12


 


26
SEGRTYSNA
12


 


32
SNAVISPNL
12


 


61
CCDLSSCDL
12


 


64
LSSCDLAWW
12


 


142
PFLGKDWGL
12


 


159
DYRELEKDL
12


 


160
YRELEKDLL
12


 


163
LEKDLLQPS
12


 


209
TQQDPELHY
12


 


240
TTPSSGEVL
12


 


245
GEVLEKEKA
12


 


300
TEHSIPTPP
12


 


385
IKQGHKQTL
12


 


387
QGHKQTLNL
12


 


416
GEGFVNVTV
12


 


441
QLQELTLPL
12


 


491
LSNLDPGNY
12


 


512
TNSTTAALI
12


 


551
NQSSDDHQI
12


 


579
GVQTPYLHL
12


 


628
VAGPDKELI
12


 


636
IFPVESATL
12


 


639
VESATLDGS
12


 


747
QRIVSYLWI
12


 


778
LTNLVEGVY
12


 


812
PRKSGLVEL
12


 


821
TLQVGVGQL
12


 


829
LTEQRKDTL
12


 


830
TEQRKDTLV
12


 


894
SKEKADFLL
12


 


906
LRVDTAGCL
12


 


918
SGHGHCDPL
12


 


933
SHLWMENLI
12


 


949
ESNCEWSIF
12


 


950
SNCEWSIFY
12


 


958
YVTVLAFTL
12


 


968
VLTGGFTWL
12


 


1002
DEQERMELR
12


 


1004
QERMELRPK
12


 


1020
TEHNSSLMV
12


 


1025
SLMVSESEF
12


 


1029
SESEFDSDQ
12


 


1032
EFDSDQDTI
12


 


1033
FDSDQDTIF
12











V2-HLA-B4402-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













4
EMSEYADDY
14



 


7
EYADDYREL
14


 


3
EEMSEYADD
13


 


2
LEEMSEYAD
12


 


6
SEYADDYRE
11


 


9
ADDYRELEK
6











V3-HLA-B4402-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













8
SPCCARKQC
5



 


4
LGWPSPCCA
4


 


6
WPSPCCARK
4


 


7
PSPCCARKQ
4


 


2
TRLGWPSPC
3


 


5
GWPSPCCAR
3











V5-HLA-B4402-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













2
EDIRKDLTF
18



 


1
PEDIRKDLT
13


 


7
DLTFLGKDW
12


 


9
TFLGKDWGL
12


 


3
DIRKDLTFL
11


















TABLE XXXIIII





Pos
123456789
score
















V1-HLA-B5101-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













517
AALIVNNAV
24



 


324
SPTTAPRTV
23


 


37
SPNLETTRI
22


 


296
TPGSTEHSI
22


 


327
TAPRTVKEL
22


 


377
HPTDYOGEI
22


 


495
DPGNYSFRL
22


 


678
IATVTGLQV
22


 


774
VALQLTNLV
22


 


881
KAAEVARNL
22


 


628
VAGPDKELI
21


 


676
KAIATVTGL
21


 


720
RARAGGRHV
21


 


858
RAHSDLSTV
21


 


897
KADFLLFKV
21


 


3
PPTGVLSSL
20


 


221
SASTPAPKL
20


 


567
GPGSEGKHV
20


 


722
RAGGRHVLV
20


 


811
DPRKSGLVE
20


 


226
APKLPERSV
19


 


277
PASLELSSV
19


 


439
SPQLQELTL
19


 


568
PGSEGKHVV
19


 


608
QSTAVVTVI
19


 


849
DSDIKVQKI
19


 


970
TGGFTWLCI
19


 


133
SPEDIRKDL
18


 


447
LPLTSALID
18


 


610
TAVVTVIVQ
18


 


618
QPENNRPPV
18


 


723
AGGRHVLVL
18


 


768
DGSDHSVAL
18


 


885
VARNLHMRL
18


 


924
DPLTKRCIC
18


 


27
EGRTYSNAV
17


 


105
RPVQRPAQL
17


 


179
SAEYTDWGL
17


 


486
SPVLRLSNL
17


 


523
NAVDYPPVA
17


 


699
QGLSSTSTL
17


 


874
RPPFKVLKA
17


 


9
SSLLLLVTI
16


 


229
LPERSVLLP
16


 


275
LPPASLELS
16


 


339
AGDNLIITL
16


 


360
PAPPVETTY
16


 


400
VGLYVFKVT
16


 


413
NAFGEGFVN
16


 


430
VNLPPVAVV
16


 


432
LPPVAVVSP
16


 


533
AGPNHTITL
16


 


582
TPYLHLSAM
16


 


593
GDYTFQLKV
16


 


637
FPVESATLD
16


 


809
QPDPRKSGL
16


 


846
NVLDSDIKV
16


 


875
PPFKVLKAA
16


 


900
FLLFKVLRV
16


 


962
LAFTLIVLT
16


 


989
IRKKTKYTI
16


 


2
APPTGVLSS
15


 


5
TGVLSSLLL
15


 


28
GRTYSNAVI
15


 


121
LNRGSPSGI
15


 


129
IWGDSPEDI
15


 


212
DPELHYLNE
15


 


236
LPLPTTPSS
15


 


306
TPPTSAAPS
15


 


317
TPSELPISP
15


 


358
VAPAPPVET
15


 


401
GLYVFKVTV
15


 


433
PPVAVVSPQ
15


 


497
GNYSFRLTV
15


 


530
VANAGPNHT
15


 


541
LPQNSITLN
15


 


626
VAVAGPDKE
15


 


687
GTYHFRLTV
15


 


701
LSSTSTLTV
15


 


731
LPNNSITLD
15


 


759
QSPAAGDVI
15


 


784
GVYTFHLRV
15


 


835
DTLVRQLAV
15


 


839
RQLAVLLNV
15


 


859
AHSDLSTVI
15


 


1
MAPPTGVLS
14


 


33
NAVISPNLE
14


 


69
LAWWFEGRC
14


 


94
GPIRSYLTF
14


 


99
YLTFVLRPV
14


 


205
VPAETQQDP
14


 


225
PAPKLPERS
14


 


287
VEKSPVLTV
14


 


290
SPVLTVTPG
14


 


337
VSAGDNLII
14


 


347
LPDNEVELK
14


 


359
APAPPVETT
14


 


387
QGHKQTLNL
14


 


415
FGEGFVNVT
14


 


416
GEGFVNVTV
14


 


426
PARRVNLPP
14


 


475
GPFIEEKTS
14


 


509
DGATNSTTA
14


 


512
TNSTTAALI
14


 


516
TAALIVNNA
14


 


527
YPPVANAGP
14


 


531
ANAGPNHTI
14


 


607
QQSTAVVTV
14


 


624
PPVAVAGPD
14


 


667
SAVEMENID
14


 


709
VAVKKENNS
14


 


761
PAAGDVIDG
14


 


762
AAGDVIDGS
14


 


800
DTDTATVEV
14


 


841
LAVLLNVLD
14


 


933
SHLWMENLI
14


 


981
CKRQKRTKI
14


 


17
IAGCARKQC
13


 


40
LETTRIMRV
13


 


47
RVSHTFPVV
13


 


88
CEPKKMGPI
13


 


141
LPFLGKDWG
13


 


159
DYRELEKDL
13


 


175
EPRGSAEYT
13


 


193
GAFNSSVGD
13


 


202
SPAVPAETQ
13


 


224
TPAPKLPER
13


 


238
LPTTPSSGE
13


 


270
MPSHSLPPA
13


 


285
VTVEKSPVL
13


 


310
SAAPSESTP
13


 


312
APSESTPSE
13


 


321
LPISPTTAP
13


 


328
APRTVKELT
13


 


336
TVSAGDNLI
13


 


338
SAGDNLIIT
13


 


362
PPVETTYNY
13


 


396
SQLSVGLYV
13


 


399
SVGLYVFKV
13


 


417
EGFVNVTVK
13


 


422
VTVKPARRV
13


 


425
KPARRVNLP
13


 


435
VAVVSPQLQ
13


 


446
TLPLTSALI
13


 


534
GPNHTITLP
13


 


566
LGPGSEGKH
13


 


623
RPPVAVAGP
13


 


630
GPDKELIFP
13


 


665
GPSAVEMEN
13


 


673
NIDKAIATV
13


 


728
VLVLPNNSI
13


 


797
GASDTDTAT
13


 


803
TATVEVQPD
13


 


818
VELTLQVGV
13


 


910
TAGCLLKCS
13


 


918
SGHGHCDPL
13


 


923
CDPLTKRCI
13


 


954
WSIFYVTVL
13


 


959
VTVLAFTLI
13


 


960
TVLAFTLIV
13


 


961
VLAFTLIVL
13


 


1007
MELRPKYGI
13


 


1010
RPKYGIKHR
13


 


1050
NPKVSMNGS
13


 


52
FPVVDCTAA
12


 


58
TAAGGDLSS
12


 


82
CPHKENCEP
12


 


89
EPKKMGPIR
12


 


116
YGDMMLNRG
12


 


136
DIRKDLPFL
12


 


169
QPSGKQEPR
12


 


188
LPGSEGAFN
12


 


240
TTPSSGEVL
12


 


241
TPSSGEVLE
12


 


248
LEKEKASQL
12


 


279
SLELSSVTV
12


 


304
IPTPPTSAA
12


 


311
AAPSESTPS
12


 


315
ESTPSELPI
12


 


329
PRTVKELTV
12


 


355
KAFVAPAPP
12


 


361
APPVETTYN
12


 


366
TTYNYEWNL
12


 


367
TYNYEWNLI
12


 


414
AFGEGFVNV
12


 


451
SALIDGSQS
12


 


457
SQSTDDTEI
12


 


465
IVSYHWEEI
12


 


510
GATNSTTAA
12


 


513
NSTTAALIV
12


 


528
PPVANAGPN
12


 


532
NAGPNHTIT
12


 


538
TITLPQNSI
12


 


605
SRQQSTAVV
12


 


655
IVFYHWEHV
12


 


682
TGLQVGTYH
12


 


718
PPRARAGGR
12


 


741
SRSTDDQRI
12


 


745
DDQRIVSYL
12


 


747
QRIVSYLWI
12


 


760
SPAAGDVID
12


 


844
LLNVLDSDI
12


 


863
LSTVIVFYV
12


 


871
VQSRPPFKV
12


 


893
LSKEKADFL
12


 


898
ADFLLFKVL
12


 


937
MENLIQRYI
12


 


1032
EFDSDQDTI
12


 


1051
PKVSMNGSI
12


 


6
GVLSSLLLL
11


 


7
VLSSLLLLV
11


 


20
CARKQCSEG
11


 


56
DCTAACCDL
11


 


59
AACCDLSSC
11


 


95
PIRSYLTFV
11


 


96
IRSYLTFVL
11


 


109
RPAQLLDYG
11


 


125
SPSGIWGDS
11


 


132
DSPEDIRKD
11


 


158
DDYRELEKD
11


 


180
AEYTDWGLL
11


 


203
PAVPAETQQ
11


 


206
PAETQQDPE
11


 


227
PKLPERSVL
11


 


264
SGKEVLMPS
11


 


276
PPASLELSS
11


 


280
LELSSVTVE
11


 


307
PPTSAAPSE
11


 


344
IITLPDNEV
11


 


350
NEVELKAFV
11


 


385
IKQGHKQTL
11


 


392
TLNLSQLSV
11


 


455
DGSQSTDDT
11


 


476
PFIEEKTSV
11


 


540
TLPQNSITL
11


 


551
NQSSDDHQI
11


 


553
SSDDHQIVL
11


 


609
STAVVTVIV
11


 


631
PDKELIFPV
11


 


636
IFPVESATL
11


 


645
DGSSSSDDH
11


 


670
EMENIDKAI
11


 


717
SPPRARAGG
11


 


730
VLPNNSITL
11


 


739
DGSRSTDDQ
11


 


757
DGQSPAAGD
11


 


766
VIDGSDHSV
11


 


798
ASDTDTATV
11


 


814
KSGLVELTL
11


 


816
GLVELTLQV
11


 


830
TEQRKDTLV
11


 


836
TLVRQLAVL
11


 


840
QLAVLLNVL
11


 


872
QSRPPFKVL
11


 


877
FKVLKAAEV
11


 


882
AAEVARNLH
11


 


901
LLFKVLRVD
11


 


906
LRVDTAGCL
11


 


951
NCEWSIFYV
11


 


953
EWSIFYVTV
11


 


958
YVTVLAFTL
11


 


1013
YGIKHRSTE
11


 


1020
TEHNSSLMV
11


 


1045
KMERGNPKV
11


 


1062
GASFSYCSK
11











V2-HLA-B5101-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













8
YADDYRELE
14



 


7
EYADDYREL
8


 


6
SEYADDYRE
6











V3-HLA-B5101-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













4
LGWPSPCCA
11



 


6
WPSPCCARK
11


 


8
SPCCARKQC
11


 


11
CARKQCSEG
11


 


2
TRLGWPSPC
5


 


7
PSPCCARKQ
5











V5-HLA-B5101-9mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.













3
DIRKDLTFL
13



 


9
TFLGKDWGL
11


 


6
KDLTFLGKD
6


















TABLE XXXIV





Pos
1234567890
score
















V1-HLA-A1-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













459
STDDTEIVSY
33



 


553
SSDDHQIVLY
33


 


743
STDDQRIVSY
31


 


649
SSDDHGIVFY
31


 


173
KQEPRGSAEY
29


 


208
ETQQDPELHY
27


 


107
VQRPAQLLDY
26


 


1019
STEHNSSLMV
25


 


894
SKEKADFLLF
23


 


949
ESNCEWSIFY
23


 


986
RTKIRKKTKY
23


 


156
YSDDYRELEK
22


 


378
PTDYQGEIKQ
22


 


160
YRELEKDLLQ
20


 


359
APAPPVETTY
20


 


769
GSDHSVALQL
20


 


860
HSDLSTVIVF
20


 


394
NLSQLSVGLY
19


 


554
SDDHQIVLYE
19


 


72
WFEGRCYLVS
18


 


182
YTDWGLLPGS
18


 


299
STEHSIPTPP
18


 


347
LPDNEVELKA
18


 


592
EGDYTFQLKV
18


 


800
DTDTATVEVQ
18


 


829
LTEQRKDTLV
18


 


882
AAEVARNLHM
18


 


907
RVDTAGCLLK
18


 


1004
QERMELRPKY
18


 


286
TVEKSPVLTV
17


 


410
SSENAFGEGF
17


 


505
VTDSDGATNS
17


 


518
ALIVNNAVDY
17


 


569
GSEGKHVVMQ
17


 


601
VTDSSRQQST
17


 


608
TVTGLQVGTY
17


 


777
QLTNLVEGVY
17


 


792
VTDSQGASDT
17


 


861
SDLSTVIVFY
17


 


1058
SIRNGASFSY
17


 


22
RKQCSEGRTY
16


 


69
LAWWFEGRCY
16


 


134
PEDIRKDLPF
16


 


190
GSEGAFNSSV
16


 


210
QQDPELHYLN
16


 


229
LPERSVLLPL
16


 


249
EKEASQLQE
16


 


313
PSESTPSELP
16


 


361
APPVETTYNY
16


 


442
LQELTLPLTS
16


 


462
DTEIVSYHWE
16


 


490
RLSNLDPGNY
16


 


507
DSDGATNSTT
16


 


575
VVMQGVQTPY
16


 


586
HLSAMQEGDY
16


 


798
ASDTDTATVE
16


 


809
QPDPRKSGLV
16











V2-HLA-A1-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
YADDYRELEK
18



 


4
EEMSEYADDY
15


 


6
MSEYADDYRE
14


 


10
ADDYRELEKD
13


 


2
GLEEMSEYAD
11


 


3
LEEMSEYADD
10











V3-HLA-A1-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
MTRLGWPSPC
6



 


6
WPSPCCARKQ
6


 


7
PSPCCARKQC
5


 


4
LGWPSPCCAR
4


 


8
SPCCARKQCS
2











V5-HLA-A1-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













2
PEDIRKDLTF
16



 


1
SPEDIRKDLT
14


 


6
RKDLTFLGKD
12


 


5
IRKDLTFLGK
9


















TABLE XXXV





Pos
1234567890
score
















V1-A0201-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













635
LIFPVESATL
27



 


343
LIITLPDNEV
25


 


345
ITLPDNEVEL
24


 


700
GLSSTSTLTV
24


 


39
NLETTRIMRV
23


 


112
QLLDYGDMML
23


 


326
TTAPRTVKEL
23


 


338
SAGDNLIITL
23


 


677
AIATVTGLQV
23


 


828
QLTEQRKDTL
23


 


862
DLSTVIVFYV
23


 


6
GVLSSLLLLV
22


 


436
AVVSPQLQEL
22


 


539
ITLPQNSITL
22


 


576
VMQGVQTPYL
22


 


729
LVLPNNSITL
22


 


820
LTLQVGVGQL
22


 


836
TLVRQLAVLL
22


 


961
VLAFTLIVLT
22


 


1000
NMDEQERMEL
22


 


11
LLLLVTIAGC
21


 


429
RVNLPPVAVV
21


 


441
QLQELTLPLT
21


 


722
RAGGRHVLVL
21


 


835
DTLVRQLAVL
21


 


843
VLLNVLDSDI
21


 


905
VLRVDTAGCL
21


 


7
VLSSLLLLVT
20


 


45
IMRVSHTFPV
20


 


120
MLNRGSPSGI
20


 


128
GIWGDSPEDI
20


 


247
VLEKEKASQL
20


 


278
ASLELSSVTV
20


 


286
TVEKSPVLTV
20


 


398
LSVGLYVFKV
20


 


431
NLPPVAVVSP
20


 


445
LTLPLTSALI
20


 


692
RLTVKDQQGL
20


 


775
ALQLTNLVEG
20


 


797
GASDTDTATV
20


 


857
IRAHSDLSTV
20


 


892
RLSKEKADFL
20


 


960
TVLAFTLIVL
20


 


988
KIRKKTKYTI
20


 


217
YLNESASTPA
19


 


269
LMPSHSLPPA
19


 


391
QTLNLSQLSV
19


 


413
NAFGEGFVNV
19


 


765
DVIDGSDHSV
19


 


773
SVALQLTNLV
19


 


776
LQLTNLVEGV
19


 


901
LLFKVLRVDT
19


 


1054
SMNGSIRNGA
19


 


2
APPTGVLSSL
18


 


8
LSSLLLLVTI
18


 


12
LLLVTIAGCA
18


 


34
AVISPNLETT
18


 


98
SYLTFVLRPV
18


 


228
KLPERSVLLP
18


 


274
SLPPASLELS
18


 


295
VTPGSTEHSI
18


 


516
TAALIVNNAV
18


 


532
NAGPNHTITL
18


 


560
VLYEWSLGPG
18


 


606
RQQSTAVVTV
18


 


627
AVAGPDKELI
18


 


654
GIVFYHWEHV
18


 


672
ENIDKAIATV
18


 


721
ARAGGRHVLV
18


 


817
LVELTLQVGV
18


 


870
YVQSRPPFKV
18


 


950
SNCEWSIFYV
18


 


967
IVLTGGFTWL
18


 


94
GPIRSYLTFV
17


 


273
HSLPPASLEL
17


 


355
KAFVAPAPPV
17


 


357
FVAPAPPVET
17


 


393
LNLSQLSVGL
17


 


423
TVKPARRVNL
17


 


444
ELTLPLTSAL
17


 


452
ALIDGSQSTD
17


 


510
GATNSTTAAL
17


 


511
ATNSTTAALI
17


 


530
VANAGPNHTI
17


 


537
HTITLPQNSI
17


 


727
HVLVLPNNSI
17


 


781
LVEGVYTFHL
17


 


811
DPRKSGLVEL
17


 


816
GLVELTLQVG
17


 


839
RQLAVLLNVL
17


 


848
LDSDIKVQKI
17


 


969
LTGGFTWLCI
17


 


1006
RMELRPKYGI
17


 


92
KMGPIRSYLT
16


 


167
LLQPSGKQEP
16


 


178
GSAEYTDWGL
16


 


186
GLLPGSEGAF
16


 


187
LLPGSEGAFN
16


 


209
TQQDPELHYL
16


 


229
LPERSVLLPL
16


 


284
SVTVEKSPVL
16


 


312
APSESTPSEL
16


 


334
ELTVSAGDNL
16


 


384
EIKQGHKQTL
16


 


400
VGLYVFKVTV
16


 


401
GLYVFKVTVS
16


 


415
FGEGFVNVTV
16


 


426
PARRVNLPPV
16


 


482
TSVDSPVLRL
16


 


518
ALIVNNAVDY
16


 


565
SLGPGSEGKH
16


 


626
VAVAGPDKEL
16


 


675
DKAIATVTGL
16


 


799
SDTDTATVEV
16


 


838
VRQLAVLLNV
16


 


856
KIRAHSDLST
16


 


879
VLKAAEVARN
16


 


896
EKADFLLFKV
16


 


899
DFLLFKVLRV
16


 


900
FLLFKVLRVD
16


 


939
NLIQRYIWDG
16


 


955
SIFYVTVLAF
16


 


959
VTVLAFTLIV
16


 


965
TLIVLTGGFT
16


 


1019
STEHNSSLMV
16


 


5
TGVLSSLLLL
15


 


10
SLLLLVTIAG
15


 


63
DLSSCDLAWW
15


 


95
PIRSYLTFVL
15


 


103
VLRPVQRPAQ
15


 


149
GLEEMSEYSD
15


 


154
SEYSDDYREL
15


 


234
VLLPLPTTPS
15


 


235
LLPLPTTPSS
15


 


255
QLQEQSSNSS
15


 


268
VLMPSHSLPP
15


 


276
PPASLELSSV
15


 


279
SLELSSVTVE
15


 


303
SIPTPPTSAA
15


 


328
APRTVKELTV
15


 


335
LTVSAGDNLI
15


 


346
TLPDNEVELK
15


 


358
VAPAPPVETT
15


 


392
TLNLSQLSVG
15


 


459
STDDTEIVSY
15


 


464
EIVSYHWEEI
15


 


488
VLRLSNLDPG
15


 


515
TTAALIVNNA
15


 


524
AVDYPPVANA
15


 


630
GPDKELIFPV
15


 


668
AVEMENIDKA
15


 


720
RARAGGRHVL
15


 


728
VLVLPNNSIT
15


 


730
VLPNNSITLD
15


 


735
SITLDGSRST
15


 


743
STDDQRIVSY
15


 


752
YLWIRDGQSP
15


 


754
WIRDGQSPAA
15


 


766
VIDGSDHSVA
15


 


767
IDGSDHSVAL
15


 


789
HLRVTDSQGA
15


 


813
RKSGLVELTL
15


 


815
SGLVELTLQV
15


 


829
LTEQRKDTLV
15


 


859
AHSDLSTVIV
15


 


873
SRPPFKVLKA
15


 


926
LTKRCICSHL
15


 


934
HLWMENLIQR
15


 


936
WMENLIQRYI
15


 


952
CEWSIFYVTV
15


 


26
SEGRTYSNAV
14


 


31
YSNAVISPNL
14


 


71
WWFEGRCYLV
14


 


91
KKMGPIRSYL
14


 


104
LRPVQRPAQL
14


 


135
EDIRKDLPFL
14


 


141
LPFLGKDWGL
14


 


143
FLGKDWGLEE
14


 


179
SAEYTDWGLL
14


 


190
GSEGAFNSSV
14


 


266
KEVLMPSHSL
14


 


323
ISPTTAPRTV
14


 


366
TTYNYEWNLI
14


 


389
HKQTLNLSQL
14


 


394
NLSQLSVGLY
14


 


438
VSPQLQELTL
14


 


451
SALIDGSQST
14


 


472
EINGPFIEEK
14


 


475
GPFIEEKTSV
14


 


494
LDPGNYSFRL
14


 


502
RLTVTDSDGA
14


 


519
LIVNNAVDYP
14


 


540
TLPQNSITLN
14


 


557
HQIVLYEWSL
14


 


584
YLHLSAMQEG
14


 


604
SSRQQSTAVV
14


 


617
VQPENNRPPV
14


 


662
HVRGPSAVEM
14


 


684
LQVGTYHFRL
14


 


702
SSTSTLTVAV
14


 


744
TDDQRIVSYL
14


 


772
HSVALQLTNL
14


 


784
GVYTFHLRVT
14


 


821
TLQVGVGQLT
14


 


832
QRKDTLVRQL
14


 


840
QLAVLLNVLD
14


 


842
AVLLNVLDSD
14


 


845
LNVLDSDIKV
14


 


880
LKAAEVARNL
14


 


913
CLLKCSGHGH
14


 


962
LAFTLIVLTG
14


 


997
ILDNMDEQER
14


 


1031
SEFDSDQDTI
14


 


1
MAPPTGVLSS
13


 


13
LLVTIAGCAR
13


 


35
VISPNLETTR
13


 


50
HTFPVVDCTA
13


 


60
ACCDLSSCDL
13


 


78
YLVSCPHKEN
13


 


119
MMLNRGSPSG
13


 


198
SVGDSPAVPA
13


 


206
PAETQQDPEL
13


 


223
STPAPKLPER
13


 


225
PAPKLPERSV
13


 


227
PKLPERSVLL
13


 


238
LPTTPSSGEV
13


 


260
SSNSSGKEVL
13


 


281
ELSSVTVEKS
13


 


285
VTVEKSPVLT
13


 


336
TVSAGDNLII
13


 


337
VSAGDNLIIT
13


 


352
VELKAFVAPA
13


 


395
LSQLSVGLYV
13


 


403
YVFKVTVSSE
13


 


411
SENAFGEGFV
13


 


414
AFGEGFVNVT
13


 


421
NVTVKPARRV
13


 


428
RRVNLPPVAV
13


 


485
DSPVLRLSNL
13


 


521
VNNAVDYPPV
13


 


547
TLNGNQSSDD
13


 


566
LGPGSEGKHV
13


 


633
KELIFPVESA
13


 


634
ELIFPVESAT
13


 


679
ATVTGLQVGT
13


 


705
STLTVAVKKE
13


 


778
LTNLVEGVYT
13


 


808
VQPDPRKSGL
13


 


844
LLNVLDSDIK
13


 


847
VLDSDIKVQK
13


 


884
EVARNLHMRL
13


 


893
LSKEKADFLL
13


 


897
KADFLLFKVL
13


 


906
LRVDTAGCLL
13


 


944
YIWDGESNCE
13


 


956
IFYVTVLAFT
13


 


957
FYVTVLAFTL
13


 


958
YVTVLAFTLI
13


 


1025
SLMVSESEFD
13


 


1004
EKMERGNPKV
13





 








V2-HLA-A0201-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













7
SEYADDYREL
15



 


2
GLEEMSEYAD
14


 


9
YADDYRELEK
10


 


1
WGLEEMSEYA
8


 


5
EMSEYADDYR
7


 


10
ADDYRELEKD
7











V3-HLA-A0201-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCCA
14



 


4
LGWPSPCCAR
6


 


9
LTFLGKDWGL
18


 


3
EDIRKDLTFL
13


















TABLE XXXVI





Pos
1234567890
score
















V1-HLA-A0203-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













51
TFPVVDCTAA
19



 


303
SIPTPPTSAA
19


 


509
DGATNSTTAA
19


 


754
WIRDGQSPAA
19


 


874
RPPFKVLKAA
19


 


352
VELKAFVAPA
18


 


524
AVDYPPVANA
18


 


620
ENNRPPVAVA
18


 


670
EMENIDKAIA
18


 


714
ENNSPPRARA
18


 


52
FPVVDCTAAC
17


 


304
IPTPPTSAAP
17


 


510
GATNSTTAAL
17


 


755
IRDGQSPAAG
17


 


875
PPFKVLKAAE
17


 


9
SSLLLLVTIA
10


 


12
LLLVTIAGCA
10


 


25
CSEGRTYSNA
10


 


50
HTFPVVDCTA
10


 


61
CCDLSSCDLA
10


 


102
FVLRPVQRPA
10


 


171
SGKQEPRGSA
10


 


185
WGLLPGSEGA
10


 


195
FNSSVGDSPA
10


 


198
SVGDSPAVPA
10


 


213
PELHYLNESA
10


 


217
YLNESASTPA
10


 


244
SGEVLEKEKA
10


 


269
LMPSHSLPPA
10


 


302
HSIPTPPTSA
10


 


319
SELPISPTTA
10


 


330
RTVKELTVSA
10


 


347
LPDNEVELKA
10


 


350
NEVELKAFVA
10


 


405
FKVTVSSENA
10


 


418
GFVNVTVKPA
10


 


427
ARRVNLPPVA
10


 


443
QELTLPLTSA
10


 


502
RLTVTDSDGA
10


 


508
SDGATNSTTA
10


 


515
TTAALIVNNA
10


 


522
NNAVDYPPVA
10


 


580
VQTPYLHLSA
10


 


602
TDSSRQQSTA
10


 


618
QPENNRPPVA
10


 


633
KELIFPVESA
10


 


659
HWEHVRGPSA
10


 


668
AVEMENIDKA
10


 


701
LSSTSTLTVA
10


 


712
KKENNSPPRA
10


 


753
LWIRDGQSPA
10


 


766
VIDGSDHSVA
10


 


789
HLRVTDSQGA
10


 


795
SQGASDTDTA
10


 


833
RKDTLVRQLA
10


 


850
SDIKVQKIRA
10


 


873
SRPPFKVLKA
10


 


877
FKVLKAAEVA
10


 


889
LHMRLSKEKA
10


 


902
LFKVLRVDTA
10


 


954
WSIFYVTVLA
10


 


1054
SMNGSIRNGA
10


 


10
SLLLLVTIAG
9


 


13
LLVTIAGCAR
9


 


26
SEGRTYSNAV
9


 


62
CDLSSCDLAW
9


 


103
VLRPVQRPAQ
9


 


172
GKQEPRGSAE
9


 


186
GLLPGSEGAF
9


 


196
NSSVGDSPAV
9


 


199
VGDSPAVPAE
9


 


214
ELHYLNESAS
9


 


218
LNESASTPAP
9


 


245
GEVLEKEKAS
9


 


270
MPSHSLPPAS
9


 


320
ELPISPTTAP
9


 


331
TVKELTVSAG
9


 


348
PDNEVELKAF
9


 


351
EVELKAFVAP
9


 


353
ELKAFVAPAP
9


 


406
KVTVSSENAF
9


 


419
FVNVTVKPAR
9


 


428
RRVNLPPVAV
9


 


444
ELTLPLTSAL
9


 


503
LTVTDSDGAT
9


 


516
TAALIVNNAV
9


 


523
NAVDYPPVAN
9


 


525
VDYPPVANAG
9


 


581
QTPYLHLSAM
9


 


603
DSSRQQSTAV
9


 


619
PENNRPPVAV
9


 


621
NNRPPVAVAG
9


 


634
ELIFPVESAT
9


 


660
WEHVRGPSAV
9


 


669
VEMENIDKAI
9


 


671
MENIDKAIAT
9


 


702
SSTSTLTVAV
9


 


713
KENNSPPRAR
9


 


715
NNSPPRARAG
9


 


767
IDGSDHSVAL
9


 


790
LRVTDSQGAS
9


 


796
QGASDTDTAT
9


 


834
KDTLVRQLAV
9


 


851
DIKVQKIRAH
9


 


878
KVLKAAEVAR
9


 


890
HMRLSKEKAD
9


 


903
FKVLRVDTAG
9


 


955
SIFYVTVLAF
9


1055
MNGSIRNGAS
9











V2-HLA-A0203-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
WGLEEMSEYA
10



 


2
GLEEMSEYAD
9


 


3
LEEMSEYADD
8











V3-HLA-A0203-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCGA
10



 


4
LGWPSPCCAR
9


 


5
GWPSPCCARK
8





 








V5-HLA-A0203-10mers-254P1D6B








No Results Found.


















TABLE XXXVII





Pos
1234567890
score
















V1-HLA-A3-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













518
ALIVNNAVDY
29



 


847
VLDSDIKVQK
27


 


907
RVDTAGCLLK
27


 


397
QLSVGLYVFK
26


 


14
LVTIAGCARK
24


 


452
ALIDGSQSTD
24


 


777
QLTNLVEGVY
24


 


878
KVLKAAEVAR
24


 


47
RVSHTFPVVD
23


 


490
RLSNLDPGNY
23


 


680
TVTGLQVGTY
23


 


791
RVTDSQGASD
23


 


1008
ELRPKYGIKH
23


 


429
RVNLPPVAVV
22


 


662
HVRGPSAVEM
22


 


872
QSRPPFKVLK
22


 


186
GLLPGSEGAF
21


 


346
TLPDNEVELK
21


 


504
TVTDSDGATN
21


 


677
AIATVTGLQV
21


 


856
KIRAHSDLST
21


 


904
KVLRVDTAGC
21


 


1058
SIRNGASFSY
21


 


34
AVISPNLETT
20


 


35
VISPNLETTR
20


 


233
SVLLPLPTTP
20


 


292
VLTVTPGSTE
20


 


472
EINGPFIEEK
20


 


493
NLDPGNYSFR
20


 


655
IVFYHWEHVR
20


 


694
TVKDQQGLSS
20


 


805
TVEVQPDPRK
20


 


825
GVGQLTEQRK
20


 


836
TLVRQLAVLL
20


 


844
LLNVLDSDIK
20


 


886
ARNLHMRLSK
20


 


888
NLHMRLSKEK
20


 


76
RCYLVSCPHK
19


 


198
SVGDSPAVPA
19


 


247
VLEKEKASQL
19


 


357
FVAPAPPVET
19


 


401
GLYVFKVTVS
19


 


423
TVKPARRVNL
19


 


431
NLPPVAVVSP
19


 


565
SLGPGSEGKH
19


 


586
HLSAMQEGDY
19


 


687
GTYHFRLTVK
19


 


729
LVLPNNSITL
19


 


865
TVIVFYVQSR
17


 


895
KEKADFLLFK
19


 


913
CLLKCSGHGH
19


 


13
LLVTIAGCAR
18


 


103
VLRPVQRPAQ
18


 


166
DLLQPSGKQE
18


 


187
LLPGSEGAFN
18


 


246
EVLEKEKASQ
18


 


359
APAPPVETTY
18


 


392
TLNLSQLSVG
18


 


406
KVTVSSENAF
18


 


487
PVLRLSNLDP
18


 


600
KVTDSSRQQS
18


 


635
LIFPVESATL
18


 


703
STSTLTVAVK
18


 


704
TSTLTVAVKK
18


 


775
ALQLTNLVEG
18


 


784
GVYTFHLRVT
18


 


819
ELTLQVGVGQ
18


 


842
AVLLNVLDSD
18


 


853
KVQKIRAHSD
18


 


919
GHGHCDPLTK
18


 


960
TVLAFTLIVL
18


 


983
RQKRTKIRKK
18


 


988
KIRKKTKYTI
18


 


1043
REKMERGNPK
18


 


7
VLSSLLLLVT
17


 


22
RKQCSEGRTY
17


 


53
PVVDCTAACC
17


 


112
QLLDYGDMML
17


 


120
MLNRGSPSGI
17


 


137
IRKDLPFLGK
17


 


228
KLPERSVLLP
17


 


279
SLELSSVTVE
17


 


286
TVEKSPVLTV
17


 


324
SPTTAPRTVK
17


 


353
ELKAFVAPAP
17


 


394
NLSQLSVGLY
17


 


446
TLPLTSALID
17


 


559
IVLYEWSLGP
17


 


575
VVMQGVQTPY
17


 


614
TVIVQPENNR
17


 


634
ELIFPVESAT
17


 


700
GLSSTSTLTV
17


 


710
AVKKENNSPP
17


 


766
VIDGSDHSVA
17


 


828
QLTEQRKDTL
17


 


840
QLAVLLNVLD
17


 


846
NVLDSDIKVQ
17


 


892
RLSKEKADFL
17


 


905
VLRVDTAGCL
17


 


934
HLWMENLIQR
17


 


955
SIFYVTVLAF
17


 


965
TLIVLTGGFT
17


 


985
KRTKIRKKTK
17


 


997
ILDNMDEQER
17


 


11
LLLLVTIAGC
16


 


12
LLLVTIAGCA
16


 


44
RIMRVSHTFP
16


 


106
PVQRPAQLLD
16


 


143
FLGKDWGLEE
16


 


219
NESASTPAPK
16


 


234
VLLPLPTTPS
16


 


268
VLMPSHSLPP
16


 


280
LELSSVTVEK
16


 


291
PVLTVTPGST
16


 


331
TVKELTVSAG
16


 


351
EVELKAFVAP
16


 


399
SVGLYVFKVT
16


 


430
VNLPPVAVVS
16


 


524
AVDYPPVANA
16


 


560
VLYEWSLGPG
16


 


598
QLKVTDSSRQ
16


 


627
AVAGPDKELI
16


 


673
NIDKAIATVT
16


 


752
YLWIRDGQSP
16


 


765
DVIDGSDHSV
16


 


780
NLVEGVYTFH
16


 


807
EVQPDPRKSG
16


 


837
LVRQLAVLLN
16


 


843
VLLNVLDSDI
16


 


879
VLKAAEVARN
16


 


900
FLLFKVLRVD
16


 


925
PLTKRCICSH
16


 


966
LIVLTGGFTW
16


 


967
IVLTGGFTWL
16


 


976
LCICCCKRQK
16


 


1007
MELRPKYGIK
16


 


6
GVLSSLLLLV
15


 


10
SLLLLVTIAG
15


 


16
TIAGCARKQC
15


 


95
PIRSYLTFVL
15


 


99
YLTFVLRPVQ
15


 


102
FVLRPVQRPA
15


 


107
VQRPAQLLDY
15


 


164
EKDLLQPSGK
15


 


173
KQEPRGSAEY
15


 


204
AVPAETQQDP
15


 


255
QLQEQSSNSS
15


 


257
QEQSSNSSGK
15


 


267
EVLMPSHSLP
15


 


284
SVTVEKSPVL
15


 


336
TVSAGDNLII
15


 


342
NLIITLPDNE
15


 


344
IITLPDNEVE
15


 


403
YVFKVTVSSE
15


 


416
GEGFVNVTVK
15


 


419
FVNVTVKPAR
15


 


444
ELTLPLTSAL
15


 


547
TLNGNQSSDD
15


 


616
IVQPENNRPP
15


 


624
PPVAVAGPDK
15


 


643
TLDGSSSSDD
15


 


816
GLVELTLQVG
15


 


817
LVELTLQVGV
15


 


884
EVARNLHMRL
15


 


901
LLFKVLRVDT
15


 


961
VLAFTLIVLT
15


 


41
ETTRIMRVSH
14


 


63
DLSSCDLAWW
14


 


156
YSDDYRELEK
14


 


214
ELHYLNESAS
14


 


274
SLPPASLELS
14


 


278
ASLELSSVTV
14


 


322
PISPTTAPRT
14


 


377
HPTDYQGEIK
14


 


459
STDDTEIVSY
14


 


488
VLRLSNLDPG
14


 


502
RLTVTDSDGA
14


 


558
QIVLYEWSLG
14


 


564
WSLGPGSEGK
14


 


574
HVVMQGVQTP
14


 


621
NNRPPVAVAG
14


 


683
GLQVGTYHFR
14


 


692
RLTVKDQQGL
14


 


720
RARAGGRHVL
14


 


727
HVLVLPNNSI
14


 


728
VLVLPNNSIT
14


 


743
STDDQRIVSY
14


 


830
TEQRKDTLVR
14


 


851
DIKVQKIRAH
14


 


979
CCCKRQKRTK
14


 


996
TILDNMDEQE
14











V2-HLA-A3-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













9
YADDYRELEK
14



 


2
GLEEMSEYAD
12


 


4
EEMSEYADDY
9


 


7
SEYADDYREL
7











V3-HLA-A3-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
RLGWPSPCCA
15



 


5
GWPSPCCARK
13


 


1
MTRLGWPSPC
8


 


4
LGWPSPCCAR
8


 


10
CCARKQCSEG
7











V5-HLA-A3-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













5
IRKDLTFLGK
17



 


2
PEDIRKDLTF
12


 


4
DIRKDLTFLG
11


 


8
DLTFLGKDWG
11


 


7
KDLTRLGKDW
8


















TABLE XXXVIII





Pos
1234567890
score
















V1-HLA-A26-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













208
ETQQDPELHY
29



 


680
TVTGLQVGTY
28


 


835
DTLVRQLAVL
28


 


884
EVARNLHMRL
28


 


365
ETTYNYEWNL
27


 


436
AVVSPQLQEL
27


 


135
EDIRKDLPFL
26


 


459
STDDTEIVSY
25


 


743
STDDQRIVSY
25


 


765
DVIDGSDHSV
24


 


960
TVLAFTLIVL
24


 


246
EVLEKEKASQ
23


 


384
EIKQGHKQTL
23


 


955
SIFYVTVLAF
23


 


326
TTAPRTVKEL
22


 


807
EVQPDPRKSG
22


 


820
LTLQVGVGQL
22


 


953
EWSIFYVTVL
22


 


151
EEMSEYSDDY
21


 


267
EVLMPSHSLP
21


 


351
EVELKAFVAP
21


 


444
ELTLPLTSAL
21


 


485
DSPVLRLSNL
21


 


729
LVLPNNSITL
21


 


949
ESNCEWSIFY
21


 


1038
DTIFSREKME
21


 


34
AVISPNLETT
20


 


41
ETTRIMRVSH
20


 


220
ESASTPAPKL
20


 


284
SVTVEKSPVL
20


 


334
ELTVSAGDNL
20


 


403
YVFKVTVSSE
20


 


406
KVTVSSENAF
20


 


423
TVKPARRVNL
20


 


480
EKTSVDSPVL
20


 


575
VVMQGVQTPY
20


 


672
ENIDKAIATV
20


 


675
DKAIATVTGL
20


 


800
DTDTATVEVQ
20


 


802
DTATVEVQPD
20


 


811
DPRKSGLVEL
20


 


865
TVIVFYVQSR
20


 


909
DTAGCLLKCS
20


 


147
DWGLEEMSEY
19


 


239
PTTPSSGEVL
19


 


331
TVKELTVSAG
19


 


464
EIVSYHWEEI
19


 


482
TSVDSPVLRL
19


 


539
ITLPQNSITL
19


 


574
HVVMQGVQTP
19


 


986
RTKIRKKTKY
19


 


1032
EFDSDQDTIF
19


 


5
TGVLSSLLLL
18


 


132
DSPEDIRKDL
18


 


159
DYRELEKDLL
18


 


472
EINGPFIEEK
18


 


611
AVVTVIVQPE
18


 


635
LIFPVESATL
18


 


781
LVEGVYTFHL
18


 


967
IVLTGGFTWL
18


 


4
PTGVLSSLLL
17


 


181
EYTDWGLLPG
17


 


286
TVEKSPVLTV
17


 


345
ITLPDNEVEL
17


 


851
DIKVQKIRAH
17


 


926
LTKRCICSHL
17


 


964
FTLIVLTGGF
17


 


6
GVLSSLLLLV
16


 


107
VQRPAQLLDY
16


 


158
DDYRELEKDL
16


 


281
ELSSVTVEKS
16


 


315
ESTPSELPIS
16


 


338
SAGDNLIITL
16


 


462
DTEIVSYHWE
16


 


483
SVDSPVLRLS
16


 


553
SSDDHQIVLY
16


 


595
YTFQLKVTDS
16


 


634
ELIFPVESAT
16


 


649
SSDDHGIVFY
16


 


772
HSVALQLTNL
16


 


786
YTFHLRVTDS
16


 


861
SDLSTVIVFY
16


 


896
EKADFLLFKV
16


 


935
LWMENLIQRY
16


 


1047
ERGNPKVSMN
16


 


1058
SIRNGASFSY
16


 


29
RTYSNAVISP
15


 


53
PVVDCTAACC
15


 


74
EGRCYLVSCP
15


 


90
PKKMGPIRSY
15


 


348
PDNEVELKAF
15


 


394
NLSQLSVGLY
15


 


417
EGFVNVTVKP
15


 


471
EEINGPFIEE
15


 


504
TVTDSDGATN
15


 


614
TVIVQPENNR
15


 


638
PVESATLDGS
15


 


668
AVEMENIDKA
15


 


694
TVKDQQGLSS
15


 


783
EGVYTFHLRV
15


 


837
LVRQLAVLLN
15


 


842
AVLLNVLDSD
15


 


846
NVLDSDIKVQ
15


 


2
APPTGVLSSL
14


 


42
TTRIMRVSHT
14


 


43
TRIMRVSHTF
14


 


50
HTFPWDCTA
14


 


162
ELEKDLLQPS
14


 


209
TQQDPELHYL
14


 


285
VTVEKSPVLT
14


 


389
HKQTLNLSQL
14


 


396
SQLSVGLYVF
14


 


429
RVNLPPVAVV
14


 


503
LTVTDSDGAT
14


 


514
STTAALIVNN
14


 


518
ALIVNNAVDY
14


 


524
AVDYPPVANA
14


 


579
GVQTPYLHLS
14


 


581
QTPYLHLSAM
14


 


609
STAVVTVIVQ
14


 


620
ENNRPPVAVA
14


 


655
IVFYHWEHVR
14


 


661
EHVRGPSAVE
14


 


705
STLTVAVKKE
14


 


744
TDDQRIVSYL
14


 


749
IVSYLWIRDG
14


 


779
TNLVEGVYTF
14


 


784
GVYTFHLRVT
14


 


791
RVTDSQGASD
14


 


804
ATVEVQPDPR
14


 


823
QVGVGQLTEQ
14


 


831
EQRKDTLVRQ
14


 


832
QRKDTLVRQL
14


 


864
STVIVFYVQS
14


 


867
IVFYVQSRPP
14


 


906
LRVDTAGCLL
14


 


1003
EQERMELRPK
14


 


1008
ELRPKYGIKH
14











V2-HLA-A26-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













4
EEMSEYADDY
21



 


5
EMSEYADDYR
12


 


8
EYADDYRELE
11











V3-HLA-A26-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
MTRLGWPSPC
9











V5-HLA-A26-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













3
EDIRKDLTFL
26



 


9
LTFLGKDWGL
20


 


4
DIRKDLTFLG
12


















TABLE XXXIX





Pos
1234567890
score
















V1-HLA-B0702-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













811
DPRKSGLVEL
25



 


226
APKLPERSVL
24


 


312
APSESTPSEL
24


 


229
LPERSVLLPL
23


 


2
APPTGVLSSL
22


 


3
PPTGVLSSLL
22


 


328
APRTVKELTV
22


 


433
PPVAWSPQL
22


 


105
RPVQRPAQLL
21


 


141
LPFLGKDWGL
20


 


317
TPSELPISPT
19


 


347
LPDNEVELKA
19


 


630
GPDKELIFPV
19


 


665
GPSAVEMENI
19


 


94
GPIRSYLTFV
18


 


495
DPGNYSFRLT
18


 


567
GPGSEGKHVV
18


 


618
QPENNRPPVA
18


 


722
RAGGRHVLVL
18


 


809
QPDPRKSGLV
18


 


874
RPPFKVLKAA
18


 


37
SPNLETTRIM
17


 


276
PPASLELSSV
17


 


475
GPFIEEKTSV
17


 


813
RKSGLVELTL
17


 


238
LPTTPSSGEV
16


 


720
RARAGGRHVL
16


 


953
EWSIFYVTVL
16


 


1050
NPKVSMNGSI
16


 


91
KKMGPIRSYL
15


 


169
QPSGKQEPRG
15


 


175
EPRGSAEYTD
15


 


241
TPSSGEVLEK
15


 


359
APAPPVETTY
15


 


386
KQGHKQTLNL
15


 


425
KPARRVNLPP
15


 


767
IDGSDHSVAL
15


 


892
RLSKEKADFL
15


 


95
PIRSYLTFVL
14


 


125
SPSGIWGDSP
14


 


270
MPSHSLPPAS
14


 


304
IPTPPTSAAP
14


 


345
ITLPDNEVEL
14


 


423
TVKPARRVNL
14


 


440
PQLQELTLPL
14


 


534
GPNHTITLPQ
14


 


576
VMQGVQTPYL
14


 


871
VQSRPPFKVL
14


 


897
KADFLLFKVL
14


 


4
PTGVLSSLLL
13


 


52
FPVVDCTAAC
13


 


70
AWWFEGRCYL
13


 


135
EDIRKDLPFL
13


 


220
ESASTPAPKL
13


 


227
PKLPERSVLL
13


 


273
HSLPPASLEL
13


 


275
LPPASLELSS
13


 


321
LPISPTTAPR
13


 


324
SPTTAPRTVK
13


 


326
TTAPRTVKEL
13


 


361
APPVETTYNY
13


 


444
ELTLPLTSAL
13


 


480
EKTSVDSPVL
13


 


482
TSVDSPVLRL
13


 


510
GATNSTTAAL
13


 


532
NAGPNHTITL
13


 


541
LPQNSITLNG
13


 


552
QSSDDHQIVL
13


 


590
MQEGDYTFQL
13


 


637
FPVESATLDG
13


 


675
DKAIATVTGL
13


 


718
PPRARAGGRH
13


 


721
ARAGGRHVLV
13


 


731
LPNNSITLDG
13


 


760
SPAAGDVIDG
13


 


769
GSDHSVALQL
13


 


781
LVEGVYTFHL
13


 


839
RQLAVLLNVL
13


 


859
AHSDLSTVIV
13


 


875
PPFKVLKAAE
13


 


931
ICSHLWMENL
13


 


967
IVLTGGFTWL
13


 


989
IRKKTKYTIL
13


 


5
TGVLSSLLLL
12


 


31
YSNAVISPNL
12


 


60
ACCDLSSCDL
12


 


82
CPHKENCEPK
12


 


89
EPKKMGPIRS
12


 


109
RPAQLLDYGD
12


 


159
DYRELEKDLL
12


 


202
SPAVPAETQQ
12


 


205
VPAETQQDPE
12


 


224
TPAPKLPERS
12


 


231
ERSVLLPLPT
12


 


239
PTTPSSGEVL
12


 


284
SVTVEKSPVL
12


 


290
SPVLTVTPGS
12


 


393
LNLSQLSVGL
12


 


427
ARRVNLPPVA
12


 


432
LPPVAVVSPQ
12


 


436
AVVSPQLQEL
12


 


438
VSPQLQELTL
12


 


494
LDPGNYSFRL
12


 


528
PPVANAGPNH
12


 


531
ANAGPNHTIT
12


 


539
ITLPQNSITL
12


 


578
QGVQTPYLHL
12


 


623
RPPVAVAGPD
12


 


624
PPVAVAGPDK
12


 


635
LIFPVESATL
12


 


662
HVRGPSAVEM
12


 


684
LQVGTYHFRL
12


 


698
QQGLSSTSTL
12


 


744
TDDQRIVSYL
12


 


772
HSVALQLTNL
12


 


835
DTLVRQLAVL
12


 


836
TLVRQLAVLL
12


 


856
KIRAHSDLST
12


 


880
LKAAEVARNL
12


 


884
EVARNLHMRL
12


 


905
VLRVDTAGCL
12


 


917
CSGHGHCDPL
12


 


960
TVLAFTLIVL
12


 


1000
NMDEQERMEL
12


 


1017
HRSTEHNSSL
12


 


1046
MERGNPKVSM
12











V2-HLA-B0702-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













7
SEYADDYREL
11



 


1
WGLEEMSEYA
6











V3-HLA-B0702-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













6
WPSPCCARKQ
13



 


8
SPCCARKQCS
10


 


3
RLGWPSPCCA
8











V5-HLA-B0702-10mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.













1
SPEDIRKDLT
16



 


3
EDIRKDLTFL
13


 


9
LTFLGKDWGL
10


 


2
PEDIRKDLTF
9


















TABLE XL





Pos
1234567890
score















V1-HLA-B08-10mers-254P1D6B





NoResultsFound.





V2-HLA-B08-10mers-254P1D6B





NoResultsFound.





V3-HLA-B08-10mers-254P1D6B





NoResultsFound.





V5-HLA-B08-10mers-254P1D6B





NoResultsFound.


















TABLE XLI





Pos
1234567890
score















V1-HLA-B1510-10mers-254P1D6B





NoResultsFound.





V2-HLA-B1510-10mers-254P1D6B





NoResultsFound.





V3-HLA-B1510-10mers-254P1D6B





NoResultsFound.





V5-HLA-B1510-10mers-254P1D6B





NoResultsFound.


















TABLE XLII





Pos
1234567890
score















V1-HLA-B2705-10mers-254P1D6B





NoResultsFound.





V2-HLA-B2705-10mers-254P1D6B





NoResultsFound.





V3-HLA-B2705-10mers-254P1D6B





NoResultsFound.





V5-HLA-B2705-10mers-254P1D6B





NoResultsFound.


















TABLE XLIII





Pos
1234567890
score
















V1-HLA-



B2709-10mers-


254P1D6B










NoResultsFound.











V2-HLA-



B2709-10mers-


254P1D6B










NoResultsFound.











V3-HLA-



B2709-10mers-


254P1D6B










NoResultsFound.











V5-HLA-



B2709-10mers-


254P1D6B










NoResultsFound.


















TABLE XLIV





Pos
1234567890
score
















V2-HLA-B4402-



10mers-254P1D6B


Each peptide is a portion


of SEQ ID NO: 5; each


start position is specified,


the length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.













4
EEMSEYADDY
24



 


7
SEYADDYREL
22











V3-HLA-



B4402-10mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.













6
WPSPCCARKQ
7



4
LGWPSPCCAR
5


 


7
PSPCCARKQC
5











V5-HLA-B4402-



10mers-254P1D6B


Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position for


each peptide is the start


position plus nine.













2
PEDIRKDLTF
23



 


3
EDIRKDLTFL
17


 


7
KDLTFLGKDW
15


 


9
LTFLGKDWGL
13


















TABLE XLV





Pos
1234567890
score
















V1-HLA-



B5101-10mers-


254P1D6B










NoResultsFound.











V2-HLA-



B5101-10mers-


254P1D6B










NoResultsFound.











V3-HLA-



B5101-10mers-


254P1D6B










NoResultsFound.











V5-HLA-



B5101-10mers-


254P1D6BTZ,1/32 







NoResultsFound.


















TABLE XLVI





Pos
123456789012345
score
















v1-HLA-B5101-



15mers-254P1D6B










NoResultsFound.











V2-HLA-DRB1-0101-



15mers-254P1D6B


Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the start


position plus fourteen.













15
ADDYRELEKDLLDPS
29



 


4
KDWGLEEMSEYADDY
14


 


5
DWGLEEMSEYADDYR
14











V3-HLA-DRB1-0101-



15mers-254P1D68


Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the start


position plus fourteen.













1
MTRLGWPSPCCARKQ
22



 


9
PCCARKQCSEGRTYS
18


 


3
RLGWPSPCCARKQCS
10


 


4
LGWPSPCCARKQCSE
10











V5-HLA-DRB1-0101-



15mers-254P1D6B


Each peptide is a portion of SEQ ID


NO: 11; each start position is specified,


the length of peptide is 15 amino acids,


and the end position for each peptide is


the start position plus fourteen.













11
RKDLTFLGKDWGLEE
19



 


7
PEDIRKDLTFLGKDW
18


 


14
LTFLGKDWGLEEMSE
18


 


5
DSPEDIRKDLTFLGK
11


 


8
EDIRKDLTFLGKDWG
11


 


12
KDLTFLGKDWGLEEM
11


 


13
DLTFLGKDWGLEEMS
10


 


15
TFLGKDWGLEEMSEY
10


 


3
WGDSPEDIRKDLTFL
9


 


6
SPEDIRKDLTFLGKD
9


 


10
IRKDLTFLGKDWGLE
9


















TABLE XLVII





Pos
123456789012345
score
















V1-HLA-DRB1-0301-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













184
DWGLLPGSEGAFNSS
29



 


903
FKVLRVDTAGCLLKC
29


 


343
LIITLPDEVELKAF
28


 


404
VFKVTVSSENAFGEG
28


 


421
NVTVKPARRVNLPPV
28


 


805
TVEVQPDPRKSGLVE
28


 


845
LNVLDSDIKVQKIRA
28


 


626
VAVAGPDKELIFPVE
27


 


1030
ESEFDSDQDTIFSRE
27


 


206
PAETQQDPELHYLNE
26


 


382
QGEIKQGHKQTLNLS
26


 


690
HFRLTVKDQQGLSST
26


 


826
VGQLTEQRKDTLVRQ
26


 


998
LDNMDEQERMELRPK
26


 


130
WGDSPEDIRKDLPFL
25


 


775
ALQLTNLVEGVYTFH
25


 


550
GNQSSDDHQIVLYEW
24


 


573
KHVVMQGVQTPYLHL
24


 


584
YLHLSAMQEGDYTFQ
24


 


134
PEDIRKDLPFLGKDW
23


 


733
NNSITLDGSRSTDDQ
23


 


866
VIVFYVQSRPPFKVL
23


 


160
YRELEKDLLQPSGKQ
22


 


442
LQELTLPLTSALIDG
22


 


834
KDTLVRQLAVLLNVL
22


 


93
MGPIRSYLTFVLRPV
21


 


110
PAQLLDYGDMMLNRG
21


 


126
PSGIWGDSPEDIRKD
21


 


114
LPFLGKDWGLEEMSE
21


 


244
SGEVLEKEKASQLQE
12


 


434
PVAVVSPQLQELTLP
12


 


633
KELIFPVESATLDGS
12


 


727
HVLVLPNNSITLDGS
12


 


765
DVIDGSDHSVALQLT
12


 


965
TLIVLTGGFTWLCIC
21


 


282
LSSVTVEKSPVLTVT
20


 


332
VKELTVSAGDNLIIT
20


 


392
TLNLSQLSVGLYVFK
20


 


485
DSPVLRLSNLDPGNY
20


 


516
TAALIVNNAVDYPPV
20


 


543
QNSITLNGNQSSDDH
20


 


612
VVTVIVQPENNRPPV
20


 


725
GRHVLVLPNNSITLD
20


 


876
PFKVLKAAEVARNLH
20


 


888
NLHMRLSKEKADFLL
20


 


953
EWSIFYVTVLAFTLI
20


 


958
YVTVLAFTLIVLTGG
20


 


62
CDLSSCDLAWWFEGR
19


 


101
TFVLRPVQRPAQLLD
19


 


152
EMSEYSDDYRELEKD
19


 


165
KDLLQPSGKQEPRGS1
19


 


245
GEVLEKEKASQLQEQ
19


 


435
VAVVSPQLQELTLPL
19


 


488
VLRLSNLDPGNYSFR
19


 


563
EWSLGPGSEGKHVVM
19


 


598
QLKVTDSSRQQSTAV
19


 


613
VTVIVQPENNRPPVA
19


 


678
IATVTGLQVGTYHFR
19


 


706
TLTVAVKKENNSPPR
19


 


788
FHLRVTDSQGASDTD
19


 


815
SGLVELTLQVGVGQL
19


 


838
VRQLAVLLNVLDSDI
19


 


882
AAEVARNLHMRLSKE
19


 


889
LHMRLSKEKADFLLF
19


 


890
HMRLSKEKADFLLFK
19


 


941
IQRYIWDGESNCEWS
19


 


975
WLCICCCKRQKRTKI
19


 


1024
SSLMVSESEFDSDQD
19


 


1056
NGSIRNGASFSYCSK
19


 


33
NAVISPNLETTRIMR
18


 


97
RSYLTFVLRPVQRPA
18


 


100
LTFVLRPVQRPAQLL
18


 


104
LRPVQRPAQLLDYGD
18


 


147
DWGLEEMSEYSDDYR
18


 


157
SDDYRELEKDLLQPS
18


 


342
NLIITLPDNEVELKA
18


 


450
TSALIDGSQSTDDTE
18


 


536
NHTITLPQNSITLNG
18


 


574
HWMQGVQTPYLHLS
18


 


588
SAMQEGDYTFQLKVT
18


 


632
DKELIFPVESATLDG
18


 


646
GSSSSDDHGIVFYHW
18


 


691
FRLTVKDQQGLSSTS
18


 


726
RHVLVLPNNSITLDG
18


 


751
SYLWIRDGQSPAAGD
18


 


779
TNLVEGVYTFHLRVT
18


 


899
DFLLFKVLRVDTAGC
18


 


996
TILDNMDEQERMELR
18


 


1002
DEQERMELRPKYGIK
18


 


1004
QERMELRPKYGIKHR
18


 


1022
HNSSLMVSESEFDSD
18


 


1037
QDTIFSREKMERGNP
18


 


77
CYLVSCPHKENGEPK
17


 


138
RKDLPFLGKDWGLEE
17


 


153
MSEYSDDYRELEKDL
17


 


202
SPAVPAETQQDPELH
17


 


212
DPELHYLNESASTPA
17


 


224
TPAPKLPERSVLLPL
17


 


334
ELTVSAGDNLIITLP
17


 


417
EGFVNVTVKPARRVN
17


 


456
GSQSTDDTEIVSYHW
17


 


490
RLSNLDPGNYSFRLT
17


 


610
TAVVTVIVQPENNRP
17


 


614
TVIVQPENNRPPVAV
17


 


625
PVAVAGPDKELIFPV
17


 


668
AVEMENIDKAIATVT
17


 


704
TSTLTVAVKKENNSP
17


 


708
TVAVKKENNSPPRAR
17


 


740
GSRSTDDQRIVSYLW
17


 


823
QVGVGQLTEQRKDTL
17


 


864
STVIVFYVQSRPPFK
17


 


984
QKRTKIRKKTKYTIL
17


 


986
RTKIRKKTKYTILDN
17


 


995
YTILDNMDEQERMEL
17


 


1052
KVSMNGSIRNGASFS
17


 


4
PTGVLSSLLLLVTIA
16


 


14
LVTIAGCARKQCSEG
16


 


66
SCDLAWWFEGRCYLV
16


 


258
EQSSNSSGKEVLMPS
16


 


361
APPVETTYNYEWNLI
16


 


363
PVETTYNYEWNLISH
16


 


374
LISHPTDYQGEIKQG
16


 


463
TEIVSYHWEEINGPF
16


 


653
HGIVFYHWEHVRGPS
16


 


688
TYHFRLTVKDQQGLS
16


 


718
PPRARAGGRHVLVLP
16


 


739
DGSRSTDDQRIVSYL
16


 


934
HLWMENLIQRYIWDG
16


 


68
DLAWWFEGRCYLVSC
15


 


156
YSDDYRELEKDLLQP
15


 


265
GKEVLMPSHSLPPAS
15


 


357
FVAPAPPVETTYNYE
15


 


436
AVVSPQLQELTLPLT
15


 


466
VSYHWEEINGPFIEE
15


 


555
DDHQIVLYEWSLGPG
15


 


811
DPRKSGLVELTLQVG
15


 


8
LSSLLLLVTIAGCAR
14


 


9
SSLLLLVTIAGCARK
14


 


89
EPKKMGPIRSYLTFV
14


 


226
APKLPERSVLLPLPT
14


 


231
ERSVLLPLPTTPSSG
14


 


232
RSVLLPLPTTPSSGE
14


 


449
LTSALIDGSQSTDDT
14


 


556
DHQIVLYEWSLGPGS
14


 


572
GKHWMQGVQTPYLH
14


 


771
DHSVALQLTNLVEGV
14


 


806
VEVQPDPRKSGLVEL
14


 


843
VLLNVLDSDIKVQKI
14


 


1015
IKHRSTEHNSSLMVS
14











V2-HLA-DRB1-0301-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













5
DWGLEEMSEYADDYR
19



 


10
EMSEYADDYRELEKD
18


 


15
ADDYRELEKDLLQPS
18


 


11
MSEYADDYRELEKDL
17


 


14
YADDYRELEKDLLQP
15


 


8
LEEMSEYADDYRELE
11


 


3
GKDWGLEEMSEYADD
10


 


7
GLEEMSEYADDYREL
9











V3-HLA-DRB1-0301-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













1
MTRLGWPSPCCARKQ
11



 


10
CCARKQCSEGRTYSN
8


 


6
WPSPCCARKQCSEGR
7


 


7
PSPCCARKQCSEGRT
7


 


5
GWPSPCCARKQCSEG
6











V5-HLA-DRB1-0301-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













3
WGDFPEDIRKDLTFL
25



 


21
PEDIRKDLTFLGKDW
23


 


14
LTFLGKDWGLEEMSE
21


 


11
RKDLTFLGKDWGLEE
18


 


13
DLTFLGKDWGLEEMS
11


















TABLE XLVIII





Pos
123456789012345
score
















V1-HLA-DR1-0401-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













68
DLAWWFEGRCYLVSC
28



 


365
ETTYNYEWNLISHPT
28


 


751
SYLWIRDGQSPAAGD
28


 


90
PKKMGPIRSYLTFVL
26


 


97
RSYLTFVLRPVQRPA
26


 


101
TFVLRPVQRPAQLLD
26


 


232
RSVLLPLPTTPSSGE
26


 


282
LSSVTVEKSPVLTVT
26


 


421
NVTVKPARRVNLPPV
26


 


574
HVVMQGVQTPYLHLS
26


 


610
TAVVTVIVQPENNRP
26


 


633
KELIFPVESATLDGS
26


 


725
GRHVLVLPNNSITLD
26


 


733
NNSITLDGSRSTDDQ
26


 


779
TNLVEGVYTFHLRVT
26


 


842
AVLLNVLDSDIKVQK
26


 


899
DFLLFKVLRVDTAGC
26


 


934
HLWMENLIQRYIWDG
26


 


28
GRTYSNAVISPNLET
22


 


49
SHTFPVVDCTAACCD
22


 


96
IRSYLTFVLRPVQRP
22


 


153
MSEYSDDYRELEKDL
22


 


157
SDDYRELEKDLLQPS
22


 


369
NYEWNLISHPTDYQG
22


 


402
LYVFKVTVSSENAFG
22


 


416
GEGFVNVTVKPARRV
22


 


467
SYHWEEINGPFIEEK
22


 


474
NGPFIEEKTSVDSPV
22


 


524
AVDYPPVANAGPNHT
22


 


657
FYHWEHVRGPSAVEM
22


 


749
IVSYLWIRDGQSPAA
22


 


874
RPPFKVLKAAEVARN
22


 


897
KADFLLFKVLRVDTA
22


 


900
FLLFKVLRVDTAGCL
22


 


943
RYIWDGESNCEWSIF
22


 


951
NCEWSIFYVTVLAFT
22


 


955
SIFYVTVLAFTLIVL
22


 


992
KTKYTILDNMDEQER
22


 


5
TGVLSSLLLLVTIAG
20


 


8
LSSLLLLVTIAGCAR
20


 


12
LLLVTIAGCARKQCS
20


 


42
TTRIMRVSHTFPVVD
20


 


43
TRIMRVSHTFPVVDC
20


 


76
RCYLVSCPHKENCEP
20


 


93
MGPIRSYLTFVLRPV
20


 


100
LTFVLRPVQRPAQLL
20


 


126
PSGIWGDSPEDIRKD
20


 


160
YRELEKDLLQPSGKQ
20


 


202
SPAVPAETQQDPELH
20


 


212
DPELHYLNESASTPA
20


 


215
LHYLNESASTPAPKL
20


 


233
SVLLPLPTTPSSGEV
20


 


245
GEVLEKEKASQLQEQ
20


 


253
ASQLQEQSSNSSGKE
20


 


272
SHSLPPASLELSSVT
20


 


279
SLELSSVTVEKSPVL
20


 


289
KSPVLTVTPGSTEHS
20


 


292
VLTVTPGSTEHSIPT
20


 


301
EHSIPTPPTSAAPSE
20


 


334
ELTVSAGDNLIITLP
20


 


341
DNLIITLPDNEVELK
20


 


355
KAFVAPAPPVETTYN
20


 


371
EWNLISHPTDYQGEI
20


 


399
SVGLYVFKVTVSSEN
20


 


432
LPPVAVVSPQLQELT
20


 


435
VAVVSPQLQELTLPL
20


 


439
SPQLQELTLPLTSAL
20


 


442
LQELTLPLTSALIDG
20


 


446
TLPLTSALIDGSQST
20


 


485
DSPVLRLSNLDPGNY
20


 


500
SFRLTVTDSDGATNS
20


 


527
YPPVANAGPNHTITL
20


 


536
NHTITLPQNSITLNG
20


 


543
QNSITLNGNQSSDDH
20


 


557
HQIVLYEWSLGPGSE
20


 


596
TFQLKVTDSSRQQST
20


 


598
QLKVTDSSRQQSTAV
20


 


614
TVIVQPENNRPPVAV
20


 


636
IFPVESATLDGSSSS
20


 


666
PSAVEMENIDKAIAT
20


 


668
AVEMENIDKAIATVT
20


 


671
MENIDKAIATVTGLQ
20


 


675
DKAIATVTGLQVGTY
20


 


698
QQGLSSTSTLTVAVK
20


 


704
TSTLTVAVKKENNSP
20


 


708
TVAVKKENNSPPRAR
20


 


726
RHVLVLPNNSITLDG
20


 


727
HVLVLPNNSITLDGS
20


 


752
YLWIRDGQSPAAGDV
20


 


764
GDVIDGSDHSVALQL
20


 


711
DHSVALQLTNLVEGV
20


 


782
VEGVYTFHLRVTDSQ
20


 


787
TFHLRVTDSQGASDT
20


 


805
TVEVQPDPRKSGLVE
20


 


815
SGLVELTLQVGVGQL
20


 


823
QVGVGQLTEQRKDTL
20


 


835
DTLVRQLAVLLNVLD
20


 


838
VRQLAVLLNVLDSDI
20


 


841
LAVLLNVLDSDIKVQ
20


 


845
LNVLDSDIKVQKIRA
20


 


860
HSDLSTVIVFYVQSR
20


 


865
TVIVFYVQSRPPFKV
20


 


877
FKVLKAAEVARNLHM
20


 


882
AAEVARNLHMRLSKE
20


 


890
HMRLSKEKADFLLFK
20


 


902
LFKVLRVDTAGCLLK
20


 


903
FKVLRVDTAGCLLKC
20


 


905
VLRVDTAGCLLKCSG
20


 


956
IFYVTVLAFTLIVLT
20


 


958
YVTVLAFTLIVLTGG
20


 


963
AFTLIVLTGGFTWLC
20


 


998
LDNMDEQERMELRPK
20


 


1024
SSLMVSESEFDSDQD
20


 


1050
NPKVSMNGSIRNGAS
20


 


1052
KVSMNGSIRNGASFS
20


 


1
MAPPTGVLSSLLLLV
18


 


2
APPTGVLSSLLLLVT
18


 


21
ARKQCSEGRTYSNAV
18


 


29
RTYSNAVISPNLETT
18


 


34
AVISPNLETTRIMRV
18


 


35
VISPNLETTRIMRVS
18


 


58
TAACCDLSSCDLAWW
18


 


130
WGDSPEDIRKDLPFL
18


 


146
KDWGLEEMSEYSDDY
18


 


169
QPSGKQEPRGSAEYT
18


 


188
LPGSEGAFNSSVGDS
18


 


208
ETQQDPELHYLNESA
18


 


225
PAPKLPERSVLLPLP
18


 


252
KASQLQEQSSNSSGK
18


 


275
LPPASLELSSVTVEK
18


 


276
PPASLELSSVTVEKS
18


 


295
VTPGSTEHSIPTPPT
18


 


298
GSTEHSIPTPPTSAA
18


 


306
TPPTSAAPSESTPSE
18


 


322
PISPTTAPRTVKELT
18


 


328
APRTVKELTVSAGDN
18


 


358
VAPAPPVETTYNYEW
18


 


368
YNYEWNLISHPTDYQ
18


 


374
LISHPTDYQGEIKQG
18


 


379
TDYQGEIKQGHKQTL
18


 


389
HKQTLNLSQLSVGLY
18


 


403
YVFKVTVSSENAFGE
18


 


413
NAFGEGFVNVTVKPA
18


 


431
NLPPVAVVSPQLQEL
18


 


438
VSPQLQELTLPLTSA
18


 


443
QELTLPLTSALIDGS
18


 


449
LTSALIDGSQSTDDT
18


 


455
DGSQSTDDTEIVSYH
18


 


478
IEEKTSVDSPVLRLS
18


 


482
TSVDSPVLRLSNLDP
18


 


505
VTDSDGATNSTTAAL
18


 


514
STTAALIVNNAVDYP
18


 


535
PNHTITLPQNSITLN
18


 


549
NGNQSSDDHQIVLYE
18


 


550
GNQSSDDHQIVLYEW
18


 


570
SEGKHVVMQGVQTPY
18


 


588
SAMQEGDYTFQLKVT
18


 


597
FQLKVTDSSRQQSTA
18


 


606
RQQSTAVVTVIVQPE
18


 


639
VESATLDGSSSSDDH
18


 


645
DGSSSSDDHGIVFYH
18


 


691
FRLTVKDQQGLSSTS
18


 


695
VKDQQGLSSTSTLTV
18


 


739
DGSRSTDDQRIVSYL
18


 


740
GSRSTDDQRIVSYLW
18


 


762
AAGDVIDGSDHSVAL
18


 


765
DVIDGSDHSVALQLT
18


 


769
GSDHSVALQLTNLVE
18


 


788
FHLRVTDSQGASDTD
18


 


813
RKSGLVELTLQVGVG
18


 


825
GVGQLTEQRKDTLVR
18


 


831
EQRKDTLVRQLAVLL
18


 


832
QRKDTLVRQLAVLLN
18


 


853
KVQKIRAHSOLSTVI
18


 


856
KIRAHSDLSTVIVFY
18


 


857
IRAHSDLSTVIVFYV
18


 


880
LKAAEVARNLHMRLS
18


 


957
FYVTVLAFTLIVLTG
18


 


996
TILDNMDEQERMELR
18


 


1009
LRPKYGIKHRSTEHN
18


 


1015
IKHRSTEHNSSLMVS
18


 


1034
DSDQDTIFSREKMER
18


 


1035
SDQDTIFSREKMERG
17


 


1053
VSMNGSIRNGASFSY
18


 


400
VGLYVFKVTVSSENA
17


 


594
DYTFQLKVTDSSRQQ
17


 


785
VYTFHLRVTDSQGAS
17


 


69
LAWWFEGRGYLVSCP
16


 


145
GKDWGLEEMSEYSDD
16


 


182
YTDWGLLPGSEGAFN
16


 


214
ELHYLNESASTPAPK
16


 


378
PTDYQGEIKQGHKQT
16


 


412
ENAFGEGFVNVTVKP
16


 


465
IVSYHWEEINGPFIE
16


 


498
NYSFRLTVTDSDGAT
16


 


559
IVLYEWSLGPGSEGK
16


 


581
QTPYLHLSAMQEGDY
16


 


634
ELIFPVESATLDGSS
16


 


654
GIVFYHWEHVRGPSA
16


 


655
IVFYHWEHVRGPSAV
16


 


688
TYHFRLTVKDQQGLS
16


 


783
EGVYTFHLRVTDSQG
16


 


866
VIVFYVQSRPPFKVL
16


 


867
IVFYVQSRPPFKVLK
16


 


941
IQRYIWDGESNCEWS
16


 


954
WSIFYVTVLAFTLIV
16


 


961
VLAFTLIVLTGGFTW
16


 


970
TGGFTWLCICCCKRQ
16


 


972
GFTWLCICCCKRQKR
16


 


1030
ESEFDSDQDTIFSRE
16


 


1038
DTIFSREKMERGNPK
16


 


475
GPFIEEKTSVDSPVL
15


 


690
HFRLTVKDQQGLSST
15


 


886
ARNLHMRLSKEKADF
15


 


1012
KYGIKHRSTEHNSSL
15


 


4
PTGVLSSLLLLVTIA
14


 


9
SSLLLLVTIAGCARK
14


 


10
SLLLLVTIAGCARKQ
14


 


11
LLLLVTIAGCARKQC
14


 


14
LVTIAGCARKQCSEG
14


 


32
SNAVISPNLETTRIM
14


 


37
SPNLETTRIMRVSHT
14


 


104
LRPVQRPAQLLDYGD
14


 


110
PAQLLDYGDMMLNRG
14


 


111
AQLLDYGDMMLNRGS
14


 


116
YGDMMLNRGSPSGIW
14


 


118
DMMLNRGSPSGIWGD
14


 


134
PEDIRKDLPFLGKDW
14


 


138
RKDLPFLGKDWGLEE
14


 


141
LPFLGKDWGLEEMSE
14


 


185
WGLLPGSEGAFNSSV
14


 


196
NSSVGDSPAVPAETQ
14


 


235
LLPLPTTPSSGEVLE
14


 


265
GKEVLMPSHSLPPAS
14


 


266
KEVLMPSHSLPPASL
14


 


267
EVLMPSHSLPPASLE
14


 


284
SVTVEKSPVLTVTPG
14


 


318
PSELPISPTTAPRTV
14


 


320
ELPISPTTAPRTVKE
14


 


329
PRTVKELTVSAGDNL
14


 


332
VKELTVSAGDNLIIT
14


 


342
NLIITLPDNEVELKA
14


 


344
IITLPDNEVELKAFV
14


 


351
EVELKAFVAPAPPVE
14


 


361
APPVETTYNYEWNLI
14


 


382
QGEIKQGHKQTLNLS
14


 


392
TLNLSQLSVGLYVFK
14


 


395
LSQLSVGLYVFKVTV
14


 


397
QLSVGLYVFKVTVSS
14


 


401
GLYVFKVTVSSENAF
14


 


406
KVTVSSENAFGEGFV
14


 


427
ARRVNLPPVAVVSPQ
14


 


429
RVNLPPVAVVSPQLQ
14


 


434
PVAVVSPQLQELTLP
14


 


450
TSALIDGSQSTDDTE
14


 


451
SALIDGSQSTDDTEI
14


 


462
DTEIVSYHWEEINGP
14


 


463
TEIVSYHWEEINGPF
14


 


470
WEEINGPFIEEKTSV
14


 


481
KTSVDSPVLRLSNLD
14


 


488
VLRLSNLDPGNYSFR
14


 


502
RLTVTDSDGATNSTT
14


 


518
ALIVNNAVDYPPVAN
14


 


522
NNAVDYPPVANAGPN
14


 


538
TITLPQNSITLNGNQ
14


 


545
SITLNGNQSSDDHQI
14


 


573
KHVVMQGVQTPYLHL
14


 


577
MQGVQTPYLHLSAMQ
14


 


587
LSAMQEGDYTFQLKV
14


 


609
STAVVTVIVQPENNR
14


 


613
VTVIVQPENNRPPVA
14


 


623
RPPVAVAGPDKELIF
14


 


625
PVAVAGPDKELIFPV
14


 


632
DKELIFPVESATLDG
14


 


641
SATLDGSSSSDDHGI
14


 


652
DHGIVFYHWEHVRGP
14


 


660
WEHVRGPSAVEMENI
14


 


678
IATVTGLQVGTYHFR
14


 


683
GLQVGTYHFRLTVKD
14


 


692
RLTVKDQQGLSSTST
14


 


735
SITLDGSRSTDDQRI
14


 


747
QRIVSYLWIRDGQSP
14


 


763
AGDVIDGSDHSVALQ
14


 


775
ALQLTNLVEGVYTFH
14


 


803
TATVEVQPDPRKSGL
14


 


814
KSGLVELTLQVGVGQ
14


 


817
LVELTLQVGVGQLTE
14


 


819
ELTLQVGVGQLTEQR
14


 


821
TLQVGVGQLTEQRKD
14


 


826
VGQLTEQRKDTLVRQ
14


 


834
KDTLVRQLAVLLNVL
14


 


844
LLNVLDSDIKVQKIR
14


 


851
DIKVQKIRAHSDLST
14


 


854
VQKIRAHSDLSTVIV
14


 


863
LSTVIVFYVQSRPPF
14


 


864
STVIVFYVQSRPPFK
14


 


876
PFKVLKAAEVARNLH
14


 


912
GCLLKCSGHGHCDPL
14


 


928
KRCICSHLWMENLIQ
14


 


932
CSHLWMENLIQRYIW
14


 


942
QRYIWDGESNCEWSI
14


 


953
EWSIFYVTVLAFTLI
14


 


959
VTVLAFTLIVLTGGF
14


 


965
TLIVLTGGFTWLCIC
14


 


966
LIVLTGGFTWLCICC
14


 


973
FTWLCICCCKRQKRT
14


 


975
WLCICCCKRQKRTKI
14


 


1023
NSSLMVSESEFDSDQ
14


 


1043
REKMERGNPKVSMNG
14


 


1056
NGSIRNGASFSYCSK
14











V2-HLA-DR1-0401-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













11
MSEYADDYRELEKDL
22



 


15
ADDYRELEKDLLQPS
22


 


4
KDWGLEEMSEYADDY
18


 


3
GKDWGLEEMSEYADD
16


 


10
EMSEYADDYRELEKD
12


 


14
YADDYRELEKDLLQP
12











V3-HLA-DR1-0401-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













3
RLGWPSPCCARKQCS
16



 


1
MTRLGWPSPCCARKQ
14


 


6
WPSPCCARKQCSEGR
12











V5-HLA-DR1-0401-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













7
PEDIRKDLTFLGKDW
20



 


3
WGDSPEDIRKDLTFL
18


 


11
RKDLTFLGKDWGLEE
14


 


14
LTFLGKDWGLEEMSE
14


 


4
GDSPEDIRKDLTFLG
12


 


8
EDIRKDLTFLGKDWG
12


















TABLE XLIX





Pos
123456789012345
score
















V1-HLA-DRB1-1101-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













668
AVEMENIDKAIATVT
27



 


42
TTRIMRVSHTFPVVD
26


 


138
RKDLPFLGKDWGLEE
26


 


654
GIVFYHWEHVRGPSA
26


 


961
VLAFTLIVLTGGFTW
26


 


157
SDDYRELEKDLLQPS
25


 


113
LLDYGDMMLNRGSPS
24


 


369
NYEWNLISHPTDYQG
24


 


49
SHTFPWDCTAACCD
23


 


97
RSYLTFVLRPVQRPA
23


 


831
EQRKDTLVRQLAVLL
23


 


900
FLLFKVLRVDTAGCL
23


 


182
YTDWGLLPGSEGAFN
22


 


242
PSSGEVLEKEKASQL
22


 


416
GEGFVNVTVKPARRV
22


 


524
AVDYPPVANAGPNHT
22


 


598
QLKVTDSSRQQSTAV
22


 


657
FYHWEHVRGPSAVEM
22


 


749
IVSYLWIRDGQSPAA
22


 


848
LDSDIKVQKIRAHSD
22


 


131
GDSPEDIRKDLPFLG
21


 


265
GKEVLMPSHSLPPAS
21


 


764
GDVIDGSDHSVALQL
21


 


887
RNLHMRLSKEKADFL
21


 


899
DFLLFKVLRVDTAGC
21


 


8
LSSLLLLVTIAGCAR
20


 


101
TFVLRPVQRPAQLLD
20


 


115
DYGDMMLNRGSPSGI
20


 


165
KDLLQPSGKQEPRGS
20


 


592
EGDYTFQLKVTDSSR
20


 


688
TYHFRLTVKDQQGLS
20


 


783
EGWTFHLRVTDSQG
20


 


805
TVEVQPDPRKSGLVE
20


 


865
TVIVFYVQSRPPFKV
20


 


908
VDTAGCLLKCSGHGH
20


 


1040
IFSREKMERGNPKVS
20


 


1052
KVSMNGSIRNGASFS
20


 


153
MSEYSDDYRELEKDL
19


 


279
SLELSSVTVEKSPVL
19


 


704
TSTLTVAVKKENNSP
19


 


747
QRIVSYLWIRDGQSP
19


 


814
KSGLVELTLQVGVGQ
19


 


866
VIVFYVQSRPPFKVL
19


 


68
DLAWWFEGRCYLVSC
18


 


99
YLTFVLRPVQRPAQL
18


 


179
SAEYTDWGLLPGSEG
18


 


192
EGAFNSSVGDSPAVP
18


 


212
DPELHYLNESASTPA
18


 


232
RSVLLPLPTTPSSGE
18


 


329
PRTVKELTVSAGDNL
18


 


378
PTDYQGEIKQGHKQT
18


 


400
VGLYVFKVTVSSENA
18


 


429
RVNLPPVAVVSPQLQ
18


 


485
DSPVLRLSNLDPGNY
18


 


594
DYTFQLKVTDSSRQQ
18


 


970
TGGFTWLCICCCKRQ
18


 


992
KTKYTILDNMDEQER
18


 


1010
RPKYGIKHRSTEHNS
18


 


1038
DTIFSREKMERGNPK
18


 


70
AWWFEGRCYLVSCPH
17


 


365
ETTYNYEWNLISHPT
17


 


417
EGFVNVTVKPARRVN
17


 


610
TAVVTVIVQPENNRP
17


 


655
IVFYHWEHVRGPSAV
17


 


740
GSRSTDDQRIVSYLW
17


 


775
ALQLTNLVEGVYTFH
17


 


874
RPPFKVLKAAEVARN
17


 


972
GFTWLCICCCKRQKR
17


 


39
NLETTRIMRVSHTFP
16


 


214
ELHYLNESASTPAPK
16


 


367
TYNYEWNLISHPTDY
16


 


465
IVSYHWEEINGPFIE
16


 


467
SYHWEEINGPFIEEK
16


 


481
KTSVDSPVLRLSNLD
16


 


559
IVLYEWSLGPGSEGK
16


 


561
LYEWSLGPGSEGKHV
16


 


578
QGVQTPYLHLSAMQE
16


 


581
QTPYLHLSAMQEGDY
16


 


656
VFYHWEHVRGPSAVE
16


 


712
KKENNSPPRARAGGR
16


 


751
SYLWIRDGQSPAAGD
16


 


826
VGQLTEQRKDTLVRQ
16


 


864
STVIVFYVQSRPPFK
16


 


882
AAEVARNLHMRLSKE
16


 


896
EKADFLLFKVLRVDT
16


 


955
SIFYVTVLAFTLIVL
16


 


956
IFYVTVLAFTLIVLT
16


 


983
RQKRTKIRKKTKYTI
16


 


1008
ELRPKYGIKHRSTEH
16


 


48
VSHTFPVVDCTAACC
15


 


100
LTFVLRPVQRPAQLL
15


 


294
TVTPGSTEHSIPTPP
15


 


500
SFRLTVTDSDGATNS
15


 


625
PVAVAGPDKELIFPV
15


 


873
SRPPFKVLKAAEVAR
15


 


879
VLKAAEVARNLHMRL
15


 


920
HGHCDPLTKRCICSH
15


 


935
LWMENLIQRYIWDGE
15


 


975
WLCICCCKRQKRTKI
15


 


1009
LRPKYGIKHRSTEHN
15


 


1037
QDTIFSREKMERGNP
15


 


14
LVTIAGCARKQCSEG
14


 


15
VTIAGCARKQCSEGR
14


 


21
ARKQCSEGRTYSNAV
14


 


76
RCYLVSCPHKENCEP
14


 


77
CYLVSCPHKENCEPK
14


 


83
PHKENCEPKKMGPIR
14


 


84
HKENCEPKKMGPIRS
14


 


87
NCEPKKMGPIRSYLT
14


 


169
QPSGKQEPRGSAEYT
14


 


244
SGEVLEKEKASQLQE
14


 


281
ELSSVTVEKSPVLTV
14


 


292
VLTVTPGSTEHSIPT
14


 


351
EVELKAFVAPAPPVE
14


 


382
QGEIKQGHKQTLNLS
14


 


398
LSVGLYVFKVTVSSE
14


 


399
SVGLYVFKVTVSSEN
14


 


421
NVTVKPARRVNLPPV
14


 


432
LPPVAVVSPQLQELT
14


 


446
TLPLTSALIDGSQST
14


 


482
TSVDSPVLRLSNLDP
14


 


518
ALIVNNAVDYPPVAN
14


 


543
QNSITLNGNQSSDDH
14


 


613
VTVIVQPENNRPPVA
14


 


678
IATVTGLQVGTYHFR
14


 


705
STLTVAVKKENNSPP
14


 


714
ENNSPPRARAGGRHV
14


 


732
PNNSITLDGSRSTDD
14


 


823
QVGVGQLTEQRKDTL
14


 


838
VRQLAVLLNVLDSDI
14


 


842
AVLLNVLDSDIKVQK
14


 


845
LNVLDSDIKVQKIRA
14


 


850
SDIKVQKIRAHSDLS
14


 


883
AEVARNLHMRLSKEK
14


 


912
GCLLKCSGHGHCDPL
14


 


914
LLKCSGHGHCDPLTK
14


 


986
RTKIRKKTKYTILDN
14


 


998
LDNMDEQERMELRPK
14


 


1004
QERMELRPKYGIKHR
14


 


1014
GIKHRSTEHNSSLMV
14


 


5
TGVLSSLLLLVTIAG
13


 


7
VLSSLLLLVTIAGCA
13


 


10
SLLLLVTIAGCARKQ
13


 


90
PKKMGPIRSYLTFVL
13


 


96
IRSYLTFVLRPVQRP
13


 


114
LDYGDMMLNRGSPSG
13


 


134
PEDIRKDLPFLGKDW
13


 


226
APKLPERSVLLPLPT
13


 


228
KLPERSVLLPLPTTP
13


 


263
SSGKEVLMPSHSLPP
13


 


272
SHSLPPASLELSSVT
13


 


287
VEKSPVLTVTPGSTE
13


 


337
VSAGDNLIITLPDNE
13


 


348
PDNEVELKAFVAPAP
13


 


390
KQTLNLSQLSVGLYV
13


 


392
TLNLSQLSVGLYVFK
13


 


401
GLYVFKVTVSSENAF
13


 


402
LYVFKVTVSSENAFG
13


 


439
SPQLQELTLPLTSAL
13


 


497
GNYSFRLTVTDSDGA
13


 


556
DHQIVLYEWSLGPGS
13


 


577
MQGVQTPYLHLSAMQ
13


 


593
GDYTFQLKVTDSSRQ
13


 


614
TVIVQPENNRPPVAV
13


 


633
KELIFPVESATLDGS
13


 


666
PSAVEMENIDKAIAT
13


 


706
TLTVAVKKENNSPPR
13


 


725
GRHVLVLPNNSITLD
13


 


784
GVYTFHLRVTDSQGA
13


 


787
TFHLRVTDSQGASDT
13


 


816
GLVELTLQVGVGQLT
13


 


835
DTLVRQLAVLLNVLD
13


 


934
HLWMENLIQRYIWDG
13


 


953
EWSIFYVTVLAFTLI
13


 


954
WSIFYVTVLAFTLIV
13


 


960
TVLAFTLIVLTGGFT
13


 


963
AFTLIVLTGGFTWLC
13


 


1043
REKMERGNPKVSMNG
13











V2-HLA-DRB1-1101-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 5; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













15
ADDYRELEKDLLQPS
25



 


11
MSEYADDYRELEKDL
19


 


5
DWGLEEMSEYADDYR
12











V3-HLA-DRB1-1101-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 7; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













6
WPSPCCARKQCSEGR
14



 


1
MTRLGWPSPCCARKQ
12


 


3
RLGWPSPCCARKQCS
12


 


5
GWPSPCCARKQCSEG
8


 


8
SPCCARKQCSEGRTY
6











V5-HLA-DRB1-1101-15mers-254P1D6B



Each peptide is a portion of


SEQ ID NO: 11; each start


position is specified, the


length of peptide is 15 amino


acids, and the end position


for each peptide is the start


position plus fourteen.













11
RKDLTFLGKDWGLEE
28



 


4
GDSPEDIRKDLTFLG
15


 


7
PEDIRKDLTFLGKDW
13
















TABLE L







Protein Characteristics of 254P1D6B











Bioinformatic Program
URL
Outcome





ORF
ORF finder

3216 bp


Protein length


1072 aa


Transmembrane
TM Pred
http://www.ch.embnet.org/
TM Helix AA 954-981


region
HMMTop
http://www.enzim.hu/hmmtop/
TM Helix AA 956-980



Sosui
http://www.genome.ad.jp/SOSui/
TM Helix AA 957-979



TMHMM
http://www.cbs.dtu.dk/services/TMHMM
TM Helix AA 956-978


Signal Peptide
Signal P
http://www.cbs.dtu.dk/services/SignalP/
Yes signal peptide


pI
pI/MW tool
http://www.expasy.ch/tools/
pI 5.34


Molecular weight
pI/MW tool
http://www.expasy.ch/tools/
1.17





  46% Plasma Membrane





  10% endoplasmic


Localization
PSORT
http://psort.nibb.ac.jp/
reticulum





33.3% Golgi





33.3% Endoplasmic





reticulum





22.2% Plasma Membrane





11.1% extracellular,



PSORT II
http://psort.nibb.ac.jp/
including cell wall





TYA transposon protein





PKD


Motifs
Blocks
http://www.blocks.fhcrc.org/
Purothionin signature



Repeats
http://dove.embl-heidelberg.de/
No Repeats
















TABLE LI







Exon compositions of 254P1D6B












Exon No.
Start position
End position
Length
















1
1
406
406



2
407
566
160



3
567
1312
746



4
1313
1505
193



5
1506
1604
99



6
1605
1702
98



7
1703
1790
88



8
1791
1883
93



9
1884
2016
133



10
2017
2245
229



11
2246
2369
124



12
2370
2502
133



13
2503
2651
149



14
2652
2803
152



15
2804
2942
139



16
2943
3102
160



17
3103
3245
143



18
3246
3368
123



19
3369
3459
91



20
3460
3551
92



21
3552
6791
3240

















TABLE LII





Nucleotide sequence of transcript variant 254P1D6B v.3 (SEQ ID NO: 269)







 














gctgccgcgg
gcggtgggcg
gggatccccc
gggggtgcaa
ccttgctcca
cctgtgctgc
60



 


cctcggcggg
cctggctggc
cccgcgcaga
gcggcggcgg
cgctcgctgt
cactgccgga
120


 


ggtgagagcg
cagcagtagc
ttcagcctgt
cttgggcttg
gtccagattc
gctcctctgg
180


 


ggctacgtcc
cggggaagag
gaagcgagga
ttttgctggg
gtggggctgt
acctcttaac
240


 


agcaggtgcg
cgcgcgaggg
tgtgaacgtg
tgtgtgtgtg
tgtgtctgtg
tgtgtgtgtg
300


 


taagacctgc
gatgacgacg
aggaggaaca
agtgggacgg
cgagtgatgc
tcagggccag
360


 


cagcaacgca
tggggcgagc
ttcagtgtcg
ccagcagtga
ccacaggtac
ggtatctact
420


 


tcccagagcg
cctggccgag
aaataggaaa
gagggcagcc
agtaggcagg
ccaataccca
480


 


acaaaagtag
aatcgagacg
ccctgagttc
agaagttctt
gaggccaaat
ctggctccta
540


 


aaaaacatca
aaggaagctt
gcaccaaact
ctcttcaggg
ccgcctcaga
agcctgccat
600


 


cacccactgt
gtggtgcaca
atggcgcccc
ccacaggtgt
gctctcttca
ttgctgctgc
660


 


tggtgacaat
tgcagtttgc
ttatggtgga
tgcactcatg
gcaaaaaaat
cactggtgag
720


 


catcatttaa
gaagacccat
gactagactg
ggctggccga
gcccatgttg
tgcccgtaag
780


 


cagtgcagcg
aggggaggac
atattccaat
gcagtcattt
cacctaactt
ggaaaccacc
840


 


agaatcatgc
gggtgtctca
caccttccct
gtcgtagact
gcacggccgc
ttgctgtgac
900


 


ctgtccagct
gtgacctggc
ctggtggttc
gagggccgct
gctacctggt
gagctgccc
960


 


cacaaagaga
actgtgagcc
caagaagatg
ggccccatca
ggtcttatct
cacttttgtg
1020


 


ctccggcctg
ttcagaggcc
tgcacagctg
ctggactatg
gggacatgat
gctgaacagg
1080


 


ggctccccct
cggggatctg
gggggactca
cctgaggata
tcagaaagga
cttgcccttt
1140


 


ctaggcaaag
attggggcct
agaggagatg
tctgagtact
cagatgacta
ccgggagctg
1200


 


gagaaggacc
tcttgcaacc
cagtggcaag
caggagccca
gagggagtgc
cgagtacacg
1260


 


gactggggcc
tactgccggg
cagcgagggg
gccttcaact
cctctgttgg
agacagtcct
1320


 


gcggtgccag
cggagacgca
gcaggaccct
gagctccatt
acctgaatga
gtcggcttca
1380


 


acccctgccc
caaaactccc
tgagagaagt
gtgttgcttc
ccttgccgac
tactccatct
1440


 


tcaggagagg
tgttggagaa
agaaaaggct
tctcagctcc
aggaacaatc
cagcaacagc
1500


 


tctggaaaag
aggttctaat
gccttcccat
agtcttcctc
cggcaagcct
ggagctcagc
1560


 


tcagtcaccg
tggagaaaag
cccagtgctc
acagtcaccc
cggggagtac
agagcacagc
1620


 


atcccaacac
ctcccactag
cgcagccccc
tctgagtcca
ccccatctga
gctacccata
1680


 


tctcctacca
ctgctcccag
gacagtgaaa
gaacttacgg
tatcggctgg
agataaccta
1740


 


attataactt
tacccgacaa
tgaagttgaa
ctgaaggcct
ttgttgcgcc
agcgccacct
1800


 


gtagaaacaa
cctacaacta
tgaatggaat
ttaataagcc
accccacaga
ctaccaaggt
1860


 


gaaataaaac
aaggacacaa
gcaaactctt
aacctctctc
aattgtccgt
cggactttat
1920


 


gtcttcaaag
tcactgtttc
tagtgaaaac
gcctttggag
aaggatttgt
caatgtcact
1980


 


gttaagcctg
ccagaagagt
caacctgcca
cctgtagcag
ttgtttctcc
ccaactgcaa
2040


 


gagctcactt
tgcctttgac
gtcagccctc
attgatggca
gccaaagtac
agatgatact
2100


 


gaaatagtga
gttatcattg
ggaagaaata
aacgggccct
tcatagaaga
gaagacttca
2160


 


gttgactctc
ccgtcttacg
cttgtctaac
cttgatcctg
gtaactatag
tttcaggttg
2220


 


actgttacag
actcggacgg
aqccactaac
tctacaactg
cagccctaat
agtgaacaat
2280


 


gctgtggact
acccaccagt
tgctaatgca
ggaccaaatc
acaccataac
tttgccccaa
2340


 


aactccatca
ctttgaatgg
aaaccagagc
agtgacgatc
accagattgt
cctctatgag
2400


 


tggtccctgg
gtcctgggag
tgagggcaaa
catgtggtca
tgcagggagt
acagacgcca
2460


 


taccttcatt
tatctgcaat
gcaggaagga
gattatacat
ttcagctgaa
ggtgacagat
2520


 


tcttcaaggc
aacagtctac
tgctgtggtg
actgtgattg
tccagcctga
aaacaataga
2580


 


cctccagtgg
ctgtggccgg
ccctgataaa
gagctgatct
tcccagtgga
aagtgctacc
2640


 


ctggatggga
gcagcagcag
cgatgaccac
ggcattgtct
tctaccactg
ggagcacgtc
2700


 


agaggcccca
gtgcagtgga
gatggaaaat
attgacaaag
caatagccac
tgtgactggt
2760


 


ctccaggtgg
ggacctacca
cttccgtttg
acagtgaaag
accagcaggg
actgagcagc
2820


 


acgtccaccc
tcactgtggc
tgtgaagaag
gaaaataata
gtcctcccag
agcccgggct
2880


 


ggtggcagac
atgttcttgt
gcttcccaat
aattccatta
ctttggatgg
ttcaaggtct
2940


 


actgatgacc
aaagaattgt
gtcctatctg
tggatccggg
atggccagag
tccagcagct
3000


 


ggagatgtca
tcgatggctc
tgaccacagt
gtggctctgc
agcttacgaa
tctggtggag
3060


 


ggggtgtaca
ctttccactt
gcgagtcacc
gacagtcagg
gggcctcgga
cacagacact
3120


 


gccactgtgg
aagtgcagcc
agaccctagg
aagagtggcc
tggtggagct
gaccctgcag
3180


 


gttggtgttg
ggcagctgac
agagcagcgg
aaggacaccc
ttgtgaggca
gctggctgtg
3240


 


ctgctgaacg
tgctggactc
ggacattaag
gtccagaaga
ttcgggccca
ctcggatctc
3300


 


agcaccgtga
ttgtgtttta
tgtacagagc
aggccgcctt
tcaaggttct
caaagctgct
3360


 


gaagtggccc
gaaatctgca
catgcggctc
tcaaaggaga
aggctgactt
cttgcttttc
3420


 


aaggtcttga
gggttgatac
agcaggttgc
cttctgaagt
gttctggcca
tggtcactgc
3480


 


gaccccctca
caaagcgctg
catttgctct
cacttatgga
tggagaacct
tatacagcgt
3540


 


tatatctggg
atggagagag
caactgtgag
tggagtatat
tctatgtgac
agtgttggct
3600


 


tttactctta
ttgtgctaac
aggaggtttc
acttggcttt
gcatctgctg
ctgcaaaaga
3660


 


caaaaaagga
ctaaaatcag
gaaaaaaaca
aagtacacca
tcctggataa
catggatgaa
3720


 


caggaaagaa
tggaactgag
gcccaaatat
ggtatcaagc
accgaagcac
agagcacaac
3780


 


tccagcctga
tggtatccga
gtctgagttt
gacagtgacc
aggacacaat
cttcagccga
3840


 


gaaaagatgg
agagagggaa
tccaaaggtt
tccatgaatg
gttccatcag
aaatggagct
3900


 


tccttcagtt
attgctcaaa
ggacagataa
tggcgcagtt
cattgtaaag
tggaaggacc
3960


 


ccttgaatcc
aagaccagtc
agtgggagtt
acagcacaaa
acccactctt
ttagaatagt
4020


 


tcattgacct
tcttccccag
tgggttagat
gtgtatcccc
acgtactaaa
agaccggttt
4080


 


ttgaaggcac
aaaacaaaaa
ctttgctctt
ttaactgaga
tgcttgttaa
tagaaataaa
4140


 


ggctgggtaa
aactctaagg
tatatactta
aaagagtttt
gagtttttgt
agctggcaca
4200


 


atctcatatt
aaagatgaac
aacgatttct
atctgtagaa
ccttagagaa
ggtgaatgaa
4260


 


acaaggtttt
aaaaagggat
gatttctgtc
ttagccgctg
tgattgcctc
taaggaacag
4320


 


cattctaaac
acggtttctc
ttgtaggacc
tgcagtcaga
tggctgtgta
tgttaaaata
4380


 


gcttgtctaa
gaggcacggg
ccatctgtgg
aggtacggag
tcttgcatgt
agcaagcttt
4440


 


ctgtgctgac
ggcaacactc
gcacagtgcc
aagccctcct
ggtttttaat
tctgtgctat
4500


 


gtcaatggca
gttttcatct
ctctcaagaa
agcagctgtt
ggccattcaa
gagctaagga
4560


 


agaatcgtat
tctaaggact
gaggcaatag
aaaggggagg
aggagcttaa
tgccgtgcag
4620


 


gttgaaggta
gcattgtaac
attatctttt
ctttctctaa
gaaaaactac
actgactcct
4680


 


ctcggtgttg
tttagcagta
tagttctcta
atgtaaacgg
atccccagtt
tacattaaat
4740


 


gcaatagaag
tgattaattc
attaagcatt
tattatgttc
tgtaggctgt
gcgtttggac
4800


 


tgccatagat
agggataacg
actcagcaat
tgtgtatata
ttccaaaact
ctgaaataca
4860


 


gtcagtctta
acttggatgg
cgtggttatg
atactctggt
ccccgacagg
tactttccaa
4920


 


aataacttga
catagatgta
ttcacttcat
atgtttaaaa
atacatttaa
gtttttctac
4980


 


cgaataaatc
ttatttcaaa
catgaaagac
aattaaaaca
ttcccaccca
caaagcagta
5040


 


ctcccgagca
attaactgga
gttaattgta
gcctgctacg
ttgactggtt
cagggtagtt
5100


 


ccccatccac
ccttggtcct
gaggctggtg
gccttggtgg
tgcccttggc
attttttgtg
5160


 


ggaagattag
aatgagagat
agaaccagtg
ttgtggtacc
aagtgtgagc
acacctaaac
5220


 


aatatcctgt
tgcacaatgc
ttttttaaca
catgggaaaa
ctaggaatgc
attgctgatg
5280


 


aagaagcaag
gtatttaaac
accagggcag
gagtgccaga
gaaaatgttt
ccccatgggt
5340


 


tcttaaaaaa
aattcagctt
ttaggtgctt
ttgtcatctc
ccggagtatt
catcctcatg
5400


 


ggaccatctt
atttttactt
attgtaattt
actggggaaa
ggcagaacta
aaaagtgtgt
5460


 


cattttattt
ttaaaataat
tgctttgctt
atgcctacac
tttctgtata
actagccaat
5520


 


tcaatactgt
ctatagtgtt
agaaggaaaa
tgtgattttt
tttttttaac
cagtattgag
5580


 


cttcataagc
ctagaatctg
ccttatcagg
tgaccagggt
tatggttgtt
tgcatgcaaa
5640


 


tgtgaatttc
tggcataggg
gacagcagcc
caaatgtaaa
gtcatcgggc
gtaatgagga
5700


 


agaagggagt
gaacatttac
cgctttatgt
acataacata
tgcagtttac
atactcattt
5760


 


gatccttata
atcaaccttg
aagaggagat
actatcattc
ttatgttgca
gatagccctc
5820


 


tgaaggccca
gagaggttaa
gtaacttccc
agaggtcatg
gccaagaagt
agtggctcca
5880


 


agaactgaat
gcaaattttt
taaactgtag
agttctgctt
tccactaaac
aaagaactcc
5940


 


tgccttgatg
gatggagggc
aaattctggt
ggaacttttg
ggccacctga
aagttctatt
6000


 


cccaggacta
agaggaattt
cttttaatgg
atccagagag
ccaaggtcag
agggagagat
6060


 


ggcctgcata
gtctcctgtg
gatcacaccc
gggccacccc
tccctctagg
tttacagtgg
6120


 


acttcttctg
cccctcctcc
ttttctgtcc
ttggccatct
cagcctggcc
tctctgatcc
6180


 


ttccatcaca
gaaggatctt
gaatctctgg
gaaatcaaac
atcacagtag
tgatcagaaa
6240


 


gtgagtcctg
tcttgtcacc
ccatttctca
tcagaacaaa
gcacgayatg
gaatgaccaa
6300


 


ccagcattct
tcatggtgga
ctgcttatca
ttgaggatct
ttgggagata
aagcacgcta
6360


 


agagctctgg
acagagaaaa
acaggcccta
gaatatggga
gtgggtgttt
gtagggctca
6420


 


taggctaaca
agcactttag
ttgctggttt
acattcaatg
aaggaggatt
catacccatg
6480


 


gcattacaag
gctaagcatg
tgtatgacta
aggaactatc
tgaaaaacat
gcagcaaggt
6540


 


aagaaaatgt
accactcaac
aagccagtga
tgccaccttt
tgtgcgcggg
gaggagagtg
6600


 


actaccattg
ttttttgtgt
gacaaagcta
tcatggacta
ttttaatctt
ggttttattg
6660


 


cttaaaatat
attatttttc
cctatgtgtt
gacaaggtat
ttctaatatc
acactattaa
6720


 


atatatgcac
taatctaaat
aaaggtgtct
gtattttctg
taatgcttat
ttttaggggg
6780


 


aaatttgttt
tctttatgct
tcagggtaga
gggattccct
tgagtatagg
tcagcaaact
6840


 


ctggcctgca
gcctgtgtgt
qcacgcccca
tgagccgaaa
agtgggtctt
atgttttcaa
6900


 


atggttaaaa
ataaataaaa
aaatttgaaa
catgtgaact
atatgacatt
cagatttgtg
6960


 


ttcataaata
aagttttatt
ggaacatatc
c


6991
















TABLE LIII





Nucleotide sequence alignment of 254P1D6B v.1 (SEQ ID NO: 270)


and 254P1D6B v.3 (SEQ ID NO: 271)















Score = 781 bits (406), Expect = 0.0Identities = 406/406 (100%) Strand =


Plus/Plus


 








 








 


Score = 314 bits (163), Expect 2e-81Identities = 165/166 (99%) Strand =


Plus/Plus


 








 


Score = 1.197e+04 bits (6225), Expect = 0.0Identities = 6225/6225 (100%) Strand =


Plus/Plus


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIV





Peptide sequences of protein coded by 254P1D6B v.3 (SEQ ID NO: 272)






















MTRLGWPSPC
CARKQCSEGR
TYSNAVISPN
LETTRIMRVS
HTFPVVDCTA
ACCDLSSCDL
60



 


AWWFEGRCYL
VSCPHKENCE
PKKMGPIRSY
LTFVLRPVQR
PAQLLDYGDM
MLNRGSPSGI
120


 


WGDSPEDIRK
DLPFLGKDWG
LEEMSEYSDD
YRELEKDLLQ
PSGKQEPRGS
AEYTDWGLLP
180


 


GSEGAFNSSV
GDSPAVPAET
QQDPELHYLN
ESASTPAPKL
PERSVLLPLP
TTPSSGEVLE
240


 


KEKASQLQEQ
SSNSSGKEVL
MPSHSLPPAS
LELSSVTVEK
SPVLTVTPGS
TEHSIPTPPT
300


 


SAAPSESTPS
ELPISPTTAP
RTVKELTVSA
GDNLIITLPD
NEVELKAFVA
PAPPVETTYN
360


 


YEWNLISHPT
DYQGEIKQGH
KQTLNLSQLS
VGLYVFKVTV
SSENAFGEGF
VNVTVKPARR
420


 


VNLPPVAVVS
PQLQELTLPL
TSALIDGSQS
TDDTEIVSYH
WEEINGPFIE
EKTSVDSPVL
480


 


RLSNLDPGNY
SFRLTVTDSD
GATNSTTAAL
IVNNAVDYPP
VANAGPNHTI
TLPQNSITLN
540


 


GNQSSDDHQI
VLYEWSLGPG
SEGKHVVMQG
VQTPYLHLSA
MQEGDYTFQL
KVTDSSRQQS
600


 


TAVVTVIVQP
ENNRPPVAVA
GPDKELIFPV
ESATLDGSSS
SDDHGIVFYH
WEHVRGPSAV
660


 


EMENIDKAIA
TVTGLQVGTY
HFRLTVKDQQ
GLSSTSTLTV
AVKKENNSPP
RARAGGRHVL
720


 


VLPNNSITLD
GSRSTDDQRI
VSYLWIRDGQ
SPAAGDVIDG
SDHSVALQLT
NLVEGVYTFH
780


 


LRVTDSQGAS
DTDTATVEVQ
PDPRKSGLVE
LTLQVGVGQL
TEQRKDTLVR
QLAVLLNVLD
840


 


SDIKVQKIRA
HSDLSTVIVF
YVQSRPPFKV
LKAAEVARNL
HMRLSKEKAD
FLLFKVLRVD
900


 


TAGCLLKCSG
HGHCDPLTKR
CICSHLWMEN
LIQRYIWDGE
SNCEWSIFYV
TVLAFTLIVL
960


 


TGGFTWLCIC
CCKRQKRTKI
RKKTKYTILD
NMDEQERMEL
RPKYGIKHRS
TEHNSSLMVS
1020


 


ESEFDSDQDT
IFSREKMERG
NPKVSMNGSI
RNGASFSYCS
KDR

1063

















TABLE LV





Amino acid sequence alignment of 254P1D6B v.1 (SEQ ID NO: 273)



and 254P1D6B v.3 (SEQ ID NO: 274)
















Score = 2124 bits (5503), Expect = 0.0 Identities = 1053/1053 (100%), Positives = 1053/1053 (100%)



 









V.1: 20
CARKQCSEGRTYSNAVISPNLETTRIMRVSHTFPVVDCTAACCDLSSCDLAWWFEGRCYL
79


 



CARKQCSEGRTYSNAVISPNLETTRIMRVSHTFPVVDCTAACCDLSSCDLAWWFEGRCYL


 


V.3: 11
CARKQCSEGRTYSNAVISPNLETTRIMRVSHTFPVVDCTAACCDLSSCDLAWWFEGRCYL
70


 


V.1: 80
VSCPHKENCEPKKMGPIRSYLTFVLRPVQRPAQLLDYGDMMLNRGSPSGIWGDSPEDIRK
139


 



VSCPHKENCEPKKMGPIRSYLTFVLRPVQRPAQLLDYGDMMLNRGSPSGIWGDSPEDIRK


 


V.3: 71
VSCPHKENCEPKKMGPIRSYLTFVLRPVQRPAQLLDYGDMMLNRGSPSGIWGDSPEDIRK
130


 


V.1: 140
DLPFLGKDWGLEEMSEYSDDYRELEKDLLQPSGKQEPRGSAEYTDWGLLPGSEGAFNSSV
199


 



DLPFLGKDWGLEEMSEYSDDYRELEKDLLQPSGKQEPRGSAEYTDWGLLPGSEGAFNSSV


 


V.3: 131
DLPFLGKDWGLEEMSEYSDDYRELEKDLLQPSGKQEPRGSAEYTDWGLLPGSEGAFNSSV
190


 


V.1: 200
GDSPAVPAETQQDPELHYLNESASTPAPKLPERSVLLPLPTTPSSGEVLEKEKASQLQEQ
259


 



GDSPAVPAETQQDPELHYLNESASTPAPKLPERSVLLPLPTTPSSGEVLEKEKASQLQEQ


 


V.3: 191
GDSPAVPAETQQDPELHYLNESASTPAPKLPERSVLLPLPTTPSSGEVLEKEKASQLQEQ
250


 


V.1: 260
SSNSSGKEVLMPSHSLPPASLELSSVTVEKSPVLTVTPGSTEHSIPTPPTSAAPSESTPS
319


 



SSNSSGKEVLMPSHSLPPASLELSSVTVEKSPVLTVTPGSTEHSIPTPPTSAAPSESTPS


 


V.3: 251
SSNSSGKEVLMPSHSLPPASLELSSVTVEKSPVLTVTPGSTEHSIPTPPTSAAPSESTPS
310


 


V.1: 320
ELPISPTTAPRTVKELTVSAGDNLIITLPDNEVELKAFVAPAPPVETTYNYEWNLISHPT
379


 



ELPISPTTAPRTVKELTVSAGDNLIITLPDNEVELKAFVAPAPPVETTYNYEWNLISHPT


 


V.3: 311
ELPISPTTAPRTVKELTVSAGDNLIITLPDNEVELKAFVAPAPPVETTYNYEWNLISHPT
370


 


V.1: 380
DYQGEIKQGHKQTLNLSQLSVGLYVFKVTVSSENAFGEGFVNVTVKPARRVNLPPVAVVS
439


 



DYQGEIKQGHKQTLNLSQLSVGLYVFKVTVSSENAFGEGFVNVTVKPARRVNLPPVAVVS


 


V.3: 371
DYQGEIKQGHKQTLNLSQLSVGLYVFKVTVSSENAFGEGFVNVTVKPARRVNLPPVAVVS
430


 


V.1: 440
PQLQELTLPLTSALIDGSQSTDDTEIVSYHWEEINGPFIEEKTSVDSPVLRLSNLDPGNY
499


 



PQLQELTLPLTSALIDGSQSTDDTEIVSYHWEEINGPFIEEKTSVDSPVLRLSNLDPGNY


 


V.3: 431
PQLQELTLPLTSALIDGSQSTDDTEIVSYHWEEINGPFIEEKTSVDSPVLRLSNLDPGNY
490


 


V.1: 500
SFRLTVTDSDGATNSTTAALIVNNAVDYPPVANAGPNHTITLPQNSITLNGNQSSDDHQI
559


 



SFRLTVTDSDGATNSTTAALIVNNAVDYPPVANAGPNHTITLPQNSITLNGNQSSDDHQI


 


V.3: 491
SFRLTVTDSDGATNSTTAALIVNNAVDYPPVANAGPNNTITLPQNSITLNGNQSSDDHQI
550


 


V.1: 560
VLYEWSLGPGSEGKHVVMQGVQTPYLHLSAMQEGDYTFQLKVTDSSRQQSTAVVTVIVQP
619


 



VLYEWSLGPGSEGKHVVMQGVQTPYLHLSAMQEGDYTFQLKVTDSSRQQSTAVVTVIVQP


 


V.3: 551
VLYEWSLGPGSEGKHVVMQGVQTPYLHLSAMQEGDYTFQLKVTDSSRQQSTAVVTVIVQP
610


 


V.1: 620
ENNRPPVAVAGPDKELIFPVESATLDGSSSSDDHGIVFYHWEHVRGPSAVEMENIDKAIA
679


 



ENNRPPVAVAGPDKELIFPVESATLDGSSSSDDHGIVFYHWEHVRGPSAVEMENIDKAIA


 


V.3: 611
ENNRPPVAVAGPDKELIFPVESATLDGSSSSDDHGIVFYHWEHVRGPSAVEMENIDKAIA
670


 


V.1: 680
TVTGLQVGTYHFRLTVKDQQGLSSTSTLTVAVKKENNSPPRARAGGRHVLVLPNNSITLD
739


 



TVTGLQVGTYHFRLTVKDQQGLSSTSTLTVAVKKENNSPPRARAGGRHVLVLPNNSITLD


 


V.3: 671
TVTGLQVGTYHFRLTVKDQQGLSSTSTLTVAVKKENNSPPRARAGGRHVLVLPNNSITLD
730


 


V.1: 740
GSRSTDDQRIVSYLWIRDGQSPAAGDVIDGSDHSVALQLTNLVEGVYTFHLRVTDSQGAS
799


 



GSRSTDDQRIVSYLWIRDGQSPAAGDVIDGSDHSVALQLTNLVEGVYTFHLRVTDSQGAS


 


V.3: 731
GSRSTDDQRIVSYLWIRDGQSPAAGDVIDGSDHSVALQLTNLVEGVYTFHLRVTDSQGAS
790


 


V.1: 800
DTDTATVEVQPDPRKSGLVELTLQVGVGQLTEQRKDTLVRQLAVLLNVLDSDIKVQKIRA
859


 



DTDTATVEVQPDPRKSGLVELTLQVGVGQLTEQRKDTLVRQLAVLLNVLDSDIKVQKIRA


 


V.3: 791
DTDTATVEVQPDPRKSGLVELTLQVGVGQLTEQRKDTLVRQLAVLLNVLDSDIKVQKIRA
850


 


V.1: 860
HSDLSTVIVFYVQSRPPFKVLKAAEVARNLHMRLSKEKADFLLFKVLRVDTAGCLLKCSG
919


 



HSDLSTVIVFYVQSRPPFKVLKAAEVARNLHMRLSKEKADFLLFKVLRVDTAGCLLKCSG


 


V.3: 851
HSDLSTVIVFYVQSRPPFKVLKAAEVARNLHMRLSKEKADFLLFKVLRVDTAGCLLKCSG
910


 


V.1: 920
HGHCDPLTKRCICSHLWMENLIQRYIWDGESNCEWSIFYVTVLAFTLIVLTGGFTWLCIC
979


 



HGHCDPLTKRCICSHLWMENLIQRYIWDGESNCEWSIFYVTVLAFTLIVLTGGFTWLCIC


 


V.3: 911
HGHCDPLTKRCICSHLWMENLIQRYIWDGESNCEWSIFYVTVLAFTLIVLTGGFTWLCIC
970


 


V.1: 980
CCKRQKRTKIRKKTKYTILDNMDEQERMELRPKYGIKHRSTEHNSSLMVSESEFDSDQDT
1039


 



CCKRQKRTKIRKKTKYTILDNMDEQERMELRPKYGIKHRSTEHNSSLMVSESEFDSDQDT


 


V.3: 971
CCKRQKRTKIRKKTKYTILDNMDEQERMELRPKYGIKHRSTEHNSSLMVSESEFDSDQDT
1030


 


V.1: 1040
IFSREKMERGNPKVSMNGSIRNGASFSYCSKDR
1072


 



IFSREKMERGNPKVSMNGSIRNGASFSYCSKDR


 


V.3: 1031
IFSREKMERGNPKVSMNGSIRNGASFSYCSKDR
1063

## Claims

1. An isolated or recombinant polypeptide having SEQ ID NO: 3 or 7.

2. A composition which comprises the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

3. A method of generating an immune response in a mammalian subject comprising exposing cells of the mammal's immune system to a polypeptide having SEQ ID NO: 3 or 7, whereby an immune response to the polypeptide is generated, and wherein said immune response is the activation of B cells.

4. A polynucleotide that encodes the polypeptide of claim 1 wherein T can also be U.

5. A polynucleotide having SEQ ID NO: 2 or 6, wherein the polynucleotide encodes an isolated or recombinant polypeptide having SEQ ID NO: 3 or 7.

6. An isolated host cell modified to contain an expression vector for expressing the polynucleotide of claim 4.

7. A method for detecting the presence of prostate cancer expressing a 254P1D6B protein in an individual comprising:
determining the level of expression of a polypeptide having SEQ ID NO: 3 or 7 in a test tissue sample from an individual; wherein the test tissue sample consists of prostate tissue; and
comparing the level so determined to the level of expression that is evidenced in a normal tissue sample,
wherein the elevated expression of said polypeptide in the test tissue sample versus the normal tissue sample is an indication of the presence of cancer in the test tissue sample.